Coinhibitory Pathways in Immunotherapy for Cancer.

The immune system is capable of recognizing tumors and eliminates many early malignant cells. However, tumors evolve to evade immune attack, and the tumor microenvironment is immunosuppressive. Immune responses are regulated by a number of immunological checkpoints that promote protective immunity and maintain tolerance. T cell coinhibitory pathways restrict the strength and duration of immune responses, thereby limiting immune-mediated tissue damage, controlling resolution of inflammation, and maintaining tolerance to prevent autoimmunity. Tumors exploit these coinhibitory pathways to evade immune eradication. Blockade of the PD-1 and CTLA-4 checkpoints is proving to be an effective and durable cancer immunotherapy in a subset of patients with a variety of tumor types, and additional combinations are further improving response rates. In this review we discuss the immunoregulatory functions of coinhibitory pathways and their translation to effective immunotherapies for cancer.

GM-CSF, IL-3, and IL-5 Family of Cytokines: Regulators of Inflammation.

The ß common chain cytokines GM-CSF, IL-3, and IL-5 regulate varied inflammatory responses that promote the rapid clearance of pathogens but also contribute to pathology in chronic inflammation. Therapeutic interventions manipulating these cytokines are approved for use in some cancers as well as allergic and autoimmune disease, and others show promising early clinical activity. These approaches are based on our understanding of the inflammatory roles of these cytokines; however, GM-CSF also participates in the resolution of inflammation, and IL-3 and IL-5 may also have such properties. Here, we review the functions of the ß common cytokines in health and disease. We discuss preclinical and clinical data, highlighting the potential inherent in targeting these cytokine pathways, the limitations, and the important gaps in understanding of the basic biology of this cytokine family.

A vaccine targeting resistant tumours by dual T cell plus NK cell attack.

Most cancer vaccines target peptide antigens, necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore, tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells, but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding, enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably, this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary, highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations.

Immune surveillance and therapy of lymphomas driven by Epstein-Barr virus protein LMP1 in a mouse model.

B cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.

Colon stroma mediates an inflammation-driven fibroblastic response controlling matrix remodeling and healing.

Chronic inflammation is often associated with the development of tissue fibrosis, but how mesenchymal cell responses dictate pathological fibrosis versus resolution and healing remains unclear. Defining stromal heterogeneity and identifying molecular circuits driving extracellular matrix deposition and remodeling stands to illuminate the relationship between inflammation, fibrosis, and healing. We performed single-cell RNA-sequencing of colon-derived stromal cells and identified distinct classes of fibroblasts with gene signatures that are differentially regulated by chronic inflammation, including IL-11-producing inflammatory fibroblasts. We further identify a transcriptional program associated with trans-differentiation of mucosa-associated fibroblasts and define a functional gene signature associated with matrix deposition and remodeling in the inflamed colon. Our analysis supports a critical role for the metalloprotease Adamdec1 at the interface between tissue remodeling and healing during colitis, demonstrating its requirement for colon epithelial integrity. These findings provide mechanistic insight into how inflammation perturbs stromal cell behaviors to drive fibroblastic responses controlling mucosal matrix remodeling and healing.

Invariant NKT cell-augmented GM-CSF-secreting tumor vaccine is effective in advanced prostate cancer model.

Invariant natural killer T cells (iNKT cells) express a semi-invariant T cell receptor that recognizes certain glycolipids (including α-galactosylceramide, αGC) bound to CD1d, and can induce potent antitumor responses. Here, we assessed whether αGC could enhance the efficacy of a GM-CSF-producing tumor cell vaccine in the transgenic SV40 T antigen-driven TRAMP prostate cancer model. In healthy mice, we initially found that optimal T cell responses were obtained with αGC-pulsed TRAMP-C2 cells secreting GM-CSF and milk fat globule epidermal growth factor protein-8 (MFG-E8) with an RGD to RGE mutation (GM-CSF/RGE TRAMP-C2), combined with systemic low dose IL-12. In a therapeutic model, transgenic TRAMP mice were then castrated at ~ 20 weeks, followed by treatment with the combination vaccine. Untreated mice succumbed to tumor by ~ 40 weeks, but survival was markedly prolonged by vaccine treatment, with most mice surviving past 80 weeks. Prostates in the treated mice were heavily infiltrated with T cells and iNKT cells, which both secreted IFNγ in response to tumor cells. The vaccine was not effective if the αGC, IL-12, or GM-CSF secretion was eliminated. Finally, immunized mice were fully resistant to challenge with TRAMP-C2 cells. Together these findings support further development of therapeutic vaccines that exploit iNKT cell activation.

The feasibility of using an autologous GM-CSF-secreting breast cancer vaccine to induce immunity in patients with stage II-III and metastatic breast cancers.

PURPOSE: The antigenic targets of immunity and the role of vaccination in breast cancer are unknown. We performed a phase I study of an autologous GM-CSF-secreting breast cancer vaccine in patients with metastatic and stage II-III breast cancer. METHODS: Tumor cells from patients with metastatic (n = 15) and stage II-III (n = 7) disease were transduced with a replication-defective adenoviral vector encoding GM-CSF, and then irradiated. Twelve and seven patients with metastatic and stage II-III disease, respectively, received weekly vaccination for three weeks, followed by every other week until disease progression or vaccine supply was exhausted (metastatic) or until six total vaccine doses were administered (stage II-III). RESULTS: Among those patients with metastatic disease who received vaccinations, eight had progressive disease at two months, three had stable disease for 4-13 months, and one has had no evidence of disease for 13 years. Of the patients with stage II-III disease, five died of metastatic disease between 1.16 and 8.49 years after the start of vaccinations (median 6.24 years) and two are alive as of September 2021. Toxicities included injection site reactions, fatigue, fever, upper respiratory symptoms, joint pain, nausea, and edema. Four of five evaluable patients with metastatic disease developed a skin reaction with immune cell infiltration after the fifth injection of unmodified, irradiated tumor cells. CONCLUSION: We conclude that tumor cells can be harvested from patients with metastatic or stage II-III breast cancer to prepare autologous GM-CSF-secreting vaccines that induce coordinated immune responses with limited toxicity. TRIAL REGISTRATION AND DATE OF REGISTRATION: clinicaltrials.gov, NCT00317603 (April 25, 2006) and NCT00880464 (April 13, 2009).

In vivo discovery of immunotherapy targets in the tumour microenvironment.

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments.

Identification and characterization of an alternative cancer-derived PD-L1 splice variant.

Therapeutic blockade of the PD-1/PD-L1 axis is recognized as an effective treatment for numerous cancer types. However, only a subset of patients respond to this treatment, warranting a greater understanding of the biological mechanisms driving immune evasion via PD-1/PD-L1 signaling and other T-cell suppressive pathways. We previously identified a head and neck squamous cell carcinoma with human papillomavirus integration in the PD-L1 locus upstream of the transmembrane domain-encoding region, suggesting expression of a truncated form of PD-L1 (Parfenov et al., Proc Natl Acad Sci USA 111(43):15544-15549, 2014). In this study, we extended this observation by performing a computational analysis of 33 other cancer types as well as human cancer cell lines, and identified additional PD-L1 isoforms with an exon 4 enrichment expressed in 20 cancers and human cancer cell lines. We demonstrate that cancer cell lines with high expression levels of exon 4-enriched PD-L1 generate a secreted form of PD-L1. Further biochemical studies of exon 4-enriched PD-L1 demonstrated that this form is secreted and maintains the capacity to bind PD-1 as well as to serve as a negative regulator on T cell function, as measured by inhibition of IL-2 and IFNg secretion. Overall, we have demonstrated that truncated forms of PD-L1 exist in numerous cancer types, and have validated that truncated PD-L1 can be secreted and negatively regulate T cell function.

Manufacturing development and clinical production of NKG2D chimeric antigen receptor-expressing T cells for autologous adoptive cell therapy.

BACKGROUND AIMS: Adoptive cell therapy employing natural killer group 2D (NKG2D) chimeric antigen receptor (CAR)-modified T cells has demonstrated preclinical efficacy in several model systems, including hematological and solid tumors. We present comprehensive data on manufacturing development and clinical production of autologous NKG2D CAR T cells for treatment of acute myeloid leukemia and multiple myeloma (ClinicalTrials.gov Identifier: NCT02203825). An NKG2D CAR was generated by fusing native full-length human NKG2D to the human CD3ζ cytoplasmic signaling domain. NKG2D naturally associates with native costimulatory molecule DAP10, effectively generating a second-generation CAR against multiple ligands upregulated during malignant transformation including MIC-A, MIC-B and the UL-16 binding proteins. METHODS: CAR T cells were infused fresh after a 9-day process wherein OKT3-activated T cells were genetically modified with replication-defective gamma-retroviral vector and expanded ex vivo for 5 days with recombinant human interleukin-2. RESULTS: Despite sizable interpatient variation in originally collected cells, release criteria, including T-cell expansion and purity (median 98%), T-cell transduction (median 66% CD8+ T cells), and functional activity against NKG2D ligand-positive cells, were met for 100% of healthy donors and patients enrolled and collected. There was minimal carryover of non-T cells, particularly malignant cells; both effector memory and central memory cells were generated, and inflammatory cytokines such as granulocyte colony-stimulating factor, RANTES, interferon-γ and tumor necrosis factor-α were selectively up-regulated. CONCLUSIONS: The process resulted in production of required cell doses for the first-in-human phase I NKG2D CAR T clinical trial and provides a robust, flexible base for further optimization of NKG2D CAR T-cell manufacturing.

Immunophenotyping of pediatric brain tumors: correlating immune infiltrate with histology, mutational load, and survival and assessing clonal T cell response.

There is little known regarding the immune infiltrate present in pediatric brain tumors and how this compares to what is known about histologically similar adult tumors and its correlation with survival. Here, we provide a descriptive analysis of the immune infiltrate of 22 fresh pediatric brain tumor tissue samples of mixed diagnoses and 40 peripheral blood samples. Samples were analyzed using a flow cytometry panel containing markers for immune cell subtypes, costimulatory markers, inhibitory signals, and markers of activation. This was compared to the standard method of immunohistochemistry (IHC) for immune markers for 89 primary pediatric brain tumors. Both flow cytometry and IHC data did not correlate with the grade of tumor or mutational load and IHC data was not significantly associated with survival for either low grade or high grade gliomas. There is a trend towards a more immunosuppressive phenotype in higher grade tumors with more regulatory T cells present in these tumor types. Both PD1 and PDL1 were present in only a small percentage of the tumor infiltrate. T cell receptor sequencing revealed up to 10% clonality of T cells in tumor infiltrates and no significant difference in clonality between low and high grade gliomas. We have shown the immune infiltrate of pediatric brain tumors does not appear to correlate with grade or survival for a small sample of patients. Further research and larger studies are needed to fully understand the interaction of pediatric brain tumors and the immune system.

Mechanisms of Acute Toxicity in NKG2D Chimeric Antigen Receptor T Cell-Treated Mice.

Targeting cancer through the use of effector T cells bearing chimeric Ag receptors (CARs) leads to elimination of tumors in animals and patients, but recognition of normal cells or excessive activation can result in significant toxicity and even death. CAR T cells based on modified NKG2D receptors are effective against many types of tumors, and their efficacy is mediated through direct cytotoxicity and cytokine production. Under certain conditions, their ligands can be expressed on nontumor cells, so a better understanding of the potential off-tumor activity of these NKG2D CAR T cells is needed. Injection of very high numbers of activated T cells expressing CARs based on murine NKG2D or DNAM1 resulted in increased serum cytokines (IFN-γ, IL-6, and MCP-1) and acute toxicity similar to cytokine release syndrome. Acute toxicity required two key effector molecules in CAR T cells-perforin and GM-CSF. Host immune cells also contributed to this toxicity, and mice with severe immune cell defects remained healthy at the highest CAR T cell dose. These data demonstrate that specific CAR T cell effector mechanisms and the host immune system are required for this cytokine release-like syndrome in murine models.

Cancer immunotherapy comes of age.

Activating the immune system for therapeutic benefit in cancer has long been a goal in immunology and oncology. After decades of disappointment, the tide has finally changed due to the success of recent proof-of-concept clinical trials. Most notable has been the ability of the anti-CTLA4 antibody, ipilimumab, to achieve a significant increase in survival for patients with metastatic melanoma, for which conventional therapies have failed. In the context of advances in the understanding of how tolerance, immunity and immunosuppression regulate antitumour immune responses together with the advent of targeted therapies, these successes suggest that active immunotherapy represents a path to obtain a durable and long-lasting response in cancer patients.

Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis.

OBJECTIVE: Accumulating evidence links the intestinal microbiota and colorectal carcinogenesis. Fusobacterium nucleatum may promote colorectal tumour growth and inhibit T cell-mediated immune responses against colorectal tumours. Thus, we hypothesised that the amount of F. nucleatum in colorectal carcinoma might be associated with worse clinical outcome. DESIGN: We used molecular pathological epidemiology database of 1069 rectal and colon cancer cases in the Nurses' Health Study and the Health Professionals Follow-up Study, and measured F. nucleatum DNA in carcinoma tissue. Cox proportional hazards model was used to compute hazard ratio (HR), controlling for potential confounders, including microsatellite instability (MSI, mismatch repair deficiency), CpG island methylator phenotype (CIMP), KRAS, BRAF, and PIK3CA mutations, and LINE-1 hypomethylation (low-level methylation). RESULTS: Compared with F. nucleatum-negative cases, multivariable HRs (95% CI) for colorectal cancer-specific mortality in F. nucleatum-low cases and F. nucleatum-high cases were 1.25 (0.82 to 1.92) and 1.58 (1.04 to 2.39), respectively, (p for trend=0.020). The amount of F. nucleatum was associated with MSI-high (multivariable odd ratio (OR), 5.22; 95% CI 2.86 to 9.55) independent of CIMP and BRAF mutation status, whereas CIMP and BRAF mutation were associated with F. nucleatum only in univariate analyses (p<0.001) but not in multivariate analysis that adjusted for MSI status. CONCLUSIONS: The amount of F. nucleatum DNA in colorectal cancer tissue is associated with shorter survival, and may potentially serve as a prognostic biomarker. Our data may have implications in developing cancer prevention and treatment strategies through targeting GI microflora by diet, probiotics and antibiotics.

High-throughput oncogene mutation profiling in human cancer.

Systematic efforts are underway to decipher the genetic changes associated with tumor initiation and progression. However, widespread clinical application of this information is hampered by an inability to identify critical genetic events across the spectrum of human tumors with adequate sensitivity and scalability. Here, we have adapted high-throughput genotyping to query 238 known oncogene mutations across 1,000 human tumor samples. This approach established robust mutation distributions spanning 17 cancer types. Of 17 oncogenes analyzed, we found 14 to be mutated at least once, and 298 (30%) samples carried at least one mutation. Moreover, we identified previously unrecognized oncogene mutations in several tumor types and observed an unexpectedly high number of co-occurring mutations. These results offer a new dimension in tumor genetics, where mutations involving multiple cancer genes may be interrogated simultaneously and in 'real time' to guide cancer classification and rational therapeutic intervention.

OX40 engagement depletes intratumoral Tregs via activating FcγRs, leading to antitumor efficacy.

Antibodies targeting checkpoint inhibitors or co-stimulatory receptors on T cells have shown significant antitumor efficacy in preclinical and clinical studies. In mouse tumor models, engagement of activating Fcγ receptor (FcγR)-expressing immune cells was recently shown to be required for the tumoricidal activity of antibodies recognizing the tumor necrosis factor superfamily receptor (TNFR) GITR (CD357) and CTLA-4 (CD152). In particular, activating FcγRs facilitated the selective elimination of intratumoral T-cell populations. However, it remains unclear whether FcγRs contribute to the antitumor efficacy of other immunomodulatory antibodies. Here, we explored the mechanism of antitumor activity mediated by an agonistic antibody (clone OX86) to the co-stimulatory TNFR OX40 (CD134). OX40 was highly expressed by intratumoral T cells, particularly those of the FoxP3(+) regulatory T-cell (Treg) lineage. OX86 administration resulted in the depletion of intratumoral regulatory T cells in an activating FcγR-dependent manner, which correlated with tumor regression. Together with previous data from our group and others, these findings support a mechanism whereby antibodies targeting antigens highly expressed by intratumoral T cells can mediate their elimination by FcγR-expressing immune cells, and facilitate subsequent antitumor immunity.

Compartment-Specific Bioluminescence Imaging platform for the high-throughput evaluation of antitumor immune function.

Conventional assays evaluating antitumor activity of immune effector cells have limitations that preclude their high-throughput application. We adapted the recently developed Compartment-Specific Bioluminescence Imaging (CS-BLI) technique to perform high-throughput quantification of innate antitumor activity and to show how pharmacologic agents (eg, lenalidomide, pomalidomide, bortezomib, and dexamethasone) and autologous BM stromal cells modulate that activity. CS-BLI-based screening allowed us to identify agents that enhance or inhibit innate antitumor cytotoxicity. Specifically, we identified compounds that stimulate immune effector cells against some tumor targets but suppressed their activity against other tumor cells. CS-BLI offers rapid, simplified, and specific evaluation of multiple conditions, including drug treatments and/or cocultures with stromal cells and highlights that immunomodulatory pharmacologic responses can be heterogeneous across different types of tumor cells. This study provides a framework to identify novel immunomodulatory agents and to prioritize compounds for clinical development on the basis of their effect on antitumor immunity.

Phase II trial of vaccination with autologous, irradiated melanoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor in patients with stage III and IV melanoma.

Background: In the era of immune checkpoint blockade, the role of cancer vaccines in immune priming has provided additional potential for therapeutic improvements. Prior studies have demonstrated delayed type hypersensitivity and anti-tumor immunity with vaccines engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). The safety, efficacy and anti-tumor immunity of GM-CSF secreting vaccine in patients with previously treated stage III or IV melanoma needs further investigation. Methods: In this phase II trial, excised lymph node metastases were processed to single cells, transduced with an adenoviral vector encoding GM-CSF, irradiated, and cryopreserved. Individual vaccines were composed of 1x106, 4x106, or 1x107 tumor cells, and were injected intradermally and subcutaneously at weekly and biweekly intervals. The primary endpoints were feasibility of producing vaccine in stage III patients and determining the proportion of patients alive at two years in stage IV patients. Results: GM-CSF vaccine was successfully developed and administered in all 61 patients. Toxicities were restricted to grade 1-2 local skin reactions. The median OS for stage III patients (n = 20) was 71.1 (95% CI, 43.7 to NR) months and 14.9 (95%CI, 12.1 to 39.7) months for stage IV patients. The median PFS in stage III patients was 50.7 (95%CI, 36.3 to NR) months and 4.1 (95% CI, 3.0-6.3) months in stage IV patients. In the overall population, the disease control rate was 39.3% (95%CI, 27.1 to 52.7%). In stage III patients, higher pre-treatment plasma cytokine levels of MMP-1, TRAIL, CXCL-11, CXCL-13 were associated with improved PFS (p<0.05 for all). An increase in post-vaccination levels of IL-15 and TRAIL for stage III patients was associated with improved PFS (p=0.03 for both). Similarly, an increase in post-vaccination IL-16 level for stage IV patients was associated with improved PFS (p=0.02) and clinical benefit. Conclusions: Vaccination with autologous melanoma cells secreting GM-CSF augments antitumor immunity in stage III and IV patients with melanoma, is safe, and demonstrates disease control. Luminex data suggests that changes in inflammatory cytokines and immune cell infiltration promote tumor antigen presentation and subsequent tumor cell destruction. Additional investigation to administer this vaccine in combination with immune checkpoint inhibitors is needed.

A molecular glue degrader of the WIZ transcription factor for fetal hemoglobin induction.

Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.