Host tissue determinants of tumour immunity
Although common evolutionary principles drive the growth of cancer cells regardless of the tissue of origin, the microenvironment in which tumours arise substantially differs across various organ sites. Recent studies have established that, in addition to cell-intrinsic effects, tumour growth regulation also depends on local cues driven by tissue environmental factors. In this Review, we discuss how tissue-specific determinants might influence tumour development and argue that unravelling the tissue-specific contribution to tumour immunity should help the development of precise immunotherapeutic strategies for patients with cancer.
An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma
Enthusiasm for the use of antibody-drug conjugates (ADCs) in cancer therapy has risen over the past few years. The success of this therapeutic approach relies on the identification of cell surface antigens that are widely and selectively expressed on tumor cells. Studies have shown that native ALK protein is expressed on the surface of most neuroblastoma cells, providing an opportunity for development of immune-targeting strategies. Clinically relevant antibodies for this target have not yet been developed. Here, we describe the development of an ALK-ADC, CDX-0125-TEI, which selectively targets both wild-type and mutated ALK-expressing neuroblastomas. CDX-0125-TEI exhibited efficient antigen binding and internalization, and cytotoxicity at picomolar concentrations in cells with different expression of ALK on the cell surface. In vivo studies showed that CDX-0125-TEI is effective against ALK wild-type and mutant patient-derived xenograft models. These data demonstrate that ALK is a bona fide immunotherapeutic target and provide a rationale for clinical development of an ALK-ADC approach for neuroblastomas and other ALK-expressing childhood cancers such as rhabdomyosarcomas.
Modulation of TAP-dependent antigen compartmentalization during human monocyte-to-DC differentiation
Dendritic cells (DCs) take up antigen in the periphery, migrate to secondary lymphoid organs, and present processed antigen fragments to adaptive immune cells and thus prime antigen-specific immunity. During local inflammation, recirculating monocytes are recruited from blood to the inflamed tissue, where they differentiate to macrophages and DCs. In this study, we found that monocytes showed high transporter associated with antigen processing (TAP)-dependent peptide compartmentalization and that after antigen pulsing, they were not able to efficiently stimulate antigen-specific T lymphocytes. Nevertheless, upon in vitro differentiation to monocyte-derived DCs, TAP-dependent peptide compartmentalization as well as surface major histocompatibility complex I turnover decreased and the cells efficiently restimulated T lymphocytes. Although TAP-dependent peptide compartmentalization decreased during DC differentiation, TAP expression levels increased. Furthermore, TAP relocated from early endosomes in monocytes to the endoplasmic reticulum (ER) and lysosomal compartments in DCs. Collectively, these data are compatible with the model that during monocyte-to-DC differentiation, the subcellular relocation of TAP and the regulation of its activity assure spatiotemporal separation of local antigen uptake and processing by monocytes and efficient T-lymphocyte stimulation by DCs.
Model to improve specificity for identification of clinically-relevant expanded T cells in peripheral blood
Current methods to quantify T-cell clonal expansion only account for variance due to random sampling from a highly diverse repertoire space. We propose a beta-binomial model to incorporate time-dependent variance into the assessment of differentially abundant T-cell clones, identified by unique T Cell Receptor (TCR) beta-chain rearrangements, and show that this model improves specificity for detecting clinically relevant clonal expansion. Using blood samples from ten healthy donors, we modeled the variance of T-cell clones within each subject over time and calibrated the dispersion parameters of the beta distribution to fit this variance. As a validation, we compared pre- versus post-treatment blood samples from urothelial cancer patients treated with atezolizumab, where clonal expansion (quantified by the earlier binomial model) was previously reported to correlate with benefit. The beta-binomial model significantly reduced the false-positive rate for detecting differentially abundant clones over time compared to the earlier binomial method. In the urothelial cancer cohort, the beta-binomial model enriched for tumor infiltrating lymphocytes among the clones detected as expanding in the peripheral blood in response to therapy compared to the binomial model and improved the overall correlation with clinical benefit. Incorporating time-dependent variance into the statistical framework for measuring differentially abundant T-cell clones improves the model's specificity for T-cells that correlate more strongly with the disease and treatment setting of-interest. Reducing background-level clonal expansion, therefore, improves the quality of clonal expansion as a biomarker for assessing the T cell immune response and correlations with clinical measures.
Dual Synthetic Peptide Conjugate Vaccine Simultaneously Triggers TLR2 and NOD2 and Activates Human Dendritic Cells
Simultaneous triggering of Toll-like receptors (TLRs) and NOD-like receptors (NLRs) has previously been shown to synergistically activate monocytes, dendritic cells and macrophages. We applied these properties in a T-cell vaccine setting by conjugating the NOD2-ligand muramyl-dipeptide (MDP) and TLR2-ligand Pam3CSK4 to a synthetic peptide derived from a model antigen. Stimulation of human DCs with the MDP-peptide-Pam3CSK4 conjugate led to a strongly increased secretion of pro-inflammatory and Th1-type cytokines and chemokines. We further show that the conjugated ligands retain their ability to trigger their respective receptors, while even improving NOD2-triggering. Also, activation of murine DCs was enhanced by the dual triggering, ultimately leading to effective induction of vaccine-specific T cells expressing IFNgamma, IL-2 and TNFalpha. Together, these data indicate that the dual MDP-SLP-Pam3CSK4 conjugate constitutes a chemically well-defined vaccine approach that holds promise for the use in the treatment of virus infections and cancer.
New epitopes in ovalbumin provide insights for cancer neoepitopes
MHC I-restricted epitopes of chicken ovalbumin (OVA) were originally identified using CD8 T cells as probes. Here, using bioinformatics tools, we identify four additional epitopes in OVA in addition to a cryptic epitope. Each new epitope is presented in vivo, as deduced from the lack of CD8 response to it in OVA-transgenic mice. In addition, CD8 responses to the known and novel epitopes are examined in C57BL/6 mice exposed to the OVA-expressing tumor E.G7 in several ways. No responses to any epitope including SIINFEKL are detected in mice with growing E.G7 or mice immunized with the tumor. Only in E.G7-bearing mice treated with an anti-CTLA4 antibody which depletes tumor-infiltrating regulatory T cells, CD8 responses to SIINFEKL and the novel epitope EKYNLTSVL are detected. Finally, all epitopes fails to treat mice with pre-existing tumors. These observations force an important re-consideration of the common assumptions about the therapeutic value of neoepitopes detected by CD8 responses in tumor-bearing hosts.
Recent clinical trials of immunotherapy in non-small-cell lung cancer
Deciphering CD4(+) T cell specificity using novel MHC-TCR chimeric receptors
alphabeta T cell antigen receptors (TCRs) bind complexes of peptide and major histocompatibility complex (pMHC) with low affinity, which poses a considerable challenge for the direct identification of alphabeta T cell cognate peptides. Here we describe a platform for the discovery of MHC class II epitopes based on the screening of engineered reporter cells expressing novel pMHC-TCR (MCR) hybrid molecules carrying cDNA-derived peptides. This technology identifies natural epitopes of CD4(+) T cells in an unbiased and efficient manner and allows detailed analysis of TCR cross-reactivity that provides recognition patterns beyond discrete peptides. We determine the cognate peptides of virus- and tumor-specific T cells in mouse disease models and present a proof of concept for human T cells. Furthermore, we use MCR to identify immunogenic tumor neo-antigens and show that vaccination with a peptide naturally recognized by tumor-infiltrating lymphocytes efficiently protects mice from tumor challenge. Thus, the MCR technology holds promise for basic research and clinical applications, allowing the personalized identification of T cell-specific neo-antigens in patients.
Enhancing the antitumor functions of invariant natural killer T cells using a soluble CD1d-CD19 fusion protein
Invariant natural killer T (iNKT) cells comprise a unique lineage of CD1d-restricted lipid-reactive T lymphocytes that potently kill tumor cells and exhibit robust immunostimulatory functions. Optimal tumor-directed iNKT cell responses often require expression of the antigen-presenting molecule CD1d on tumors; however, many tumor cells downregulate CD1d and thus evade iNKT cell recognition. We generated a soluble bispecific fusion protein designed to direct iNKT cells to the site of B-cell cancers in a tumor antigen-specific but CD1d-independent manner. This fusion protein is composed of a human CD1d molecule joined to a single chain antibody FV fragment specific for CD19, an antigen widely expressed on B-cell cancers. The CD1d-CD19 fusion protein binds specifically to CD19-expressing, but not CD19-negative cells. Once loaded with the iNKT cell lipid agonist alpha-galactosyl ceramide (alphaGC), the CD1d-CD19 fusion induces robust in vitro activation of and cytokine production by human iNKT cells. iNKT cells stimulated by the alphaGC-loaded CD1d-CD19 fusion also strongly transactivate T-, B-, and NK-cell responses and promote dendritic cell maturation. Importantly, the alphaGC-loaded fusion induces robust lysis of CD19(+)CD1d(-) Epstein-Barr virus immortalized human B-lymphoblastoid cell lines that are otherwise resistant to iNKT cell killing. Consistent with these findings; administration of the alphaGC-loaded fusion protein controlled the growth of CD19(+)CD1d(-) tumors in vivo, suggesting that it can "link" iNKT cells and CD19(+)CD1d(-) targets in a therapeutically beneficial manner. Taken together, these preclinical studies demonstrate that this B cell-directed fusion protein can be used to effectively induce iNKT cell antitumor responses in vitro and in vivo.
Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells
A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA-E in tumor cells, which suppresses natural killer (NK) cell activity via ligation of the NK inhibitory receptor CD94/NKG2A. Gene expression data from approximately 10,000 tumor samples showed widespread HLAE expression, with levels correlating with those of KLRC1 (NKG2A) and KLRD1 (CD94). To bypass HLA-E inhibition, we developed a way to generate highly functional NK cells lacking NKG2A. Constructs containing a single-chain variable fragment derived from an anti-NKG2A antibody were linked to endoplasmic reticulum-retention domains. After retroviral transduction in human peripheral blood NK cells, these NKG2A Protein Expression Blockers (PEBLs) abrogated NKG2A expression. The resulting NKG2Anull NK cells had higher cytotoxicity against HLA-E-expressing tumor cells. Transduction of anti-NKG2A PEBL produced more potent cytotoxicity than interference with an anti-NKG2A antibody and prevented de novo NKG2A expression, without affecting NK cell proliferation. In immunodeficient mice, NKG2Anull NK cells were significantly more powerful than NKG2A+ NK cells against HLA-E-expressing tumors. Thus, NKG2A downregulation evades the HLA-E cancer immune-checkpoint, and increases the anti-tumor activity of NK cell infusions. Because this strategy is easily adaptable to current protocols for clinical-grade immune cell processing, its clinical testing is feasible and warranted.