The W6/32 monoclonal antibody reacts with the human major histocompatibility complex (MHC) class I, HLA-A, B, and C. All human nucleated cell express MHC class I antigens associated with β2-microglobulin. MHC class I plays a central role in cell-mediated immune responses and tumor surveillance.
Valenzuela, N. M., et al. (2013). "Blockade of p-selectin is sufficient to reduce MHC I antibody-elicited monocyte recruitment in vitro and in vivo" Am J Transplant 13(2): 299-311. PubMed
Donor-specific HLA antibodies significantly lower allograft survival, but as yet there are no satisfactory therapies for prevention of antibody-mediated rejection. Intracapillary macrophage infiltration is a hallmark of antibody-mediated rejection, and macrophages are important in both acute and chronic rejection. The purpose of this study was to investigate the Fc-independent effect of HLA I antibodies on endothelial cell activation, leading to monocyte recruitment. We used an in vitro model to assess monocyte binding to endothelial cells in response to HLA I antibodies. We confirmed our results in a mouse model of antibody-mediated rejection, in which B6.RAG1(-/-) recipients of BALB/c cardiac allografts were passively transferred with donor-specific MHC I antibodies. Our findings demonstrate that HLA I antibodies rapidly increase intracellular calcium and endothelial presentation of P-selectin, which supports monocyte binding. In the experimental model, donor-specific MHC I antibodies significantly increased macrophage accumulation in the allograft. Concurrent administration of rPSGL-1-Ig abolished antibody-induced monocyte infiltration in the allograft, but had little effect on antibody-induced endothelial injury. Our data suggest that antagonism of P-selectin may ameliorate accumulation of macrophages in the allograft during antibody-mediated rejection.
Functional Assays
Valenzuela, N. M., et al. (2013). "HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcgammaRs" J Immunol 190(12): 6635-6650. PubMed
Ab-mediated rejection (AMR) of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor-specific Ab binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the Ab. We investigated the mechanisms underlying monocyte recruitment by HLA class I (HLA I) Ab-activated endothelium. We used a panel of murine mAbs of different subclasses to crosslink HLA I on human aortic, venous, and microvascular endothelial cells and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine (m)IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. mIgG2a but not mIgG1 could bind human FcgammaRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcgammaRI, and, to a lesser extent, FcgammaRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during AMR. We confirmed these observations using human HLA allele-specific mAbs and IgG purified from transplant patient sera. HLA I Abs universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during AMR. Importantly, the subclass of donor-specific Ab may influence its pathogenesis. These results imply that human IgG1 and human IgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcgammaR interactions.
T-cell-based immunotherapies hold promise in treating cancer by leveraging the immune system’s recognition of cancer-specific antigens. 1 However, their efficacy is often limited in tumors with fewer somatic mutations and significant intratumoral heterogeneity, such as glioblastoma. 2–5 Here we introduce a previously uncharacterized class of tumor-wide and public neoantigens, originating from RNA-splicing aberrations in various cancer types. Notably, we identified T-cell receptor clones capable of recognizing and targeting neoantigens derived from aberrant splicing in GNAS and RPL22 . In multi-site-directed biopsies across various cancer types, we detected the tumor-wide expression of the GNAS neojunction within glioma, mesothelioma, prostate cancer, and liver cancer patients. Importantly, these neoantigens were proven to be endogenously generated and presented by tumor cells under physiological conditions, which was sufficient in triggering the eradication of cancer cells by neoantigen-specific CD8+ T-cells. Moreover, our study unravels the complex interplay of dysregulated splicing factor expression in specific cancer subtypes, which leads to recurrent patterns of neojunction upregulation. These findings offer a robust molecular basis for T-cell-based immunotherapy that targets a newfound class of tumor-wide public neoantigens, addressing the challenges of intratumoral heterogeneity. By characterizing this unique class of tumor-wide and public neoantigens, our research emphasizes the need to consider intratumoral heterogeneity in the quest for effective cancer immunotherapies. These findings have significant implications for the development of targeted treatments and mark a pivotal step in the ongoing journey to uncover neoantigens for cancer immunotherapy.
Isolation of MHC ligands and subsequent analysis by mass spectrometry is considered the gold standard for defining targets for TCR-T immunotherapies. However, as many targets of high tumor-specificity are only presented at low abundance on the cell surface of tumor cells, the efficient isolation of these peptides is crucial for their successful detection. Here, we demonstrate how different isolation strategies, which consider hydrophobicity and post-translational modifications, can improve the detection of MHC ligands, including cysteinylated MHC ligands from cancer germline antigens or point-mutated neoepitopes. Furthermore, we developed a novel MHC class I ligand prediction algorithm (ARDisplay-I) that outperforms the current state-of-the-art and facilitates the assignment of peptides to the correct MHC allele. The model has other applications, such as the identification of additional MHC ligands not detected from mass spectrometry or determining whether the MHC ligands can be presented on the cell surface via MHC alleles not included in the study. The implementation of these strategies can augment the development of T cell receptor-based therapies (i.a. TIL 1 -derived T cells, genetically engineered T cells expressing tumor recognizing receptors or TCR-mimic antibodies) by facilitating the identification of novel immunotherapy targets and by enriching the resources available in the field of computational immunology. Significance: This study demonstrates how the isolation of different tumor-specific MHC ligands can be optimized when considering their hydrophobicity and post-translational modification status. Additionally, we developed a novel machine-learning model for the probability prediction of the MHC ligands’ presentation on the cell surface. The algorithm can assign these MHC ligands to their respective MHC alleles which is essential for the design of TCR-T immunotherapies.
ABSTRACT Euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/2), which catalyze demethylation of histone H3 lysine 9 (H3K9me2), contribute to tumorigenesis and therapy resistance through unknown mechanisms of action. In ovarian cancer, EHMT1/2 and H3K9me2 are directly linked to acquired resistance to poly-ADP-ribose polymerase (PARP) inhibitors and are correlated with poor clinical outcomes. Using a combination of experimental and bioinformatic analyses in several PARP inhibitor resistant ovarian cancer models, we demonstrate that combinatory inhibition of EHMT and PARP is effective in treating PARP inhibitor resistant ovarian cancers. Our in vitro studies show that combinatory therapy reactivates transposable elements, increases immunostimulatory dsRNA formation, and elicits several immune signaling pathways. Our in vivo studies show that both single inhibition of EHMT and combinatory inhibition of EHMT and PARP reduces tumor burden, and that this reduction is dependent on CD8 T cells. Together, our results uncover a direct mechanism by which EHMT inhibition helps to overcome PARP inhibitor resistance and shows how an epigenetic therapy can be used to enhance anti-tumor immunity and address therapy resistance.
Combining multiple therapeutic strategies in NRAS/BRAF mutant melanoma-namely MEK/BRAF kinase inhibitors, immune checkpoint inhibitors (ICIs), and targeted immunotherapies-may offer an improved survival benefit by overcoming limitations associated with any individual therapy. Still, optimal combination, order, and timing of administration remains under investigation. Here, we measure how MEK inhibition (MEKi) alters anti-tumor immunity by utilizing quantitative immunopeptidomics to profile changes in the peptide major histocompatibility molecules (pMHC) repertoire. These data reveal a collection of tumor antigens whose presentation levels are selectively augmented following therapy, including several epitopes present at over 1,000 copies per cell. We leveraged the tunable abundance of MEKi-modulated antigens by targeting four epitopes with pMHC-specific T cell engagers and antibody drug conjugates, enhancing cell killing in tumor cells following MEK inhibition. These results highlight drug treatment as a means to enhance immunotherapy efficacy by targeting specific upregulated pMHCs and provide a methodological framework for identifying, quantifying, and therapeutically targeting additional epitopes of interest.
Phosphopeptides derived from dysregulated protein phosphorylation in cancer cells can be processed and presented by MHC class I and class II molecules and, therefore, represent an untapped class of tumor-specific antigens that could be used as widely expressed "public" cancer neoantigens (NeoAgs). We generated a TCR mimic (TCRm) mAb, 6B1, specific for a phosphopeptide derived from insulin receptor substrate 2 (pIRS2) presented by HLA-A*02:01. The pIRS2 epitope's presentation by HLA-A*02:01 was confirmed by mass spectrometry. The TCRm 6B1 specifically bound to pIRS2/HLA-A2 complex on tumor cell lines that expressed pIRS2 in the context of HLA-A*02:01. Bispecific mAbs engaging CD3 of T cells were able to kill tumor cell lines in a pIRS2- and HLA-A*02:01-restricted manner. Structure modeling shows a prerequisite for an arginine or lysine at the first position to bind mAb. Therefore, 6B1 could recognize phosphopeptides derived from various phosphorylated proteins with similar amino acid compositions. This raised the possibility that a TCRm specific for the pIRS2/HLA-A2 complex could target a range of phosphopeptides presented by HLA-A*02:01 in various tumor cells. This is the first TCRm mAb to our knowledge targeting a phosphopeptide/MHC class I complex; the potential of this class of agents for clinical applications warrants further investigation.
h4>ABSTRACT/h4> Combining multiple therapeutic strategies in NRAS/BRAF mutant melanoma – namely MEK/BRAF kinase inhibitors, immune checkpoint inhibitors, and targeted immunotherapies – may offer an improved survival benefit by overcoming limitations associated with any individual therapy. Still, optimal combination, order, and timing of administration remains under investigation. Here, we measure how MEK inhibition (MEKi) alters anti-tumor immunity by utilizing quantitative immunopeptidomics to profile changes in the peptide MHC (pMHC) repertoire. These data reveal a collection of tumor antigens whose presentation levels are selectively augmented following therapy, including several epitopes present at over 1000 copies-per-cell. We leveraged the tunable abundance of MEKi-modulated antigens by targeting 4 epitopes with pMHC-specific T cell engagers and antibody drug conjugates, enhancing cell killing in tumor cells following MEK inhibition. These results highlight drug treatment as a means to enhance immunotherapy efficacy by targeting specific upregulated pMHCs and provide a methodological framework for identifying, quantifying, and therapeutically targeting additional epitopes of interest. h4>SIGNIFICANCE/h4> Kinase inhibitor treatment in NRAS/BRAF mutant melanoma can sensitize tumors to immunotherapy, in part through an increase in average surface presentation of peptide MHC molecules. Here, we demonstrate that MEK inhibition selectively boosts epitope abundance of select tumor-associated antigens in vitro and in vivo , enhancing targeted immunotherapy efficacy against these treatment-modulated epitopes.
We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.Published by Elsevier Inc.
Immune checkpoint inhibitor (ICI) therapy has been a paradigm shift in the treatment of cancer. ICI therapy results in durable responses and survival benefit for a large number of tumor types. Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has shown great efficacy treating EGFR mutant lung cancers; however, all patients eventually develop resistance. ICI therapy has not benefitted EGFR mutant lung cancer. Herein, we employed stable isotope labeling by amino acids in cell culture (SILAC) quantitative mass spectrometry-based proteomics to investigate potential immune escape molecular mechanisms in osimertinib resistant EGFR mutant lung adenocarcinoma by interrogating the alterations in the human leukocyte antigen (HLA) Class I-presented immunopeptidome, Class I-interactome, and the whole cell proteome between isogenic osimertinib-sensitive and -resistant human lung adenocarcinoma cells. Our study demonstrates an overall reduction in HLA class I-presented immunopeptidome and downregulation of antigen presentation core complex (e.g., TAP1 and ERAP1/2) and immunoproteasome in osimertinib resistant lung adenocarcinoma cells. Several key components in autophagy pathway are differentially altered. S100 proteins and SLC3A2 may play critical roles in reduced antigen presentation. Our dataset also includes ~1000 novel HLA class I interaction partners and hundreds of Class I-presented immunopeptides in EGFR mutant lung adenocarcinoma. This large-scale unbiased proteomics study provides novel insights and potential mechanisms of immune evasion of EGFR mutant lung adenocarcinoma.
Exploring the repertoire of peptides presented on major histocompatibility complexes (MHC) has been utilized to identify targets for immunotherapy in many hematological malignancies. However, such data have not been described systematically for diffuse large B-cell lymphomas (DLBCL), which might be explained by the profound downregulation of MHC expression in many DLBCLs, and in particular in the EZH2-mutated subgroup. Epigenetic drug treatment, especially in the context of interferon gamma (IFNg), restored MHC expression in DLBCL. DLBCL MHC-presented peptides were identified via mass spectrometry following tazemetostat or decitabine treatments alone, or in combination with IFNg. Such treatment synergistically increased MHC class I surface protein expression up to 50-fold and class II expression up to 3-fold. Peptides presented on MHC complexes increased to a similar extent for MHC class I and remained constant for class II. Overall, these treatments restored the diversity of the immunopeptidome to levels described in healthy B cells and allowed the systematic search for new targets for immunotherapy. Consequently, we identified multiple MHC ligands from regulator of G protein signaling 13 (RGS13) and E2F transcription factor 8 (E2F8) on different MHC alleles, none of which have been described in healthy tissues and therefore represent tumor-specific MHC ligands, which are unmasked only after drug treatment. Overall, our results show that EZH2 inhibition in combination with decitabine and IFNg can expand the repertoire of MHC ligands presented on DLBCLs by revealing cryptic epitopes, thus allowing the systematic analysis and identification of new potential immunotherapy targets. h4>Key points/h4> Combination therapy of interferon gamma with epigenetic regulators leads to large increases in the immunopeptidome of DLBCL. HLA ligands from proteins RGS13 and E2F8 may provide DLBCL-specific targets for immunotherapy.
Immune checkpoint inhibitors and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential in cancers with high tumor mutational burden (TMB), such as melanoma, but not in cancers with low TMB, such as mutant epidermal growth factor receptor (EGFR)-driven lung adenocarcinoma. Precision immunotherapy is an unmet need for most cancers, particularly for cancers that respond inadequately to immune checkpoint inhibitors. Here, we employed large-scale MS-based proteogenomic profiling to identify potential immunogenic human leukocyte antigen (HLA) class I-presented peptides in melanoma and EGFR-mutant lung adenocarcinoma. Similar numbers of peptides were identified from both tumor types. Cell line and patient-specific databases (DBs) were constructed using variants identified from whole-exome sequencing. A de novo search algorithm was used to interrogate the HLA class I immunopeptidome MS data. We identified 12 variant peptides and several classes of tumor-associated antigen-derived peptides. We constructed a cancer germ line (CG) antigen DB with 285 antigens. This allowed us to identify 40 class I-presented CG antigen-derived peptides. The class I immunopeptidome comprised more than 1000 post-translationally modified (PTM) peptides representing 58 different PTMs, underscoring the critical role PTMs may play in HLA binding. Finally, leveraging de novo search algorithm and an annotated long noncoding RNA (lncRNA) DB, we developed a novel lncRNA-encoded peptide discovery pipeline to identify 44 lncRNA-derived peptides that are presented by class I. We validated tandem MS spectra of select variant, CG antigen, and lncRNA-derived peptides using synthetic peptides and performed HLA class I-binding assays to demonstrate binding to class I proteins. In summary, we provide direct evidence of HLA class I presentation of a large number of variant and tumor-associated peptides in both low and high TMB cancer. These results can potentially be useful for precision immunotherapies, such as vaccine or adoptive cell therapies in melanoma and EGFR-mutant lung cancers.Published by Elsevier Inc.
