InVivoMAb anti-human MHC Class I (HLA-A, HLA-B, HLA-C)

Catalog #BE0079
Product Citations:
37
Clone:
W6/32
Reactivities:
Human

$164.00 - $4,280.00

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Product Details

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.

Specifications

Isotype Mouse IgG2a, κ
Recommended Isotype Control(s) InVivoMAb mouse IgG2a isotype control, unknown specificity
Recommended Dilution Buffer InVivoPure pH 6.5 Dilution Buffer
Conjugation This product is unconjugated. Conjugation is available via our Antibody Conjugation Services.
Immunogen Human tonsil cell membrane
Reported Applications Functional assays
Formulation PBS, pH 6.5
Contains no stabilizers or preservatives
Endotoxin <2EU/mg (<0.002EU/μg)
Determined by LAL gel clotting assay
Purity >95%
Determined by SDS-PAGE
Sterility 0.2 µm filtration
Production Purified from cell culture supernatant in an animal-free facility
Purification Protein G
RRID AB_1107730
Molecular Weight 150 kDa
Storage The antibody solution should be stored at the stock concentration at 4°C. Do not freeze.

Additional Formats

Functional Assays
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.

    • Cancer Research
    • ,
    • Genetics
    Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens

    Preprint on Research Square on 16 November 2023 by Okada, H., Kwok, D., et al.

    PubMed

    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.

    • Biochemistry and Molecular biology
    • ,
    • Cancer Research
    • ,
    • Immunology and Microbiology
    Identification of tumor-specific MHC ligands through improved biochemical isolation and incorporation of machine learning

    Preprint on BioRxiv : the Preprint Server for Biology on 10 June 2023 by Mecklenbräuker, S., Skoczylas, P., et al.

    PubMed

    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.

    • Homo sapiens (Human)
    • ,
    • Cancer Research
    Single-cell protein profiling defines cell populations associated with triple-negative breast cancer aggressiveness.

    In Molecular Oncology on 1 June 2023 by Kvokačková, B., Fedr, R., et al.

    PubMed

    Triple-negative breast cancer (TNBC) is an aggressive and complex subtype of breast cancer that lacks targeted therapy. TNBC manifests characteristic, extensive intratumoral heterogeneity that promotes disease progression and influences drug response. Single-cell techniques in combination with next-generation computation provide an unprecedented opportunity to identify molecular events with therapeutic potential. Here, we describe the generation of a comprehensive mass cytometry panel for multiparametric detection of 23 phenotypic markers and 13 signaling molecules. This single-cell proteomic approach allowed us to explore the landscape of TNBC heterogeneity, with particular emphasis on the tumor microenvironment. We prospectively profiled freshly resected tumors from 26 TNBC patients. These tumors contained phenotypically distinct subpopulations of cancer and stromal cells that were associated with the patient's clinical status at the time of surgery. We further classified the epithelial-mesenchymal plasticity of tumor cells, and molecularly defined phenotypically diverse populations of tumor-associated stroma. Furthermore, in a retrospective tissue-microarray TNBC cohort, we showed that the level of CD97 at the time of surgery has prognostic potential. © 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

    • COVID-19
    • ,
    • Immunology and Microbiology
    The T-cell-directed vaccine BNT162b4 encoding conserved non-spike antigens protects animals from severe SARS-CoV-2 infection.

    In Cell on 25 May 2023 by Arieta, C. M., Xie, Y. J., et al.

    PubMed

    T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861). Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

    Proteomic discovery of chemical probes that perturb protein complexes in human cells.

    In Molecular Cell on 18 May 2023 by Lazear, M. R., Remsberg, J. R., et al.

    PubMed

    Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells. Copyright © 2023 Elsevier Inc. All rights reserved.

    • IHC
    • ,
    • Cancer Research
    • ,
    • Immunology and Microbiology
    Combinatory EHMT and PARP inhibition induces an interferon response and a CD8 T cell-dependent tumor regression in PARP inhibitor-resistant models

    Preprint on BioRxiv : the Preprint Server for Biology on 23 February 2023 by Nguyen, L. L., Watson, Z. L., et al.

    PubMed

    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.

    • Functional
    • ,
    • Homo sapiens (Human)
    • ,
    • Cancer Research
    Neutrophil-activating therapy for the treatment of cancer.

    In Cancer Cell on 13 February 2023 by Linde, I. L., Prestwood, T. R., et al.

    PubMed

    Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils in vitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and T cell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer. Copyright © 2023 Elsevier Inc. All rights reserved.

    • Immunology and Microbiology
    • ,
    • Cancer Research
    The ectonucleotidase CD39 identifies tumor-reactive CD8+ T cells predictive of immune checkpoint blockade efficacy in human lung cancer.

    In Immunity on 10 January 2023 by Chow, A., Uddin, F. Z., et al.

    PubMed

    Improved identification of anti-tumor T cells is needed to advance cancer immunotherapies. CD39 expression is a promising surrogate of tumor-reactive CD8+ T cells. Here, we comprehensively profiled CD39 expression in human lung cancer. CD39 expression enriched for CD8+ T cells with features of exhaustion, tumor reactivity, and clonal expansion. Flow cytometry of 440 lung cancer biospecimens revealed weak association between CD39+ CD8+ T cells and tumoral features, such as programmed death-ligand 1 (PD-L1), tumor mutation burden, and driver mutations. Immune checkpoint blockade (ICB), but not cytotoxic chemotherapy, increased intratumoral CD39+ CD8+ T cells. Higher baseline frequency of CD39+ CD8+ T cells conferred improved clinical outcomes from ICB therapy. Furthermore, a gene signature of CD39+ CD8+ T cells predicted benefit from ICB, but not chemotherapy, in a phase III clinical trial of non-small cell lung cancer. These findings highlight CD39 as a proxy of tumor-reactive CD8+ T cells in human lung cancer. Copyright © 2022 Elsevier Inc. All rights reserved.

    • Cancer Research
    • ,
    • Immunology and Microbiology
    MEK inhibition enhances presentation of targetable MHC-I tumor antigens in mutant melanomas.

    In Proceedings of the National Academy of Sciences of the United States of America on 6 December 2022 by Stopfer, L. E., Rettko, N. J., et al.

    PubMed

    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.

    • PLA
    • ,
    • ELISA
    • ,
    • Homo sapiens (Human)
    • ,
    • Cancer Research
    • ,
    • Immunology and Microbiology
    A covalent inhibitor of K-Ras(G12C) induces MHC class I presentation of haptenated peptide neoepitopes targetable by immunotherapy.

    In Cancer Cell on 12 September 2022 by Zhang, Z., Rohweder, P. J., et al.

    PubMed

    Immunotargeting of tumor-specific antigens is a powerful therapeutic strategy. Immunotherapies directed at MHC-I complexes have expanded the scope of antigens and enabled the direct targeting of intracellular oncoproteins at the cell surface. We asked whether covalent drugs that alkylate mutated residues on oncoproteins could act as haptens to generate unique MHC-I-restricted neoantigens. Here, we report that KRAS G12C mutant cells treated with the covalent inhibitor ARS1620 present ARS1620-modified peptides in MHC-I complexes. Using ARS1620-specific antibodies identified by phage display, we show that these haptenated MHC-I complexes can serve as tumor-specific neoantigens and that a bispecific T cell engager construct based on a hapten-specific antibody elicits a cytotoxic T cell response against KRAS G12C cells, including those resistant to direct KRAS G12C inhibition. With multiple K-RAS G12C inhibitors in clinical use or undergoing clinical trials, our results present a strategy to enhance their efficacy and overcome the rapidly arising tumor resistance.Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

    • Cancer Research
    • ,
    • Cardiovascular biology
    • ,
    • Immunology and Microbiology
    A TCR mimic CAR T cell specific for NDC80 is broadly reactive with solid tumors and hematologic malignancies.

    In Blood on 25 August 2022 by Klatt, M. G., Dao, T., et al.

    PubMed

    Target identification for chimeric antigen receptor (CAR) T-cell therapies remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through T-cell receptor mimic antibodies. Mass spectrometry (MS) of HLA ligands from 8 hematologic and nonhematologic cancer cell lines identified a shared, non-immunogenic, HLA-A*02-restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene. CAR T cells directed against the ALNEQIARL:HLA-A*02 complex exhibited high sensitivity and specificity for recognition and killing of multiple cancer types, especially those of hematologic origin, and were efficacious in mouse models against a human leukemia and a solid tumor. In contrast, no toxicities toward resting or activated healthy leukocytes as well as hematopoietic stem cells were observed. This shows how MS can inform the design of broadly reactive therapeutic T-cell receptor mimic CAR T-cell therapies that can target multiple cancer types currently not druggable by small molecules, conventional CAR T cells, T cells, or antibodies. © 2022 by The American Society of Hematology.

    • Cancer Research
    • ,
    • Immunology and Microbiology
    Unmasking the suppressed immunopeptidome of EZH2-mutated diffuse large B-cell lymphomas through combination drug treatment.

    In Blood Advances on 26 July 2022 by Bourne, C. M., Mun, S. S., et al.

    PubMed

    Exploring the repertoire of peptides presented on major histocompatibility complexes (MHCs) helps identify targets for immunotherapy in many hematologic malignancies. However, there is a paucity of such data for diffuse large B-cell lymphomas (DLBCLs), which might be explained by the profound downregulation of MHC expression in many DLBCLs, and in particular in the enhancer of zeste homolog 2 (EZH2)-mutated subgroup. Epigenetic drug treatment, especially in the context of interferon-γ (IFN-γ), restored MHC expression in DLBCL. In DLBCL, peptides presented on MHCs were identified via mass spectrometry after treatment with tazemetostat or decitabine alone or in combination with IFN-γ. Such treatment synergistically increased the expression of MHC class I surface proteins up to 50-fold and the expression of class II surface proteins up to threefold. Peptides presented on MHCs increased to a similar extent for both class I and class II MHCs. Overall, these treatments restored the diversity of the immunopeptidome to levels described in healthy B cells for 2 of 3 cell lines and allowed the systematic search for new targets for immunotherapy. Consequently, we identified multiple MHC ligands from the 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 that are unmasked only after drug treatment. Overall, our results show that EZH2 inhibition in combination with decitabine and IFN-γ can expand the repertoire of MHC ligands presented on DLBCLs by revealing suppressed epitopes, thus allowing the systematic analysis and identification of new potential immunotherapy targets. © 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

    • Immu-puri
    • ,
    • Immunology and Microbiology
    Immunogenicity and therapeutic targeting of a public neoantigen derived from mutated PIK3CA.

    In Nature Medicine on 1 May 2022 by Chandran, S. S., Ma, J., et al.

    PubMed

    Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrently mutated driver genes. Here we describe a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant PIK3CA, among the most frequently genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this strategy, we developed a panel of TCRs that recognize an endogenously processed neopeptide encompassing a common PIK3CA hotspot mutation restricted by the prevalent human leukocyte antigen (HLA)-A*03:01 allele. Mechanistically, immunogenicity to this public NeoAg arises from enhanced neopeptide/HLA complex stability caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a comparatively 'featureless' surface dominated by the peptide's backbone. To bind this epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface facilitated by an unusually long CDR3β loop. In patients with diverse malignancies, we observed NeoAg clonal conservation and spontaneous immunogenicity to the neoepitope. Finally, adoptive transfer of TCR-engineered T cells led to tumor regression in vivo in mice bearing PIK3CA-mutant tumors but not wild-type PIK3CA tumors. Together, these findings establish the immunogenicity and therapeutic potential of a mutant PIK3CA-derived public NeoAg. © 2022. The Author(s).

    • In Vitro
    • ,
    • Homo sapiens (Human)
    A TCR mimic monoclonal antibody reactive with the "public" phospho-neoantigen pIRS2/HLA-A*02:01 complex.

    In JCI Insight on 8 March 2022 by Dao, T., Mun, S. S., et al.

    PubMed

    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.

    • IP
    • ,
    • Cancer Research
    • ,
    • Immunology and Microbiology
    MEK inhibition enhances presentation of targetable MHC-I tumor antigens in mutant melanomas

    Preprint on BioRxiv : the Preprint Server for Biology on 12 January 2022 by Stopfer, L. E., Rettko, N. J., et al.

    PubMed

    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.

    • IP
    • ,
    • Homo sapiens (Human)
    Immuno-transcriptomic profiling of extracranial pediatric solid malignancies.

    In Cell Reports on 23 November 2021 by Brohl, A. S., Sindiri, S., et al.

    PubMed

    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.

    • Cancer Research
    • ,
    • Immunology and Microbiology
    • ,
    • Neuroscience
    Characteristics of Immune Memory and Effector Activity to Cancer-Expressed MHC Class I Phosphopeptides Differ in Healthy Donors and Ovarian Cancer Patients.

    In Cancer Immunology Research on 1 November 2021 by Lulu, A. M., Cummings, K. L., et al.

    PubMed

    Elevated immunity to cancer-expressed antigens can be detected in people with no history of cancer and may contribute to cancer prevention. We have previously reported that MHC-restricted phosphopeptides are cancer-expressed antigens and targets of immune recognition. However, the extent to which this immunity reflects prior or ongoing phosphopeptide exposures was not investigated. In this study, we found that preexisting immune memory to cancer-expressed phosphopeptides was evident in most healthy donors, but the breadth among donors was highly variable. Although three phosphopeptides were recognized by most donors, suggesting exposures to common microbial/infectious agents, most of the 205 tested phosphopeptides were not recognized by peripheral blood mononuclear cells (PBMC) from any donor and the remainder were recognized by only 1 to 3 donors. In longitudinal analyses of 2 donors, effector immune response profiles suggested active reexposures to a subset of phosphopeptides. These findings suggest that the immunogens generating most phosphopeptide-specific immune memory are rare infectious agents or incipient cancer cells with distinct phosphoproteome dysregulations, and that repetitive immunogenic exposures occur in individual donors. Phosphopeptide-specific immunity in PBMCs and tumor-infiltrating lymphocytes from ovarian cancer patients was limited, regardless of whether the phosphopeptide was expressed on the tumor. However, 4 of 10 patients responded to 1 to 2 immunodominant phosphopeptides, and 1 showed an elevated effector response to a tumor-expressed phosphopeptide. As the tumors from these patients displayed many phosphopeptides, these data are consistent with lack of prior exposure or impaired ability to respond to some phosphopeptides and suggest that enhancing phosphopeptide-specific T-cell responses could be a useful approach to improve tumor immunotherapy. ©2021 American Association for Cancer Research.

    • Immu-puri
    • ,
    • Homo sapiens (Human)
    • ,
    • Cancer Research
    Alterations in HLA Class I-Presented Immunopeptidome and Class I-Interactome upon Osimertinib Resistance in EGFR Mutant Lung Adenocarcinoma.

    In Cancers on 4 October 2021 by Qi, Y. A., Maity, T. K., et al.

    PubMed

    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.

    • Cancer Research
    • ,
    • Immunology and Microbiology
    Unmasking the cryptic immunopeptidome of EZH2 mutated diffuse large B-cell lymphomas through combination drug treatment

    Preprint on BioRxiv : the Preprint Server for Biology on 3 September 2021 by Bourne, C. M., Mun, S. S., et al.

    PubMed

    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.

    • IP
    • ,
    • Homo sapiens (Human)
    • ,
    • Biochemistry and Molecular biology
    • ,
    • Cancer Research
    Proteogenomic Analysis Unveils the HLA Class I-Presented Immunopeptidome in Melanoma and EGFR-Mutant Lung Adenocarcinoma.

    In Molecular & Cellular Proteomics : MCP on 16 August 2021 by Qi, Y. A., Maity, T. K., et al.

    PubMed

    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.

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