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

Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options and a dismal prognosis. While immunotherapy has shown promise in treating some solid tumors, the treatment of GBM has been mostly unsuccessful because of a lack of targetable tumor antigens and high tumor heterogeneity. Here, we report RCAN1-4 as a novel tumor antigen derived from alternative splicing induced by the transcription factor C/EBPβ. Both C/EBPβ and RCAN1-4 are highly expressed in GBM and glioma stem cells as mesenchymal subtype hallmarks. We report an immunogenic HLA-A24-specific splicing junction epitope within exon 4 and exon 5 that is unique to RCAN1-4. This epitope was validated for its ability to stimulate T cell responses in HLA-A24+ donors and GBM patients, leading us to identify RCAN1-4-reactive T cell receptors (TCRs) for the construction of TCR-engineered T cells (TCR-T cells). Functional studies of TCR-Ts demonstrated the in vitro and in vivo killing of RCAN1-4pos GBM tumor cells, highlighting its potential as an immunotherapeutic target in mesenchymal GBM.

Covalent inhibitors of oncoprotein KRAS have initial efficacy, but responses lack durability. Covalently modified oncoproteins are presented as MHC-restricted hapten-peptides (p*MHC) on the cancer cell surface, enabling combination of targeted therapy with immunotherapy to overcome drug resistance. Building on indirect evidence of KRASG12C-derived p*MHCs, we use immunopeptidomics to identify and directly quantify these synthetic neoantigens. To address challenges by their low copy number, we develop AETX-R114, a T cell engaging bispecific antibody with picomolar affinity for MHC-restricted sotorasib-modified KRASG12C peptides presented by three HLA-A3 supertype alleles. AETX-R114 dramatically increases the half-life and thereby the number of presented p*MHCs, enabling selective and potent killing of resistant cancer cells both in vitro and in vivo. To broaden the therapeutic potential of creating and targeting synthetic neoantigens, we further develop AETX-R302, which recognizes divarasib-modified KRASG12C peptides presented on alleles from the HLA-A2 and A3 supertypes. Cryo-EM structure determination reveals the molecular basis for breaking HLA supertype restriction. Collectively, our study illustrates how engineered antibodies can transform synthetic neoantigens into actionable cancer immunotherapy targets.

Immune checkpoint blockade for the treatment of malignancies has been focused on reversing inhibitory pathways in T lymphocytes. NK cells are a potent innate defense against tumors and virally infected cells, but their therapeutic manipulation for anticancer immunity has been inadequately explored. Considerable attention has been focused on approaches to blocking inhibitory receptors on NK and myeloid cells. Most effort has been directed to the killer immunoglobulin-like receptors and CD94/NKG2A on NK cells. Another set of receptors with similar function in both NK cells and myeloid cells is the leukocyte immunoglobulin-like receptors (LILR) that interact with a wide variety of HLA molecules. Using pan-anti-HLA mAbs that recognize a conserved epitopic region on HLA also seen by LILRs, we investigated their functional effects in several models of tumor immunity. The pan-anti-HLA mAbs blocked the binding of most LILRs and did not block killer cell immunoglobulin-like receptors or CD94/NKG2A/C or T-cell receptor recognition. They also activated dysfunctional NK cells explanted from a variety of human cancers and resulted in enhancement of tumor immunity in humanized mice. The mAbs also exert direct antitumor effects. These results suggest that activation of innate immunity via disruption of HLA/LILR interactions is a potent approach for control of both primary tumors and potentially tumor metastases.

Ovarian cancer represents the most lethal gynecological cancer with poor response to checkpoint inhibitors. Human endogenous retroviruses (HERVs) are aberrantly expressed by tumor cells and may represent a source of shared T-cell epitopes for cancer immunotherapy regardless of the tumor mutational burden.

Amyotrophic lateral sclerosis (ALS) is the most common and fatal motor neuron disease. Approximately 90% of ALS patients exhibit pathology of the master RNA regulator, transactive response DNA binding protein (TDP-43). Despite the prevalence TDP-43 pathology in ALS motor neurons, recent findings suggest immune dysfunction is a determinant of disease progression in patients. Whether TDP-43 aggregates elicit immune responses remains underexplored. In this study, we demonstrate that TDP-43 aggregates are internalized by antigen-presenting cell populations, cause vesicle rupture, and drive innate and adaptive immune cell activation by way of antigen presentation. Using a multiplex imaging platform, we observed enrichment of activated microglia/macrophages in ALS white matter that correlated with phosphorylated TDP-43 accumulation, CD8 T cell infiltration, and major histocompatibility complex expression. Taken together, this study sheds light on a novel cellular response to TDP-43 aggregates through an immunological lens.

Translation of the noncoding genome in cancer can generate cryptic (noncanonical) peptides capable of presentation by human leukocyte antigen class I (HLA-I); however, the cancer specificity and immunogenicity of noncanonical HLA-I-bound peptides (ncHLAp) are incompletely understood. Using high-resolution immunopeptidomics, we discovered that cryptic peptides are abundant in the pancreatic cancer immunopeptidome. Approximately 30% of ncHLAp exhibited cancer-restricted translation, and a substantial subset were shared among patients. Cancer-restricted ncHLAp displayed robust immunogenic potential in a sensitive ex vivo T cell priming platform. ncHLAp-reactive, T cell receptor-redirected T cells exhibited tumoricidal activity against patient-derived pancreatic cancer organoids. These findings demonstrate that pancreatic cancer harbors cancer-restricted ncHLAp that can be recognized by cytotoxic T cells. Future therapeutic strategies for pancreatic cancer, and potentially other solid tumors, may include targeting cryptic antigens.

T cell-based immunotherapies hold promise in treating cancer by leveraging the immune system's recognition of cancer-specific antigens1. However, their efficacy is limited in tumours with few somatic mutations and substantial intratumoural heterogeneity2-4. Here we introduce a previously uncharacterized class of tumour-wide public neoantigens originating from RNA splicing aberrations in diverse cancer types. We identified T cell receptor clones capable of recognizing and targeting neoantigens derived from aberrant splicing in GNAS and RPL22. In cases with multi-site biopsies, we detected the tumour-wide expression of the GNAS neojunction in glioma, mesothelioma, prostate cancer and liver cancer. These neoantigens are endogenously generated and presented by tumour cells under physiologic conditions and are sufficient to trigger cancer cell eradication by neoantigen-specific CD8+ T cells. Moreover, our study highlights a role for dysregulated splicing factor expression in specific cancer types, leading to recurrent patterns of neojunction upregulation. These findings establish a molecular basis for T cell-based immunotherapies addressing the challenges of intratumoural heterogeneity.

Poly ADP-ribose polymerase inhibitors (PARPi) are first-line maintenance therapy for ovarian cancer and an alternative therapy for several other cancer types. However, PARPi-resistance is rising, and there is currently an unmet need to combat PARPi-resistant tumors. Here, we created an immunocompetent, PARPi-resistant mouse model to test the efficacy of combinatory PARPi and euchromatic histone methyltransferase 1/2 inhibitor (EHMTi) in the treatment of PARPi-resistant ovarian cancer. We discovered that inhibition of EHMT1/2 resensitizes cells to PARPi. Markedly, we show that single EHMTi and combinatory EHMTi/PARPi significantly reduced PARPi-resistant tumor burden and that this reduction is partially dependent on CD8 T cells. Altogether, our results show a low-toxicity drug that effectively treats PARPi-resistant ovarian cancer in an immune-dependent manner, supporting its entry into clinical development and potential incorporation of immunotherapy. Implications: Targeting the epigenome of therapy-resistant ovarian cancer induces an antitumor response mediated in part through an antitumor immune response.

Next-generation T-cell-directed vaccines for COVID-19 focus on establishing lasting T-cell immunity against current and emerging SARS-CoV-2 variants. Precise identification of conserved T-cell epitopes is critical for designing effective vaccines. Here we introduce a comprehensive computational framework incorporating a machine learning algorithm-MHCvalidator-to enhance mass spectrometry-based immunopeptidomics sensitivity. MHCvalidator identifies unique T-cell epitopes presented by the B7 supertype, including an epitope from a + 1-frameshift in a truncated Spike antigen, supported by ribosome profiling. Analysis of 100,512 COVID-19 patient proteomes shows Spike antigen truncation in 0.85% of cases, revealing frameshifted viral antigens at the population level. Our EpiTrack pipeline tracks global mutations of MHCvalidator-identified CD8 + T-cell epitopes from the BNT162b4 vaccine. While most vaccine epitopes remain globally conserved, an immunodominant A*01-associated epitope mutates in Delta and Omicron variants. This work highlights SARS-CoV-2 antigenic features and emphasizes the importance of continuous adaptation in T-cell vaccine development.

The major histocompatibility complex class I antigen presentation pathways play pivotal roles in orchestrating immune responses. Recent studies have begun to explore the therapeutic potential of cysteines within the immunopeptidome, such as the use of covalent ligands to generate haptenated peptide neoepitopes for immunotherapy. In this work, we report a platform for mapping reactive cysteines on MHC-I-bound peptide antigens. We develop cell-impermeable sulfonated maleimide probes capable of capturing reactive cysteines on these antigens. Using these probes in chemoproteomic experiments, we discover that cysteines on MHC-I-bound antigens exhibit various degrees of reactivity. Moreover, interferon-gamma stimulation enhances the reactivity of cysteines at position 8 of 9-mer MHC-I-bound antigens. Finally, we demonstrate that targeting reactive cysteines on MHC-I-bound antigens with a maleimide-conjugated Fc-binding cyclic peptide contributes to the induction of antibody-dependent cellular phagocytosis.

Antibody-based CD47 blockade aims to activate macrophage phagocytosis of tumor cells. However, macrophages possess a high degree of phenotype heterogeneity that likely influences phagocytic capacity. In murine models, proinflammatory (M1) activation increases macrophage phagocytosis of tumor cells, but in human models, results have been conflicting. Here, we investigated the effects of proinflammatory polarization on the phagocytic response of human monocyte-derived macrophages in an in vitro model. Using both flow cytometry-based and fluorescence live-cell imaging-based phagocytosis assays, we observed that mouse monoclonal anti-CD47 antibody (B6H12) induced monocyte-derived macrophage phagocytosis of cancer cells in vitro. Proinflammatory (M1) macrophage polarization with IFN-γ+LPS resulted in a severe reduction in phagocytic response to CD47 blockade. This reduction coincided with increased expression of the antiphagocytic membrane proteins LILRB1 and Siglec-10 but was not rescued by combination blockade of the corresponding ligands. However, matrix metalloproteinase inhibitors (TAPI-0 or GM6001) partly restored response to CD47 blockade in a dose-dependent manner. In summary, these data suggest that proinflammatory (M1) activation reduces phagocytic response to CD47 blockade in human monocyte-derived macrophages.

In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.