Immunoproteasome Activation Expands the MHC Class I Immunopeptidome, Unmasks Neoantigens, and Enhances T-cell Anti-Myeloma Activity.

Proteasomes generate antigenic peptides that are presented on the tumor surface to cytotoxic T-lymphocytes. Immunoproteasomes are highly specialized proteasome variants that are expressed at higher levels in antigen-presenting cells and contain replacements of the three constitutive proteasome catalytic subunits to generate peptides with a hydrophobic C-terminus that fit within the groove of MHC class I (MHC-I) molecules. A hallmark of cancer is the ability to evade immunosurveillance by disrupting the antigen presentation machinery and downregulating MHC-I antigen presentation. High-throughput screening was performed to identify compound A, a novel molecule that selectively increased immunoproteasome activity and expanded the number and diversity of MHC-I-bound peptides presented on multiple myeloma cells. Compound A increased the presentation of individual MHC-I-bound peptides by >100-fold and unmasked tumor-specific neoantigens on myeloma cells. Global proteomic integral stability assays determined that compound A binds to the proteasome structural subunit PSMA1 and promotes association of the proteasome activator PA28α/β (PSME1/PSME2) with immunoproteasomes. CRISPR/Cas9 silencing of PSMA1, PSME1, or PSME2 as well as treatment with immunoproteasome-specific suicide inhibitors abolished the effects of compound A on antigen presentation. Treatment of multiple myeloma cell lines and patient bone marrow-derived CD138+ cells with compound A increased the anti-myeloma activity of allogenic and autologous T cells. Compound A was well-tolerated in vivo and co-treatment with allogeneic T cells reduced the growth of myeloma xenotransplants in NOD/SCID gamma mice. Taken together, our results demonstrate the paradigm shifting impact of immunoproteasome activators to diversify the antigenic landscape, expand the immunopeptidome, potentiate T-cell-directed therapy, and reveal actionable neoantigens for personalized T-cell immunotherapy.

Rana PS ，Ignatz-Hoover JJ ，Guo C ，Mosley AL ，Malek E ，Federov Y ，Adams DJ ，Driscoll JJ ... -  《-》

HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity.

Proteasomes degrade intracellular proteins to generate antigenic peptides that are recognized by the adaptive immune system and promote anticancer immunity. However, tumors subvert the antigen presentation machinery to escape immunosurveillance. We hypothesized that proteasome activation could concomitantly increase antigen abundance and diversity in multiple myeloma cells. High-throughput screens revealed that histone deacetylase 6 (HDAC6) inhibitors activated proteasomes to unmask neoantigens and amplify the tumor-specific antigenic landscape. Treatment of patient CD138+ cells with HDAC6 inhibitors significantly promoted the antimyeloma activity of autologous CD8+ T cells. Pharmacologic blockade and genetic ablation of the HDAC6 ubiquitin-binding domain released HR23B, which shuttles ubiquitinylated cargo to proteasomes, while silencing HDAC6 or HR23B in multiple myeloma cells abolished the effect of HDAC6 inhibitors on proteasomes, antigen presentation, and T-cell cytotoxicity. Taken together, our results demonstrate the paradigm-shifting translational impact of proteasome activators to expand the myeloma immunopeptidome and have revealed novel, actionable antigenic targets for T cell-directed immunotherapy. The elimination of therapy-resistant tumor cells remains a major challenge in the treatment of multiple myeloma. Our study identifies and functionally validates agents that amplify MHC class I-presented antigens and pave the way for the development of proteasome activators as immune adjuvants to enhance immunotherapeutic responses in patients with multiple myeloma.

Rana PS ，Ignatz-Hoover JJ ，Kim BG ，Malek E ，Federov Y ，Adams D ，Chan T ，Driscoll JJ ... -  《-》

Anti-cancer immune effect of human colorectal cancer neoantigen peptide based on MHC class I molecular affinity screening.

Tumor antigen peptide vaccines have shown remarkable efficacy, safety, and reliability in recent studies. However, the screening process for immunopotent antigenic peptides is cumbersome, limiting their widespread application. Identifying neoantigen peptides that can effectively trigger an immune response is crucial for personalized cancer treatment. Whole exome sequencing was performed on patient-derived colon cancer cells to predict 9-amino-acid (9aa) neoantigen peptides. In vitro simulation of endogenous antigen presentation by antigen-presenting cells (dendritic cells) to CD8+ T cells was conducted, aiming to activate the CD8+ immune response to the predicted antigens. The immunological effects of each neoantigen were assessed using flow cytometry and ELISpot assays, while the relationship between neoantigen immunogenicity and MHC molecular affinity was examined. 1. Next-generation sequencing (NGS) predicted 9-amino acid (9aa) neoantigen peptides for subsequent immunological analysis.2. Higher mDC Levels in Experimental Group: CD11c+CD83+ mature dendritic cells (mDCs) were 96.6% in the experimental group, compared to 0.051% in the control group. CD80 fluorescence intensity was also significantly higher in the experimental group, confirming a greater mDC presence.3. Neoantigen Peptides Promote CD4+, CD8+ T, and NK Cell Proliferation: After 14 days, flow cytometry showed higher percentages of CD4+ T (37.41% vs 7.8%), CD8+ T (16.67% vs 14.63%), and NK cells (33.09% vs 7.81%) in the experimental group, indicating that the neoantigen peptides induced proliferation of CD4+, CD8+ T cells, and NK cells. 4. The results, analyzed using two-way ANOVA, showed that the standardized T-value for HLA molecular affinity variation in the 1-4 range (Group B) was significantly higher than for ≤1 (Group A, p < 0.0001) and >4 (Group C, p < 0.05). Regarding HLA-allele genotypes, HLA-Type 1 had a significantly higher standardized T-value than HLA-Type 2 (p < 0.05) and HLA-Type 3 (p < 0.0001). HLA-Type 1 was identified as the allele associated with the highest T-value. 1. The most immunogenic neoantigens typically exhibit an MHC molecular affinity variation between 1 and 4, indicating that stronger immunogenicity correlates with higher MHC molecular affinity variation. 2. Each patient's HLA molecules were classified into Types 1, 2, and 3, with Type 1 showing the highest binding capacity for neoantigens. Our findings indicate that the most immunogenic neoantigens were associated with HLA Type 1. 3. Neoantigen peptides were shown to activate the proliferation of both CD8+ T-cells and induce proliferation of CD4+ T-cells and NK cells. 4. Variation in MHC molecular affinity and HLA neoantigen genotype are anticipated to serve as valuable variables for screening highly immunogenic neoantigens, facilitating more efficient preparation of effective polypeptide tumor vaccines.

Zhang S ，Huang C ，Li Y ，Li Z ，Zhu Y ，Yang L ，Hu H ，Sun Q ，Liu M ，Cao S ... -  《Frontiers in Immunology》

Alanine-based spacers promote an efficient antigen processing and presentation in neoantigen polypeptide vaccines.

Neoantigens are tumor-specific antigens that are mostly particular for each patient. Since the immune system is able to mount a specific immune response against these neoantigens, they are a promising tool for the development of therapeutic personalized cancer vaccines. Neoantigens must be presented to T cells by antigen presenting cells (APC) in the context of MHC-I or MHC-II molecules. Therefore, the strategy of vaccine delivery may have a major impact on the magnitude and quality of T cell responses. Neoantigen-based vaccines are frequently administered as a pool of individual synthetic peptides that induce mainly CD4+ T cell responses. MHC-I-mediated presentation and the elicitation of CD8+ T cell responses may be improved using DNA or RNA sequences that code for a unique long polypeptide that concatenates the different neoantigens spaced by linker sequences. When administered this way, the selection of the spacer between neoantigens is of special interest, as it might influence the processing and presentation of the right peptides by APCs. Here, we evaluate the impact of such linker regions on the MHC-I-dependent antigen presentation using an in vitro assay that assesses the MHC-I presentation of SIINFEKL, a H-2 Kb-restricted OVA peptide. Our results show that spacers used to generate epitope concatenates have a large impact on the efficiency of neoantigen processing and presentation by MHC-I molecules; in contrast, the peptide position and the flanking regions have a minimal impact. Moreover, linkers based on alanine residues promote a more efficient peptide presentation than the commonly used GGGS linker.

Aguilar-Gurrieri C ，Barajas A ，Rovirosa C ，Ortiz R ，Urrea V ，de la Iglesia N ，Clotet B ，Blanco J ，Carrillo J ... -  《-》

Alternate MHC I Antigen Presentation Pathways Allow CD8+ T-cell Recognition and Killing of Cancer Cells in the Absence of β2M or TAP.

MHC I antigen presentation allows CD8+ T cells to detect and eliminate cancerous or virally infected cells. The MHC I pathway is not essential for cell growth and viability, so cancers and viruses can evade control by CD8+ T cells by inactivating antigen presentation. In cancers, two common ways for this evasion are the loss of either the MHC I light chain [β2 microglobulin (β2M)] or the transporter-associated with antigen processing (TAP). β2M-null cells are generally thought to lack the MHC I pathway because the MHC I heavy chain by itself lacks the proper conformation for peptide display. TAP-null cells are thought to have severely defective MHC I antigen presentation because they are incapable of supplying peptides from the cytosol to MHC I molecules in the endoplasmic reticulum (ER). However, we have found that highly reactive memory CD8+ T cells could still recognize cells that completely lacked β2M or TAP. This was at least in part because in TAP-null cells, the Sec62 component of the Sec61 translocon supported the transfer of cytosolic peptides into the ER. In β2M-negative cells, free MHC I heavy chains were able to bind peptides and assume a conformation that was sufficiently recognized by CD8+ T cells. This process required ER chaperones and the peptide-loading complex. We found that these mechanisms supported antigen presentation at a level that was sufficient for memory CD8+ T cells to kill melanoma cells both in vitro and in tumor-bearing mice. The implications for tumor immunotherapy are discussed.

Cruz FM ，Orellano LAA ，Chan A ，Rock KL ... -  《-》