Tumor Antigen-Based Nanovaccines for Cancer Immunotherapy: A Review

As an important means of tumor immunotherapy, tumor vaccines have achieved exciting results in the past few decades. However, there are still many obstacles that hinder tumor vaccines from achieving maximum efficacy, including lack of tumor antigens, low antigen immunogenicity and poor delivery efficiency. To overcome these challenges, researchers have developed and investigated various new types of tumor antigens with higher antigenic specificity and broader antigen spectrum, such as tumor-specific peptide antigens, tumor lysates, tumor cell membrane, tumor associated exosomes, etc. At the same time, different nanoparticulate delivery platforms have been developed to increase the immunogenicity of the tumor antigens, for example by increasing their targeting efficiency of antigen-presenting cells and lymph nodes, and by co-delivering antigens with adjuvants. In this review, we summarized different types of the tumor antigens that have been reported, and introduced several nanovaccine strategies for increasing the immunogenicity of tumor antigens. The review of recent progress in these fields may provide reference for the follow-up studies of tumor antigen-based cancer immunotherapy.

Recognition of Multiple Hybrid Insulin Peptides by a Single Highly Diabetogenic T-Cell Receptor

The mechanisms underlying the major histocompatibility complex class II (MHCII) type 1 diabetes (T1D) association remain incompletely understood. We have previously shown that thymocytes expressing the highly diabetogenic, I-Ag7-restricted 4.1-T-cell receptor (TCR) are MHCII-promiscuous, and that, in MHCII-heterozygous mice, they sequentially undergo positive and negative selection/Treg deviation by recognizing pro- and anti-diabetogenic MHCII molecules on cortical thymic epithelial cells and medullary hematopoietic antigen-presenting cells (APCs), respectively. Here, we use a novel autoantigen discovery approach to define the antigenic specificity of this TCR in the context of I-Ag7. This was done by screening the ability of random epitope–GS linker–I-Aβg7chain fusion pools to form agonistic peptide–MHCII complexes on the surface of I-Aαd chain-transgenic artificial APCs. Pool deconvolution, I-Ag7-binding register-fixing, TCR contact residue mapping, and alanine scanning mutagenesis resulted in the identification of a 4.1-TCR recognition motif XL(G/A)XEXE(D/E)X that was shared by seven agonistic hybrid insulin peptides (HIPs) resulting from the fusion of several different chromogranin A and/or insulin C fragments, including post-translationally modified variants. These data validate a novel, highly sensitive MHCII-restricted epitope discovery approach for orphan TCRs and suggest thymic selection of autoantigen-promiscuous TCRs as a mechanism for the murine T1D–I-Ag7-association.

Occurrence and Antigenic Specificity of Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (P-ANCA) in Systemic Autoimmune Diseases

Perinuclear anti-neutrophilic cytoplasmic antibodies (P-ANCA) recognize heterogeneous antigens, including myeloperoxidase (MPO), lactoferrin, elastase, cathepsin-G and bactericidal/permeability-increasing protein. Although P-ANCA have diagnostic utility in vasculitides, they may also be found in patients with various other systemic autoimmune rheumatic diseases (SARDs). Nevertheless, the clinical significance and the targets recognized by P-ANCA in such patients remain unclear. For this purpose, herein we investigated the occurrence of ANCA-related antigenic specificities in 82 P-ANCA-positive sera by multiplex ELISA, as well as their association with other autoantibodies. The P-ANCA-positive sera corresponded to patients with vasculitides (n = 24), systemic lupus erythematosus (n = 28), antiphospholipid syndrome (n = 5), Sjögren’s syndrome (n = 7), rheumatoid arthritis (n = 3), systemic scleroderma (n = 1), sarcoidosis (n = 1) and Hashimoto′s thyroiditis (n = 13). In most P-ANCA-positive patients studied (51/82, 62.3%), these autoantibodies occurred in high titers (>1:160). The analysis of P-ANCA-positive sera revealed reactivity to MPO in only 50% of patients with vasculitides, whereas it was infrequent in the other disease groups studied. Reactivity to other P-ANCA-related autoantigens was also rarely detected. Our findings support that high P-ANCA titers occur in SARD. The P-ANCA-positive staining pattern is associated with MPO specificity in vasculitides, while in other autoimmune diseases, it mostly involves unknown autoantigens.

Antimitochondrial Antibodies and Primary Biliary Cholangitis in Patients with Polymyalgia Rheumatica/Giant Cell Arteritis

Background and Objectives: Laboratory liver abnormalities can be observed in patients affected with polymyalgia rheumatica (PMR) and/or giant cell arteritis (GCA), especially with a cholestatic pattern. The first objective of our review article is to discuss the potential link between antimitochondrial antibodies (AMA) and/or primary biliary cholangitis (PBC) and PMR/GCA, according to the evidences of literature. The second objective is to discuss the association of PMR/GCA with the other rheumatic diseases having PBC as a common manifestation. Materials and Methods: A literature search was performed on PubMed and Medline (OVID interface) using these terms: polymyalgia rheumatica, giant cell arteritis, antimitochondrial antibodies, primary biliary cholangitis, primary Sjogren’s syndrome, systemic sclerosis, and systemic lupus erythematosus. The search was restricted to all studies and case reports published in any language. Reviews, conference abstracts, comments, and non-original articles were excluded; however, each review’s reference list was scanned for additional publications meeting this study’s aim. When papers reported data partially presented in previous articles, we referred to the most recent published data. Results and Conclusions: Our literature search highlighted that cases reporting an association between AMA, PBC and PMR/GCA were very uncommon; AMA antigenic specificity had never been detected and biopsy-proven PBC was reported only in one patient with PMR/GCA. Finally, the association of PMR/GCA with autoimmune rheumatic diseases in which PBC is relatively common was anecdotal.

Evolving Antibody Therapies for the Treatment of Type 1 Diabetes

Type 1 diabetes (T1D) is widely considered to be a T cell driven autoimmune disease resulting in reduced insulin production due to dysfunction/destruction of pancreatic β cells. Currently, there continues to be a need for immunotherapies that selectively reestablish persistent β cell-specific self-tolerance for the prevention and remission of T1D in the clinic. The utilization of monoclonal antibodies (mAb) is one strategy to target specific immune cell populations inducing autoimmune-driven pathology. Several mAb have proven to be clinically safe and exhibit varying degrees of efficacy in modulating autoimmunity, including T1D. Traditionally, mAb therapies have been used to deplete a targeted cell population regardless of antigenic specificity. However, this treatment strategy can prove detrimental resulting in the loss of acquired protective immunity. Nondepleting mAb have also been applied to modulate the function of immune effector cells. Recent studies have begun to define novel mechanisms associated with mAb-based immunotherapy that alter the function of targeted effector cell pools. These results suggest short course mAb therapies may have persistent effects for regaining and maintaining self-tolerance. Furthermore, the flexibility to manipulate mAb properties permits the development of novel strategies to target multiple antigens and/or deliver therapeutic drugs by a single mAb molecule. Here, we discuss current and potential future therapeutic mAb treatment strategies for T1D, and T cell-mediated autoimmunity.

613 HER2-XPAT, a novel protease-activatable prodrug T cell engager (TCE), with potent T-cell activation and efficacy in solid tumors and large predicted safety margins in non-human primate (NHP)

BackgroundTCEs are effective in leukemias but have been challenging in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent CRS include step-up dosing and/or complex designs but are unsuccessful due to toxicity and/or enhanced immunogenicity. HER2-XPAT, or XTENylated Protease-Activated bispecific T-Cell Engager, is a prodrug TCE that exploits the protease activity present in tumors vs. healthy tissue to expand the therapeutic index (TI). The core of the HER2-XPAT (PAT) consists of 2 tandem scFvs targeting CD3 and HER2. Attached to the core, two unstructured polypeptide masks (XTEN) sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, unleashing a potent TCE with short T1/2, further improving the TI. HER2-XPAT, a tumor protease-activatable prodrug with wide safety margins, can co-opt T-cells regardless of antigenic specificity to induce T-cell killing of HER2+ tumors.MethodsPreclinical studies were conducted to characterize the activity of HER2-XPAT, HER2-PAT (cleaved XPAT), and HER2-NonClv (a non-cleavable XPAT) for cytotoxicity in vitro, for anti-tumor efficacy in xenograft models, and for safety in NHPs.ResultsHER2-PAT demonstrated potent in vitro T-cell cytotoxicity (EC50 1-2pM) and target-dependent T-cell activation and cytokine production by hPBMCs. HER2-XPAT provided up to 14,000-fold protection against killing of HER2 tumor cells and no cytotoxicity against cardiomyocytes up to 1uM. In vivo, HER2-XPAT induced complete tumor regressions in BT-474 tumors with equimolar dosing to HER2-PAT, whereas HER2-NonClv had no efficacy, supporting requirement of protease cleavage for T-cell activity. In NHP, HER2-XPAT has been dose-escalated safely up to 42mg/kg (MTD). HER2-XPAT demonstrated early T-cell margination at 2 mg/kg but largely spared CRS, cytokine production, and tissue toxicity up to 42 mg/kg. PK profiles of HER2-XPAT and HER2-NonClv were comparable, consistent with ex vivo stability for cleavage when incubated in cancer pts plasma for 7 days at 37°C. HER2-PAT by continuous infusion induced lethal CRS and cytokine spikes at 0.3 mg/kg/d but was tolerated at 0.25 mg/kg/d, providing HER2-XPAT with >1300-fold protection in tolerability vs. HER2-PAT, >4 logs over cytotoxicity EC50s for HER2 cell lines, and a 20-fold safety margin over the dose required for pharmacodynamic activity.ConclusionsHER2-XPAT is a potent prodrug TCE with no CRS and a wide TI based on NHPs. With XTEN’s clinical data demonstrating low immunogenicity, the XPATs are a promising solution. IND studies are ongoing. Additional PK/PD, cytokines, safety, and efficacy data will be presented.

Antigenic properties of sARs-CoV-2/human/RUs/nsk-FRCFtM-1/2020 coronavirus isolate from a patient in novosibirsk

Objective: isolation of coronavirus SARS-CoV-2 from clinical sample of patient with COVID-19 in Novosibirsk; obtaining a purified and inactivated viral antigen and study of its antigenic properties. Materials and methods: virus isolation was carried out in Vero cell culture from nasopharyngeal swab positive on SARS-CoV-2 RNA. The efficiency of SARSCoV-2 replication in cell culture was assessed on the appearance of cytopathic effect (CPE) and the presence of viral RNA in cultural medium with reverse transcription – polymerase chain reaction (RT-PCR). Purification, concentration and inactivation of the viral preparation were carried out according to standard methods. The purity of the purified preparation and the profile of viral proteins were determined by electrophoresis in 10% polyacrylamide gel (PAG) with the addition of sodium dodecyl sulfate (SDS). The presence and specificity of viral proteins were detected using COVID-19 convalescent’s sera with enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results: SARS-CoV-2/human/ RUS/Nsk-FRCFTM-1/2020 isolate was obtained after passage on Vero cells from a virus-containing clinical sample. A purified, concentrated, inactivated, whole-virion antigen was obtained. It contains three structural proteins: glycoprotein S (approximately 200 kDa), nucleoprotein N (48 kDa), and matrix protein M (20-25 kDa). All viral proteins were detected with serum antibodies of COVID-19 convalescents. Conclusion: SARS-CoV-2 coronavirus can be isolated in Vero cell culture. The antigenic specificity of the three structural viral proteins (S, N, and M) is preserved in the purified inactivated viral preparation. The inactivated whole-virion antigen of SARS-CoV-2/human/RUS/Nsk-FRCFTM-1/2020 isolate can be used to study the antigenic immunomodulating properties of viral proteins, to obtain immune sera of laboratory animals, and also as a component of test systems for the detection of specific antibodies with ELISA and immunoblotting.

HLAs, TCRs, and KIRs, a Triumvirate of Human Cell-Mediated Immunity

In all human cells, human leukocyte antigen (HLA) class I glycoproteins assemble with a peptide and take it to the cell surface for surveillance by lymphocytes. These include natural killer (NK) cells and γδ T cells of innate immunity and αβ T cells of adaptive immunity. In healthy cells, the presented peptides derive from human proteins, to which lymphocytes are tolerant. In pathogen-infected cells, HLA class I expression is perturbed. Reduced HLA class I expression is detected by KIR and CD94:NKG2A receptors of NK cells. Almost any change in peptide presentation can be detected by αβ CD8+ T cells. In responding to extracellular pathogens, HLA class II glycoproteins, expressed by specialized antigen-presenting cells, present peptides to αβ CD4+ T cells. In comparison to the families of major histocompatibility complex (MHC) class I, MHC class II and αβ T cell receptors, the antigenic specificity of the γδ T cell receptors is incompletely understood.

SAT0347 AUTOANTIBODIES TO UNCOUPLED RO52 PROTEIN IN PATIENTS WITH ANTI-SYNTHETASE SYNDROME; A POTENTIAL MARKER FOR SUBCLASSIFICATION.

Background:Anti-Ro/SS-A (anti-SS-A) antibody is one of myositis-associated antibodies, and is found in patients with anti-synthetase antibodies1. Anti-SS-A antibody targets the complex consisting of Ro52 and Ro60 proteins coupled with cytoplasmic non-coding Y-RNAs. Autoantibody against Ro52 uncoupled with Y-RNAs (anti-uncoupled Ro52) is also present in patients with a variety of connective tissue diseases, including myositis2. However, the majority of previous studies used enzyme-linked immunoassay (EIA) for detection of anti-Ro52 antibodies, resulting in failure to discriminate between anti-Ro52 antibodies coupled and uncoupled with Y-RNAs. The prevalence and clinical significance of anti-uncoupled Ro52 antibodies still remain unclear in patients with anti-synthetase antibodies.Objectives:To elucidate clinical relevance of anti-uncoupled Ro52 antibodies in a cohort of anti-synthetase syndrome employing RNA immunoprecipitation assay (RNA-IP) in combination with EIA.Methods:This is a single-center, cross-sectional study involving 80 patients positive for anti-synthetase antibodies by RNA-IPP. Complete clinical information was obtained from a medical chart review. Serum samples were obtained at first visit and stored at -20°C until use. Anti-SS-A and anti-SS-B antibodies were detected by RNA-IP, and anti-Ro52 and anti-Ro60 antibodies were measured by commercial EIA kits (ORGENTIC, Mainz, Germany). Autoantibodies that immunoprecipitated Y-RNAs regardless of results of anti-anti-Ro52 or anti-Ro60 EIAs were regarded as anti-SS-A antibody, while antibodies that did not immunoprecipitate Y-RNAs but reacted with anti-Ro52 antibodies by EIA were regarded as anti-uncoupled Ro52 antibody. Student’s t-test, Mann-Whitney’s U test, and Fisher’s exact test were employed to compare the clinical features between each group. Cumulative survival rates were compared using log-rank test.Results:In our cohort of 80 patients with anti-synthetase antibody, mean age at diagnosis was 61 ± 12 years, and 76% were female. Clinical diagnosis was classic dermatomyositis (cDM) in 11, clinically amyopathic dermatomyositis (CADM) in 21, polymyositis (PM) in 11, systemic sclerosis (SSc) in 3, myositis-SSc overlap in 5, interstitial lung disease (ILD) alone in 29, and unclassified in 3. The antigenic specificity of anti-synthetase antibodies included Jo-1 in 19, PL-7 in 12, PL-12 in 9, EJ in 21, OJ in 4, and KS in 16. Anti-SS-A anti-SS-B antibodies were found in 14 (17%) and 2 (2.5%) patients, respectively. The presence of anti-Ro60 and anti-SS-A antibodies was almost concordant (P < 0.0001), although the presence of anti-Ro52 and anti-SS-A antibodies was not correlated (P = 0.8). This was primarily because of high prevalence (40%) of autoantibodies to uncoupled Ro52. Interestingly, prevalence of anti-uncoupled Ro52 antibodies was different among antigenic specificities of anti-synthetase antibodies: high in Jo-1 (58%) and EJ (55%), and low in PL-7 (8%) and OJ (0%). Gottron’s sign/papule was more frequent in patients with anti-uncoupled Ro52 than in those without (61% versus 28%, P = 0.005), resulting in clinical diagnosis of cDM or CADM more common in patients with anti-uncoupled Ro52 than in those without (59% versus 26%; P = 0.003). The prevalence and extent of ILD tended to be greater in anti-uncoupled Ro52-positive versus negative patients, but difference did not reach statistical significance. There were no differences in cumulative survival rates between patients stratified by the presence or absence of anti-uncoupled Ro52 antibodies.Conclusion:Autoantibodies to uncoupled Ro52 were commonly found in patients with anti-synthetase antibodies. Anti-Ro52 positivity might be useful for subclassifying anti-synthetase syndrome.References:[1]McHugh NJ et al.Nat Rev Rheumatol. 2018;14(5): 290-302.[2]Schulte-Pelkum J et al.Autoimmun Rev. 2009;8(7): 632-7.Disclosure of Interests:Akira Yoshida: None declared, Yuka Okazaki: None declared, Takahisa Gono Speakers bureau: Astellas, and Medical and Biological Laboratories, Masataka Kuwana Grant/research support from: Acetelion, Consultant of: Acetelion, Bayer, Chugai, Corbus Pharmaceuticals, CSL Behring and Reata Pharmaceuticals. He was a member of the SENSCIS trial Steering Committee (Boehringer Ingelheim)