Human HLA Research Articles

Generation of a HuTCR mouse platform-derived MAGE-A1-directed high-affinity TCR with superior potency versus human-derived TCRs.

e14515 Background: As cancer-testis antigens are self-antigens, T cells expressing high-affinity TCRs against such antigens are suppressed via negative thymic selection. Therefore, patient- or donor-derived TCRs are typically of low affinity and result in a reduced antitumor effect. Using our proprietary HuTCR platform, which consists of mouse lines carrying the full human TCR α/β loci in combination with common human HLA alleles, we have isolated high-affinity TCRs specific for the cancer-testis antigen MAGE-A1 and compared them to human-derived MAGE-A1-specific TCRs that are currently reported to be in clinical development. Furthermore, we validated MAGE-A1 as a potential cancer therapy target by using immunohistochemistry to evaluate expression in several major tumor types and healthy tissue. Methods: Using scRNAseq, TCRs were isolated from HuTCR mice. Human-derived MAGE-A1-specific TCR sequences were obtained from publicly available databases. All TCRs were expressed in primary human T cells as verified using peptide-MHC-multimer staining. Functional avidity of the TCRs was analyzed by coculture with T2 target cells loaded with titrated amounts of epitope peptides and measuring cytokine concentration by ELISA. Reactivity of TCRs to endogenously processed MAGE-A1 protein was assessed by co-culture with a panel of tumor cell lines varying in MAGE-A1 and/or MHC-class-I expression. MAGE-A1 expression on protein level was evaluated by immunohistochemistry. Results: Immunization of HuTCR mice with the antigen resulted in robust CD8+ T cell responses and several TCR clonotypes were identified by scRNAseq, with the majority of clonotypes being specific to the MAGE-A1-derived peptide KVLEYVIKV and TCR affinities ranging from 0.3 nM to 3 nM. By comparison, human-derived TCRs exhibited generally lower functional avidity from 3 nM to 60 nM. In addition, HuTCR-mouse-derived TCRs were more sensitive in recognition of tumor cell lines expressing low MAGE-A1 and/or HLA-A2. Immunohistochemical analysis of MAGE-A1 expression in healthy tissues demonstrated highly selective expression of MAGE-A1 in testis, only. Screening for expression confirmed that a significant proportion of several major cancer types expresses MAGE-A1 as reported by various other groups [reviewed in Curr Opin Cell Biol. 2015 December; 37: 1–8]. Conclusions: The HuTCR mouse platform allows for the generation of high-affinity MAGE-A1-specific TCRs with increased anti-tumor efficacy as compared to human-derived TCRs against the same cancer antigen. In addition, it was confirmed that MAGE-A1 has a highly selective expression pattern in healthy tissues (testis, only), but shows distinct expression in several major human tumor types.

Comprehensive mutagenesis identifies the peptide repertoire of a p53 T-cell receptor mimic antibody that displays no toxicity in mice transgenic for human HLA-A*0201.

T-cell receptor mimic (TCRm) antibodies have expanded the repertoire of antigens targetable by monoclonal antibodies, to include peptides derived from intracellular proteins that are presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface. We have previously used this approach to target p53, which represents a valuable target for cancer immunotherapy because of the high frequency of its deregulation by mutation or other mechanisms. The T1-116C TCRm antibody targets the wild type p5365-73 peptide (RMPEAAPPV) presented by HLA-A*0201 (HLA-A2) and exhibited in vivo efficacy against triple receptor negative breast cancer xenografts. Here we report a comprehensive mutational analysis of the p53 RMPEAAPPV peptide to assess the T1-116C epitope and its peptide specificity. Antibody binding absolutely required the N-terminal arginine residue, while amino acids in the center of the peptide contributed little to specificity. Data mining the immune epitope database with the T1-116C binding consensus and validation of peptide recognition using the T2 stabilization assay identified additional tumor antigens targeted by T1-116C, including WT1, gp100, Tyrosinase and NY-ESO-1. Most peptides recognized by T1-116C were conserved in mice and human HLA-A2 transgenic mice showed no toxicity when treated with T1-116C in vivo. We conclude that comprehensive validation of TCRm antibody target specificity is critical for assessing their safety profile.

Molecular Signatures of Natural Killer Cells in CMV-Associated Anterior Uveitis, A New Type of CMV-Induced Disease in Immunocompetent Individuals

Cytomegalovirus (CMV) causes clinical issues primarily in immune-suppressed conditions. CMV-associated anterior uveitis (CMV-AU) is a notable new disease entity manifesting recurrent ocular inflammation in immunocompetent individuals. As patient demographics indicated contributions from genetic background and immunosenescence as possible underlying pathological mechanisms, we analyzed the immunogenetics of the cohort in conjunction with cell phenotypes to identify molecular signatures of CMV-AU. Among the immune cell types, natural killer (NK) cells are main responders against CMV. Therefore, we first characterized variants of polymorphic genes that encode differences in CMV-related human NK cell responses (Killer cell Immunoglobulin-like Receptors (KIR) and HLA class I) in 122 CMV-AU patients. The cases were then stratified according to their genetic features and NK cells were analyzed for human CMV-related markers (CD57, KLRG1, NKG2C) by flow cytometry. KIR3DL1 and HLA class I combinations encoding strong receptor–ligand interactions were present at substantially higher frequencies in CMV-AU. In these cases, NK cell profiling revealed expansion of the subset co-expressing CD57 and KLRG1, and together with KIR3DL1 and the CMV-recognizing NKG2C receptor. The findings imply that a mechanism of CMV-AU pathogenesis likely involves CMV-responding NK cells co-expressing CD57/KLRG1/NKG2C that develop on a genetic background of KIR3DL1/HLA-B allotypes encoding strong receptor–ligand interactions.

Corona Viruses and Human Leukocyte Antigen (HLA) alleles

Major histocompatibility complex (MHC) is consisted of cluster of genes known as human leukocyte antigen HLA, these genes are committed to process and present antigens to T lymphocyte. Corona viruses, belonging to a genus of the corona viridae family, are enveloped viruses containing single stranded RNA 27-32 kb. This study was conducted to highlight some observations that may help other researcher for rapid research advances; the new corona virus genome sequence was obtained from Virus pathogen database, the spike protein (surface glycoprotein) was aligned with SPIKE_CVHSA Spike glycoprotein OS=Human SARS coronavirus OX=694009 GN=S PE=1 SV=1 which was retrieved from Uniprot data base Distribution of HLA alleles was obtained from allele frequencies database, IEDB server was used for MHC 1 prediction. The distribution of HLA-A alleles among population revealed that, the most common wasHLA-A*11:01. The affinity prediction of HLA-A*03:01(AAAYFVGYLK) and HLA-A*11:01(AAYFVGYLK) alleles to the spike peptide were positive. The two positive predicted peptides for both 2 alleles showed mutation of K245Q, This mutation may affect the binding affinity of the protein peptides to HLA-A alleles.
 Keywords: HLA. MHC, alleles, Corona viruses, SARS, APS

In-silico identification of subunit vaccine candidates against lung cancer-associated oncogenic viruses

Globally, ~20% of cancer malignancies are associated with virus infections. Lung cancer is the most prevalent cancer and has a 10% 5-year survival rate when diagnosed at stage IV. Cancer vaccines and oncolytic immunotherapy are promising treatment strategies for better clinical outcomes in advanced-stage cancer patients. Here, we used a reverse vaccinology approach to devise subunit vaccine candidates against lung cancer-causing oncogenic viruses. Protein components (945) from nine oncogenic virus species were systematically analyzed to identify epitope-based subunit vaccine candidates. Best vaccine candidates were identified based on their predicted ability to stimulate humoral and cell-mediated immunity and avoid self-tolerance. Using a rigorous integrative approach, we identified 125 best antigenic epitopes with predicted B-cell, T-cell, and/or MHC-binding capability and vaccine adjuvant potential. Thirty-two of these antigenic epitopes were predicted to have IL-4/IFN-gamma inducing potential and IL-10 non-inducing potential and were predicted to bind 15 MHC-type I and 49 MHC-type II alleles. All 32 epitopes were non-allergenic and 31 were non-toxic. The identified epitopes showed good conservancy and likely bind a broad class of human HLA alleles, indicating promiscuous potential. The majority of best antigenic epitopes were derived from Human papillomavirus and Epstein-Barr virus proteins. Of the 32 epitopes, 25 promiscuous epitopes were related to E1 and E6 envelope genes and were present in multiple viral strains/species, potentially providing heterologous immunity. Further validating our results, 38 antigenic epitopes were also present in the largest experimentally-validated epitope resource, Immune Epitope Database and Analysis Resource. We further narrowed the selection to 29 antigenic epitopes with the highest immunogenic/immune-boosting potential. These epitopes possess tremendous therapeutic potential as vaccines against lung cancer-causing viruses and should be validated in future experiments. All findings are available at https://webs.iiitd.edu.in/raghava/vlcvirus/.

Distribution of HLA class Ⅰ and class Ⅱ haplotypes in Chinese Han population based on family segregation

HLA haplotype analysis has important application value in human population genetics, anthropological research and HLA matching transplantation. Based on HLA-A, -B, -C, -DRB1 and -DQB1 genotyping data from 663 families including 663 leukemia patients and 991 related donors, the allele frequency (AF) and haplotype frequency (HF) of two-, three- and five-locus haplotype distribution patterns in the Chinese Han population were determined by family segregation. A total of 38 alleles at A locus, 75 alleles at B locus, 35 alleles at C locus, 53 alleles at DRB1 locus and 22 alleles at DQB1 locus were discovered in this population. The frequencies of these alleles were basically consistent with those of previous reports except for some tiny differences. The study found 11 A-C, 15 C-B, 4 B-DRB1 and 11 DRB1-DQB1 two-locus haplotypes with a frequency over 2%. The number of A-C-B and A-B-DRB1 three-locus haplotype with a frequency over 1% were 11 and 3 respectively. The most common HLA-A-C-B-DRB1-DQB1 haplotype (HF>1%) were A*3001-C*0602-B*1302-DR*0701-DQ*0202 (4.30%), A*0207-C*0102-B*4601-DR*0901-DQ*0303 (3.07%), A*3303-C*0302-B*5801-DR*0301-DQ*0201 (1.49%) and A*1101-C*0102-B*4601-DR*0901-DQ*0303 (1.01%). The results are helpful for finding matching donors for hematopoietic stem cell transplant patients and also contribute to transplant immunology, HLA-related diseases, research of human genetics and other fields.

Dendritic Cells Prime Anti-Tumor CD8 <sup>+</sup> T Cell Responses Through MHC-Dressing

Antigen cross-presentation, wherein dendritic cells (DC) present exogenous antigen on MHC-I molecules, is considered the primary mechanism by which DCs initiate tumor-specific CD8+ T cell responses. Here, we demonstrate that MHC-dressing, an antigen presentation pathway in which DCs acquire and display intact peptide:MHC-I molecules, is critical for anti-tumor immunity. Cancer cell MHC-I expression was required for optimal CD8+ T cell activation in wo subcutaneous tumor models. In vivo acquisition of tumor-derived peptide:MHC-I molecules by DCs was sufficient to induce antigen-specific CD8+ T cell priming, which was not ablated upon genetic inactivation of tumor antigen cross-presentation. Transfer of human tumor-derived HLA molecules to myeloid cells was detected in vitro and in human tumor xenografts. Inconclusion, MHC-I-dressing is crucial for anti-tumor CD8+ T cell priming by DCs. In addition to quantitatively enhancing tumor antigen presentation, MHC-dressing might also enable DCs to more faithfully and efficiently mirror the cancer cell peptidome.

Mycobacterium Tuberculosis'in Sitotoksik T Lenfosit epitoplarının Hesaplamalı Haritalaması ve Aşı Tasarımındaki Potansiyel Rolü

Objective: Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) is one of the deadliest diseases causing millions of deaths worldwide. Bacillus Calmette-Guérin (BCG) is the only vaccine that has been used in many countries where TB is prevalent. Despite vaccination, this disease prevails in many of the developing countries, necessitating the development of an effective vaccine against TB. Since M. tb acts as an intracellular pathogen, cell-mediated immune response plays an important role in disease control. Therefore, screening of CD8+ T cell epitopes of M. tb antigens could aid in the development of an effective vaccine against TB. In the current study, a reverse vaccinology approach was utilized to predict and map cytotoxic T lymphocyte (CTL) epitopes in the virulent proteins that are also essential for M. tb. Materials and Methods: Database of Essential Genes and Virulence Factor Database were used for identifying the virulent proteins of M. tb and their antigenicity was assessed using VaxiJen server. Various immunoinformatics tools were used to predict MHC class I binding, MHC processing, immunogenicity, toxicity and allergenicity. Results: Twelve M. tb antigens were selected for the prediction analyses using various tools. The results indicated the presence of 20 novel CTL epitopes predicted against human HLA-A alleles. This study has also screened for multiple allele binding epitopes that could be used as a vaccine component. Conclusion: This study has yielded a few hitherto unreported CTL epitopes binding to class I HLA-A alleles. Further experimental validation is necessary for confirming their potential as vaccine candidates.

In vivo delivery of a multiepitope peptide and Nef protein using novel cell-penetrating peptides for development of HIV-1 vaccine candidate.

ObjectivesA potent HIV vaccine should overcome some limitations such as polymorphism of human HLA, the diversity of HIV-1 virus, and the lack of an effective delivery system. In this study, a DNA construct encoding Nef60–84, Nef126–144, Vpr34–47, Vpr60–75, Gp16030–53, Gp160308–323, and P248–151 epitopes was designed using bioinformatics tools. The pcDNA3.1-nef-vpr-gp160-p24 and pcDNA3.1-nef constructs were prepared in large scale as endotoxin-free form. Moreover, the recombinant Nef-Vpr-Gp160-p24 polypeptide and Nef protein were generated inE. coli. These constructs were delivered using cell penetrating peptides (CPPs) in vivo, and immune responses were assessed for different modalities in BALB/c mice.ResultsThe recombinant DNA constructs were confirmed as the ~ 867 bp and ~ 648 bp bands related tonef-vpr-gp160-p24 andnef genes on agarose gel. Moreover, the purified Nef-Vpr-Gp160-p24 polypeptide and Nef protein showed the ~ 32 kDa and ~ 30 kDa bands on SDS-PAGE, respectively. The results of immune responses indicated that the heterologous prime/boost regimens using both Nef-Vpr-Gp160-P24 and Nef antigens induced significantly the secretion of IgG2a, IgG2b, IFN-γ and Granzyme B compared to other groups. The levels of Granzyme B in mice immunized with Nef antigen were higher than those immunized with Nef-Vpr-Gp160-P24 antigen. The CPPs showed the same potency with Montanide adjuvant for eliciting immune responses.ConclusionsThe heterologous prime/boost regimens for both antigens could significantly direct immune responses toward Th1 and CTL activity compared to other regimens. Comparing the efficiency of Nef-Vpr-Gp160-P24 and Nef constructs, the Nef-Vpr-Gp160-P24 constructs delivered by CPPs showed promising results as an HIV vaccine candidate.

Specific Inhibition of Tumor Growth by T Cell Receptor–Drug Conjugates Targeting Intracellular Cancer-Testis Antigen NY-ESO-1/LAGE-1

Despite the significant therapeutic advances in T-cell immunotherapy, many malignancies remain unresponsive, which might be because of the negative regulation of T cells by the tumor microenvironment (TME). T cells discriminate tumor cells and normal cells through T-cell receptors (TCRs); therefore, we generated a novel type of TCR-drug conjugates (TDCs) by referring antibody-drug conjugations (ADCs) to overcome the effects of the TME on T cells while preserving the specificity of TCR for tumor recognition. We selected HLA-A2/NY-ESO-1157-165 (peptide NY-ESO-1157-165 in complex with human leukocyte antigen serotype HLA-A*02:01) as the antigen and the antigen-specific TCR (1G4113) as the carrier. By sortase A-mediated ligation, we obtained three NY-TCR-vcMMAEs and further studied their properties, antitumor activity, and toxicity in vitro and in vivo. We found that all the NY-TCR-vcMMAEs had high endocytosis efficiency and specifically killed HLA-A2/NY-ESO-1157-165 positive tumor cells. In xenograft models, one of the TDCs, NY-TCR-2M, was effectively and specifically distributed into tumor tissues and inhibited tumor growth without inducing obvious toxicity. Our results demonstrated that TCRs can be carriers of toxic payloads and that the TDCs thus formed can specifically inhibit tumor growth, neglecting the immune microenvironment.

Japanese Encephalitis Virus Vaccination Elicits Cross-Reactive HLA-Class I-Restricted CD8 T Cell Response Against Zika Virus Infection.

Japanese encephalitis virus (JEV) exposure or vaccination could elicit cross-reactive CD8 T cell immunity against heterologous flaviviruses in humans. In addition, cross-reactive CD8 T cells induced by dengue virus (DENV) have been shown to play a protective role against Zika virus (ZIKV). However, how JEV exposure or vaccination affects ZIKV infection in humans remains unclear. In this report, epitope prediction algorithms were used to predict the cross-reactive CD8 T cell epitope restricted to human HLA between JEV and ZIKV. We found that these predicted CD8 T cell epitopes are immunogenic and cross-reactive in humanized HLA transgenic mice. Moreover, JEV vaccine immunization provided cross-protection against ZIKV infection. Furthermore, CD8 T cells were involved in the protection against ZKIV infection in vivo. Our results have an important clinical implication that vaccination with JEV SA14-14-2 may provide protection against ZIKV infection in humans.

Abstract PO-049: SARS-CoV-2 multiepitope vaccine constructs designed to drive long-term immunity in the majority of the population

Abstract The current SARS-CoV-2 pandemic has precipitated an urgent need for a safe and effective vaccine to be developed and deployed in a highly accelerated timeframe as compared to standard vaccine development processes. Upfront selection of epitopes most likely to induce a safe and effective immune response can accelerate these efforts. Optimally designed vaccines maximize immunogenicity towards regions of proteins that contribute most to protective immunity, while minimizing the antigenic load contributed by unnecessary protein domains that may result in autoimmunity, reactogenicity, or enhanced infectivity. Adopting tools developed for population-scale characterization of HLA presentation of tumor antigens and cross-reactivity of TCRs with tumor self-antigens, we have generated an immunogenicity map of SARS-CoV-2 to inform vaccine design based on analyses across five parameters: 1) stimulation of CD4 and CD8 T cells; 2) immunogenicity across the majority of human HLA alleles; 3) targeting both evolutionarily conserved regions, as well as newly divergent regions of the virus that increase infectivity; 4) targeting linear and conformational B-cell epitopes; and 5) targeting viral regions with the highest degree of dissimilarity to the self-immunopeptidome such as to maximize safety and immunogenicity. Using these analyses, we have generated 11 SARS-CoV-2 vaccine constructs optimized for long-term immunity across the majority of the population. These constructs contain combinations of epitopes selected from our analysis such as to drive affinity-enhanced memory response in combination with current spike protein vaccine strategies, for use as T-cell vaccines, and a stand-alone vaccine designed to drive memory B- and T-cell responses in the majority of the population. Epitopes were optimized using our immunogenicity algorithm to minimize immunogenicity across the junctions between epitopes and cloned into pVAX vectors, using signal peptides targeting the lysosome, ER, and cytoplasmic secretion, such as to promote presentation to CD4, CD8, and B cells, respectively. Finally, we describe methods for identifying immunodominant epitopes arising from vaccination using barcoded, multiplexed tetramers. Vaccine constructs are currently undergoing testing in transgenic mice expressing human HLA-A2. We expect that these constructs will help drive long-term immunity across the population, targeting conserved regions across multiple coronaviruses. Citation Format: Mark Yarmarkovich, John M. Warrington, Alvin Farrel, John M. Maris. SARS-CoV-2 multiepitope vaccine constructs designed to drive long-term immunity in the majority of the population [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr PO-049.

OnionMHC: A deep learning model for peptide — HLA-A*02:01 binding predictions using both structure and sequence feature sets

The peptide binding to Major Histocompatibility Complex (MHC) proteins is an important step in the antigen-presentation pathway. Thus, predicting the binding potential of peptides with MHC is essential for the design of peptide-based therapeutics. Most of the available machine learning-based models predict the peptide-MHC binding based on the sequence of amino acids alone. Given the importance of structural information in determining the stability of the complex, here we have utilized both the complex structure and the peptide sequence features to predict the binding affinity of peptides to human receptor HLA-A*02:01. To our knowledge, no such model has been developed for the human HLA receptor before that incorporates both structure and sequence-based features. Results: We have applied machine learning techniques through the natural language processing (NLP) and convolutional neural network to design a model that performs comparably with the existing state-of-the-art models. Our model shows that the information from both sequence and structure domains results in enhanced performance in the binding prediction compared to the information from one domain alone. The testing results in 18 weekly benchmark datasets provided by the Immune Epitope Database (IEDB) as well as experimentally validated peptides from the whole-exome sequencing analysis of the breast cancer patients indicate that our model has achieved state-of-the-art performance. Conclusion: We have developed a deep-learning model (OnionMHC) that incorporates both structure as well as sequence-based features to predict the binding affinity of peptides with human receptor HLA-A*02:01. The model demonstrates state-of-the-art performance on the IEDB benchmark dataset as well as the experimentally validated peptides. The model can be used in the screening of potential neo-epitopes for the development of cancer vaccines or designing peptides for peptide-based therapeutics. OnionMHC is freely available at https://github.com/shikhar249/OnionMHC .

Strict Assembly Restriction of Peptides from Rabbit Hemorrhagic Disease Virus Presented by Rabbit Major Histocompatibility Complex Class I Molecule RLA-A1.

Rabbits are pivotal domestic animals for both the economy and as an animal model for human diseases. A large number of rabbits have been infected by rabbit hemorrhagic disease virus (RHDV) in natural and artificial pandemics in the past. Differences in presentation of antigenic peptides by polymorphic major histocompatibility complex (MHC) molecules to T-cell receptors (TCR) on T lymphocytes are associated with viral clearance in mammals. Here, we screened and identified a series of peptides derived from RHDV binding to the rabbit MHC class I molecule, RLA-A1. The small, hydrophobic B and F pockets of RLA-A1 capture a peptide motif analogous to that recognized by human class I molecule HLA-A*0201, with more restricted aliphatic anchors at P2 and PΩ positions. Moreover, the rabbit molecule is characterized by an uncommon residue combination of Gly53, Val55, and Glu56, making the 310 helix and the loop between the 310 and α1 helices closer to the α2 helix. A wider A pocket in RLA-A1 can induce a special conformation of the P1 anchor and may play a pivotal role in peptide assembly and TCR recognition. Our study broadens the knowledge of T-cell immunity in domestic animals and also provides useful insights for vaccine development to prevent infectious diseases in rabbits.IMPORTANCE We screened rabbit MHC class I RLA-A1-restricted peptides from the capsid protein VP60 of rabbit hemorrhagic disease virus (RHDV) and determined the structures of RLA-A1 complexed with three peptides, VP60-1, VP60-2, and VP60-10. From the structures, we found that the peptide binding motifs of RLA-A1 are extremely constraining. Thus, there is a generally restricted peptide selection for RLA-A1 compared to that for human HLA-A*0201. In addition, uncommon residues Gly53, Val55, and Glu56 of RLA-A1 are located between the 310 helix and α1 helix, which makes the steric position of the 310 helix in RLA-A1 much closer to the α2 helix than that found in other mammalian MHC class I molecules. This special conformation between the 310 helix and α1 helix plays a pivotal role in rabbit MHC class I assembly. Our results provide new insights into MHC class I molecule assembly and peptide presentation of domestic mammals. Furthermore, these data also broaden our knowledge on T-cell immunity in rabbits and may also provide useful information for vaccine development to prevent infectious diseases in rabbits.

Discovery of New Targets to Control Metastasis in Pancreatic Cancer by Single-cell Transcriptomics Analysis of Circulating Tumor Cells.

Metastasis development is the leading cause of cancer-related mortality in pancreatic ductal adenocarcinoma (PDAC) and yet, few preclinical systems to recapitulate its full spreading process are available. Thus, modeling of tumor progression to metastasis is urgently needed. In this work, we describe the generation of highly metastatic PDAC patient-derived xenograft (PDX) mouse models and subsequent single-cell RNA-sequencing (RNA-seq) of circulating tumor cells (CTC), isolated by human HLA sorting, to identify altered signaling and metabolic pathways, as well as potential therapeutic targets. The mouse models developed liver and lung metastasis with a high reproducibility rate. Isolated CTCs were highly tumorigenic, had metastatic potential, and single-cell RNA-seq showed that their expression profiles clustered separately from those of their matched primary and metastatic tumors and were characterized by low expression of cell-cycle and extracellular matrix-associated genes. CTC transcriptomics identified survivin (BIRC5), a key regulator of mitosis and apoptosis, as one of the highest upregulated genes during metastatic spread. Pharmacologic inhibition of survivin with YM155 or survivin knockdown promoted cell death in organoid models as well as anoikis, suggesting that survivin facilitates cancer cell survival in circulation. Treatment of metastatic PDX models with YM155 alone and in combination with chemotherapy hindered the metastatic development resulting in improved survival. Metastatic PDX mouse model development allowed the identification of survivin as a promising therapeutic target to prevent the metastatic dissemination in PDAC.

Incorporation of a Novel CD4+ Helper Epitope Identified from Aquifex aeolicus Enhances Humoral Responses Induced by DNA and Protein Vaccinations.

SummaryCD4+ T cells play an important role in the maturation of the antibody responses. Conjugation of identified CD4+ T cell helper epitope to the target antigen has been developed as a strategy to enhance vaccine-induced humoral immunity. In this work, we reported the identification of a novel HLA-IAb helper epitope LS-3 from Aquifex aeolicus. In silico analysis predicted this epitope to have high binding affinity to common human HLA alleles and have complementary binding coverage to the established PADRE epitope. Introduction of HLA-IAb knockout mutations to the LS-3 epitope significantly attenuated humoral responses induced by a vaccine containing this epitope. Finally, engineered fusion of the epitope to a model antigen, influenza hemagglutinin, significantly improved both binding and hemagglutination inhibition antibody responses in mice receiving DNA or protein vaccines. In summary, LS-3 and additional identified CD4+ helper epitopes may be further explored to improve vaccine responses in translational studies.

Enhanced Immunogenicity of Engineered HER2 Antigens Potentiates Antitumor Immune Responses.

For cancer vaccines, the selection of optimal tumor-associated antigens (TAAs) that can maximize the immunogenicity of the vaccine without causing unwanted adverse effects is challenging. In this study, we developed two engineered Human epidermal growth factor receptor 2 (HER2) antigens, K965 and K1117, and compared their immunogenicity to a previously reported truncated HER2 antigen, K684, within a B cell and monocyte-based vaccine (BVAC). We found that BVAC-K965 and BVAC-K1117 induced comparable antigen-specific antibody responses and antigen-specific T cell responses to BVAC-K684. Interestingly, BVAC-K1117 induced more potent antitumor activity than the other vaccines in murine CT26-HER2 tumor models. In addition, BVAC-K1117 showed enhanced antitumor effects against truncated p95HER2-expressing CT26 tumors compared to BVAC-K965 and BVAC-K684 based on the survival analysis by inducing T cell responses against intracellular domain (ICD) epitopes. The increased ICD epitope-specific T cell responses induced by BVAC-K1117 compared to BVAC-K965 and BVAC-K684 were recapitulated in human leukocyte antigen (HLA)-untyped human PBMCs and HLA-A*0201 PBMCs. Furthermore, we also observed synergistic antitumor effects between BVAC-K1117 and anti-PD-L1 antibody treatment against CT26-HER2 tumors. Collectively, our findings demonstrate that inclusion of a sufficient number of ICD epitopes of HER2 in cellular vaccines can improve the antitumor activity of the vaccine and provide a way to optimize the efficacy of anticancer cellular vaccines targeting HER2.

Abstract IA04: Novel approaches to study the tumor-stroma interaction and translate immune-directed therapies

Abstract Cancer research has long been hampered by the limitations of the current model systems. Both cultured cells and mouse xenografts grow in an environment highly dissimilar to that of their originating tumor, frequently resulting in promising treatments that are ultimately clinically ineffective. The development of highly immunodeficient mouse strains into which human immune systems can be engrafted can help bridge this gap. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse (HM) models. HM allow researchers to examine xenograft growth in the context of a human immune system and resultant tumor microenvironment, and recent studies have highlighted the increased similarities in attendant tumor structure, metastasis, and signaling to those features in cancer patients. This setting also facilitates the examination of investigational cancer therapies, including new immunotherapies. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient’s tumor, suggesting an enhanced capability to accurately recapitulate a human TME. This review presentation also discusses other recent advancements in the generation and application of HM models, such as engineering mouse strains expressing human cytokines or HLA proteins, and implanting human bone, liver, and thymus tissue to facilitate immune cell maturation and trafficking. Finally, we discuss how these improvements may help direct future HM model cancer studies, and their potential in generating clinically relevant treatments. Citation Format: Antonio Jimeno. Novel approaches to study the tumor-stroma interaction and translate immune-directed therapies [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA04.

Computational approaches in epitope design using DNA binding proteins as vaccine candidate in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is a successful pathogen in the history of mankind. A high rate of mortality and morbidity raises the need for vaccine development. Mechanism of pathogenesis, survival strategy and virulence determinant are needed to be explored well for this pathogen. The involvement of DNA binding proteins in the regulation of virulence genes, transcription, DNA replication, repair make them more significant. In present work, we have identified 1453 DNA binding proteins (DBPs) in the 4173 genes of Mtb through the DNABIND tool and they were subjected for further screening by incorporating different bioinformatics tools. The eighteen DBPs were selected for the B-cell epitope prediction by using ABCpred server. Moreover, the B-cell epitope bearing the antigenic and non- allergenic property were selected for T-cell epitope prediction using ProPredI, and ProPred server. Finally, DGIGSAVSV (Rv1088), IRALPSSRH (Rv3923c), LTISPIANS (Rv3235), VQPSGKGGL (Rv2871) VPRPGPRPG (Rv2731) and VGQKINPHG (Rv0707) were identified as T-cell epitopes. The structural modelling of these epitopes and DBPs was performed to ensure the localization of these epitopes on the respective proteins. The interaction studies of these epitopes with human HLA confirmed their validation to be used as potential vaccine candidates. Collectively, these results revealed that the DBPs– Rv2731, Rv3235, Rv1088, Rv0707, Rv3923c and Rv2871 are the most appropriate vaccine candidates. In our knowledge, it is the first report of using the DBPs of Mtb for epitope prediction. Significantly, this study also provides evidence to be useful for designing a peptide-based vaccine against tuberculosis.

Applying MAPPs Assays to Assess Drug Immunogenicity.

Immunogenicity against biotherapeutic proteins (BPs) and the potential outcome for the patient are difficult to predict. In vitro assays that can help to assess the immunogenic potential of BPs are not yet used routinely during drug development. MAPPs (MHC-associated peptide proteomics) is one of the assays best characterized regarding its value for immunogenicity potential assessment. This review is focusing on recent studies that have employed human HLA class II-MAPPs assays to rank biotherapeutic candidates, investigate clinical immunogenicity, and understand mechanistic root causes of immunogenicity. Advantages and challenges of the technology are discussed as well as the different areas of application.