Generation of novel complete HLA class I monoallelic cell lines used in an MHC stabilization assay for neoantigen evaluation.

Immunogenic neoantigens derived from somatic mutations in cancer have been identified through clinical studies with the cloning of tumor-infiltrating T cells, and cancer driver gene mutation-derived epitopes have been reported; however, these are rare. At present, the validation of epitopes predicted in silico is difficult as human T-cell clonal diversity cannot be reproduced in vitro or in experimental animal models. To confirm the epitope peptides presented by human leukocyte antigen (HLA) class I molecules predicted in silico, biochemical methods such as major histocompatibility complex (MHC) stabilization assays and mass spectrometry-mediated identification have been developed based on HLA-A*02:01 monoallelic T2 cells and HLA-C*01:02 monoallelic LCL721.221 cells. Therefore, in the present study, to prevent confusion due to peptide cross-presentation among HLA molecules, HLA class I monoallelic B-cell clones were generated from the TISI cell line by knocking out HLA-ABC and TAP2, and knocking in HLA alleles. To explore cancer driver mutations as potential targets for immunotherapy, exome sequencing data from 5,143 patients with cancer enrolled in a comprehensive genome analysis project at the Shizuoka Cancer Center were used to identify somatic amino acid substituted mutations and the 50 most frequent mutations in five genes, TP53, EGFR, PIK3CA, KRAS and BRAF, were identified. Using NetMHC4.1, the present study predicted whether epitopes derived from these mutations are presented on major HLA-ABC alleles in Japanese individuals and synthesized 138 peptides for MHC stabilization assays. The authors also attempted to examine the candidate epitopes at physiological temperatures by using antibody clone G46-2.6, which can detect HLA-ABC, independent of ß2-microglobulin association. In the assays, although the peptide-induced HLA expression levels were associated with the predicted affinities, the respective HLA alleles exhibited varying degrees of responsiveness, and unexpectedly, p53-mutant epitopes with predicted weak affinities exhibited strong responses. These results suggested that MHC stabilization assays using completely monoallelic HLA-expressing B-cell lines are useful for evaluating the presentation of neoantigen epitopes.

Development of Novel Small Antitumor Compounds Inhibiting PD-1/PD-L1 Binding.

BACKGROUND/AIM: Anti-programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) antibody is a successful treatment for patients with solid cancers; however, there are several disadvantages that need to be resolved. Oral small molecule anti-PD-1/PD-L1 inhibitors have been developed and have good bioavailability. MATERIALS AND METHODS: Potent anti-PD-1/PD-L1 inhibitor candidates from the Shizuoka small compound library were screened and investigated for their antitumor activities in vitro and in vivo using a humanized mouse model. A search for small compounds that inhibit PD-1/PD-L1 binding among 67,395 compounds through three rounds of screening procedures identified six compounds. RESULTS: The two compounds (SCL-1 and SCL-2), which have as a key chemical structure of triazolopyridazin backbone with a piperazine residue on the aromatic ring and 1,3-diphenyl pyrazoline with hydrazinylphthalazine were selected based on in vitro assays and absorption, distribution, metabolism, and excretion (ADME) scoring and subjected to in vivo experiments using a humanized NOG mouse model. SCL-1 and SCL-2 exhibited moderate inhibitory activities against PD-1/PD-L1 binding compared to an anti-PD-1 antibody, with SCL-1 exerting markedly weaker cytotoxic effects on target cells than the other compounds. In in vivo experiments, SCL-1 exerted significant antitumor effects on PD-L1+ SCC-3 tumors, which were dependent on CD8+ T cell infiltration and PD-L1 expression in tumors. A pharmacokinetic study revealed that it has good bioavailability and distribution as an oral reagent. CONCLUSION: SCL-1 is a novel small compound that inhibits PD-1/PD-L1 binding and exerts potent antitumor effects. Thus, it has potential as an oral reagent for cancer immunotherapy.

Characterization of the Immunological Status of Hypermutated Solid Tumors in the Cancer Genome Analysis Project HOPE.

BACKGROUND: Many reports demonstrate that a high tumor mutation burden (TMB-H) is closely associated with good prognosis of cancer. However, specific studies investigating the association of various TMB statuses with overall survival in patients with solid tumors are scarce. PATIENTS AND METHODS: In the present study, we investigated the association of TMB status with overall survival in 5,072 patients with cancer from the HOPE project and clarified the specific mechanism responsible for the good prognosis of the TMB-H group. All tumors were classified into one of four groups based on TMB: ultralow (UL), low (L), intermediate (I) and high (H). RESULTS: The TMB-H group had a better prognosis than the TMB-I and TMB-L groups, but not than the TMB-UL group. Analyzing the expression of 293 immune response-associated genes, 17 genes were up-regulated in the TMB-H group compared to the TMB-I and TNB-L groups, and two genes [CD274 and interferon-γ (IFNG)] were identified as good prognostic factors. Analysis of immune cell populations inside tumors demonstrated that the frequencies of exhausted CD8+ T-cells, activated effector CD8+ T-cells and natural killer cells were significantly higher in the TMB-H group. The T-cell receptor repertoire numbers and the diversity evenness score (DE50) were lower in the TMB-H group than in TMB-UL group; however, no association of the DE50 value with the binding or elution affinity of epitope peptides from neoantigens was found. CONCLUSION: One possible mechanism for the good prognosis of the TMB-UL group compared to the TMB-H group might be that the TMB-UL group features a balance between immunosuppression and immunostimulation.

Development of an Automatic Measurement Method for CD8 and PD-1 Positive T Cells Using Image Analysis Software.

BACKGROUND/AIM: With the progress in cancer immunotherapy using immune checkpoint blockade (ICB) therapy, histological observations of tumor-infiltrating lymphocyte (TIL) status are needed to evaluate the antitumor effect of ICB using imaging analysis software. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded sections obtained from colorectal cancer and gastric cancer patients with more than 500 single nucleotide variants were stained with anti-CD8 and anti-PD-1 antibodies. Based on our own algorithm and imaging analysis software, an automatic TIL measurement method was established and compared to the manual counting methods. RESULTS: In the CD8+ T cell number measurement, there was a good correlation (r=0.738 by Pearson test) between the manual and automated counting methods. However, in the PD-1+ T cell measurement, there was a large difference in TIL numbers in both groups. After adjustment of the parameter settings, the correlation between the manual and automated methods in the PD-1+ T cell measurements improved (r=0.668 by Pearson test). CONCLUSION: An imaging software-based automatic measurement could be a simple and useful tool for evaluating the therapeutic effect of cancer immunotherapies in terms of TIL status.

Identification of tumor microenvironment-associated immunological genes as potent prognostic markers in the cancer genome analysis project HOPE.

Project High-tech Omics-based Patient Evaluation (HOPE), which used whole-exome sequencing and gene expression profiling, was launched in 2014. A total of ~2,000 patients were enrolled until March 2016, and the survival time was observed up to July 2019. In our previous study, a tumor microenvironment immune type classification based on the expression levels of the programmed death-ligand 1 (PD-L1) and CD8B genes was performed based on four types: A, adaptive immune resistance; B, intrinsic induction; C, immunological ignorance; and D, tolerance. Type A (PD-L1+ and CD8B+) exhibited upregulated features of T helper 1 antitumor responses. In the present study, survival time analysis at 5 years revealed that patients in type A had a better prognosis than those in other categories [5 year survival rate (%); A (80.5) vs. B (73.9), C (73.4) and D (72.6), P=0.0005]. Based on the expression data of 293 immune response-associated genes, 62 specific genes were upregulated in the type A group. Among these genes, 18 specific genes, such as activated effector T-cell markers (CD8/CD40LG/GZMB), effector memory T-cell markers (PD-1/CD27/ICOS), chemokine markers (CXCL9/CXCL10) and activated dendritic cell markers (CD80/CD274/SLAMF1), were significantly associated with a good prognosis using overall survival time analysis. Finally, multivariate Cox proportional hazard regression analyses of overall survival demonstrated that four genes (GZMB, HAVCR2, CXCL9 and CD40LG) were independent prognostic markers, and GZMB, CXCL9 and CD40LG may contribute to the survival benefit of patients in the immune type A group.

Alternative Evaluation of an ELISPOT Assay Using Cytokine Activity as a Novel Parameter.

BACKGROUND/AIM: The enzyme-linked immunospot (ELISPOT) assay is a well-established method used to evaluate the strength of T cell-mediated immune activity, and accepted as a standard functional immunological assay. Cytokine activity is a novel parameter reflecting spot size and intensity, which has not been used in ELISPOT assay before. MATERIALS AND METHODS: In the present study, from 113 ELISPOT assay data derived from previous clinical trials with dendritic cell vaccines, both spot number count and cytokine activity data for IFN-γ secretion were obtained using an ELISPOT reader. Comparing the new parameter cytokine activity with the existing parameter spot number, the feasibility of cytokine activity was investigated. RESULTS: There were no significant differences in sensitivity and specificity between spot number and cytokine activity among ELISPOT assay data from CMVpp65 and other antigen peptide-stimulated cytotoxic T lymphocytes. CONCLUSION: Although cytokine activity is a novel parameter unreported so far, it did not show any advantages in the evaluation T cell immune responses compared to the existing spot number parameter.

Alpha-type-1 Polarized Dendritic Cell-based Vaccination in Newly Diagnosed High-grade Glioma: A Phase II Clinical Trial.

BACKGROUND/AIM: Glioblastoma multiforme (GBM) is an intractable tumor that has a very poor prognosis despite intensive treatment with temozolomide plus radiotherapy. PATIENTS AND METHODS: Sixteen newly diagnosed patients with high-grade gliomas were enrolled in a phase II study of the α-type-1 DC vaccine. Briefly, DCs obtained from the culture of enriched monocytes in the presence of a cytokine cocktail, were pulsed with a cocktail of 5 synthetic peptides and cryopreserved until injection into patients. RESULTS: The amount of IL-12 produced by activated DCs was higher than that previously reported. Among 15 evaluable patients, 10 showed positive CTL responses to any peptides in an ELISPOT assay. After 6 years of observation, five patients were still alive, and two of these patients were relapse-free. Moreover, a significant survival-prolonging effect was verified in DC-treated glioma patients. CONCLUSION: Peptide-cocktail-pulsed α-type-1 DC vaccines have a potential therapeutic effect on survival when used in combination with the standard regimen, which is partly based on IL-12-IFN-γ-mediated T-cell activation.