ImmuHub®技术平台发表文献

ImmuHub® Platform Publications

Deep Sequencing of the T-cell Receptors for Monitoring Peripheral CD8+ T Cells in Chinese Advanced Non-small-cell Lung Cancer Patients Treated with anti-PD-L1 antibody

Background: Atezolizumab, a high-affinity engineered human anti-PD-L1 antibody, has produced a clinical benefit for patients with advanced non-small-cell lung cancer (NSCLC). However, associated with T cell regulation, the immuno-modulatory effect of PD-L1 blockade and its biomarker in peripheral immunity remains illustrated. Methods: In a prospective cohort with 12 Chinese advanced NSCLC patients who received atezolizumab 1200 mg every three weeks as a second-line treatment, blood samples were obtained before and six weeks after atezolizumab initiation, and when disease progression confirmed. Patients were classified into a response or progression group according to Response Evaluation Criteria in Solid Tumors (RECIST)1.1. Fresh peripheral blood mononuclear cells (PBMCs) from patients were stained with anti-human CD3, CD8, and PD-1 antibodies for flow cytometry analysis. T cell receptor (TCR) β chains of CD8+ T cells were analyzed by Next-Generation Sequencing (NGS) at the deep level. Diversity, clonality, and similarity of TCR have been calculated before and after treatment in both groups. Results: Clonal expansion with high PD1 expression was detected in all patients’ peripheral CD8+ T cells before the treatment of atezolizumab. Unlike the progression group, the diversity of TCR repertoire and singletons in the TCRβ pool, increased over time with atezolizumab administration, and the TCR repertoire dynamically changes in the response group. The percentage of CD8+ PD1high terminal exhausted T cells declined in the response group after the PD-L1 blockade. Two patterns of TCR changes among patients who receive PD-L1 targeted immunotherapy were observed. Conclusions: Deep sequencing of the T-cell receptors confirmed the existence of CD8+ PD1high T cells with an exhaustion phenotype in Chinese NSCLC patients. Our study demonstrated that efficient anti-PD-L1 therapy could reshape the TCR repertoire for anti-tumor. Furthermore, singleton frequency may help us select patients who are sensitive to anti-PD-L1 immunotherapy.

Profiling T cell receptor β-chain in responders after immunization with recombinant hepatitis B vaccine

Background: T cells with edited T cell receptor β-chain variable (TRBV) involve in the immune response to recombinant hepatitis B surface antigen (rHBsAg) vaccine and the production of hepatitis B surface antibody (HBsAb). The immune repertoire (IR) profile and mechanism of vaccination positive responder (VPR) with rHBsAg is not fully understood. Methods: The IR of 6 VPRs (HBsAb+, HBsAg-) with rHBsAg vaccination was established by high throughput sequencing (HTS) technique and bioinformatics analysis, and comparing to those in 5 vaccination negative responders (VNRs) (HBsAb-, HBsAg-) who were also inoculated with rHBsAg. The repertoire features of BV, BJ, and V (CDR3)J genes selected and immune diversity in peripheral blood mononuclear cells (PBMC) of each subject were analyzed, respectively. Results: There is no significant difference in sequencing amplification indexes of each sample. However, there are TRBV15, BJ2-3 with significantly high expression levels in VPR compared with those in VNR group (both P <0.05). Further results show that BV15/BJ2-5 level was significantly increased from VPR than that of VNR group. Interestingly, the motif of CDR3 in TRBV15/BJ2-5 is mostly expressed as "GGETQ" or "GETQ". Additionally, there is no remarkable difference between the two groups of distribution of different clone expressed levels of V (CDR3) J. Conclusions: The features of IR in the VPR and VNR will contribute to explore the mechanism of positive response to rHBsAg, and contribute to development of optimized hepatitis B vaccine, and also provide partial interpretation of the VNR who is relatively few infected with HBV. Keywords: Complementarity determining region 3; Hepatitis B vaccine; High throughput sequencing; TR repertoire; Vaccination positive responder.

Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq

B cell response plays a critical role against SARS-CoV-2 infection. However, little is known about the diversity and frequency of the paired SARS-CoV-2 antigen-specific BCR repertoire after SARS-CoV-2 infection. Here, we performed single-cell RNA sequencing and VDJ sequencing using the memory and plasma B cells isolated from five convalescent COVID-19 patients, and analyzed the spectrum and transcriptional heterogeneity of antibody immune responses. Via linking BCR to antigen specificity through sequencing (LIBRA-seq), we identified a distinct activated memory B cell subgroup (CD11chigh CD95high) had a higher proportion of SARS-CoV-2 antigen-labeled cells compared with memory B cells. Our results revealed the diversity of paired BCR repertoire and the non-stochastic pairing of SARS-CoV-2 antigen-specific immunoglobulin heavy and light chains after SARS-CoV-2 infection. The public antibody clonotypes were shared by distinct convalescent individuals. Moreover, several antibodies isolated by LIBRA-seq showed high binding affinity against SARS-CoV-2 receptor-binding domain (RBD) or nucleoprotein (NP) via ELISA assay. Two RBD-reactive antibodies C14646P3S and C2767P3S isolated by LIBRA-seq exhibited high neutralizing activities against both pseudotyped and authentic SARS-CoV-2 viruses in vitro. Our study provides fundamental insights into B cell response following SARS-CoV-2 infection at the single-cell level.

Natural evolution mechanisms of the IDH mutant glioma revealed by multi-omics sequencing of a long-term survivor

Results: The center and edge of the tumor were diagnosed as LGG and GBM, respectively. They shared the same trunk mutations including IDH, TP53, and ATRX. They both have mixing cell origins and they have a highly correlated methylation level at the probe level. CIC, BRCA2, and RPA4 mutation which occurred only in G4 with mutant allele frequency(MAF) higher than 15% may contribute to evolution. NAF1 of which the MAF increased by 70% and the mutant RNA reads nearly doubled in G4 may also involve in the evolution. In the pathway level, the MSP-RON pathway was strongly up-regulated in G4. Concomitant with the tumor evolution, we discovered enhanced inflammatory signals represented by the up-regulation of the NF-κB pathway and the recruitment of mast cell, of which the absolute cell proportion increased from 3.9% to 6.9%. Contradictorily, the adaptive immune response was suppressed as we found pathways associated with IL17, dendritic cells, and cytotoxic T cells were down-regulated and the infiltration level of CD4+ and CD8+ T cells were both decreased. As for the TCR profile, only about 2% of blood clonotypes were detected in the tumor microenvironment. Nevertheless, two clonotypes were found significantly expanded. Clone frequencies of them were 38% and 19% in G2, respectively. And these were 24% and 11% in G4, respectively. Conclusion: Mutation of CIC, BRCA2, RPA4, and NAF1 and activation of the MSP-RON pathway could promote glioma evolution under natural conditions. Increased inflammatory response and decreased adaptive immune response may also contribute to the process. Besides, two highly expanded TCR clonotypes discovered in this case may serve as a potential adoptive cell therapy source in glioma.

T cells expanded from PD-1+ peripheral blood lymphocytes share more clones with paired tumor-infiltrating lymphocytes

Both tumor-infiltrating lymphocytes (TIL) and PD-1+ peripheral blood lymphocytes (PBL) are enriched for tumor-reactive clones recognizing known and unknown tumor antigens. However, the relationship between the T-cell receptor (TCR)-β repertoires of the TIL and T cells expanded from paired PD-1+ PBL and whether T cells expanded from PD-1+ PBL can be used to treat patients with cancer as TIL substitutes remains unclear. Here we established a highly efficient protocol to prepare polyclonal T cells from PD-1+ PBL. A functional T-cell assay and tetramer staining revealed that cells from PD-1+ PBL were relatively enriched for tumor-reactive T cells. Furthermore, deep TCR-β sequencing data revealed that an average of 11.29% (1.32%-29.06%, p=0.015, n=8) tumor-resident clonotypes were found in T cells expanded from paired PD-1+ PBL, and the mean accumulated frequency of TIL clones found in T cells expanded from PD-1+ PBL was 35.11% (7.23%-78.02%, p=0.017, n=8). Moreover, treatment of four patients, who failed multi-line therapy and developed acquired resistance to anti-PD-1, with autologous T cells expanded from PD-1+ PBL combined with anti-PD-1 antibody elicited objective responses from three of them. These results indicate that T cells expanded from PD-1+ PBL share more clones with paired TIL and could be used to treat patients with cancer as TIL substitutes.

Restricted TcR β chain CDR3 clonotype is associated with resolved acute hepatitis B subjects

Background: T cells play an important role in the prognosis of hepatitis B virus (HBV) infection, and are involved in the seroconversion of a patient from HBsAb negative to positive. To compare the T-cell receptor β-chain variable region (TcRBV) complementarity-determining region 3 (CDR3) in subjects with or without hepatitis B surface antigen (HBsAg) convert to hepatitis B surface antibody (HBsAb), the TcRBV was determined using high throughput sequencing (HTS). Methods: The clonotype and diversity of CDR3 in peripheral blood mononuclear cells of subjects with resolved acute hepatitis B (AHB, HBsAb+, HBsAg-) (n = 5), chronic hepatitis B (CHB, HBsAb-, HBsAg+) (n = 5), and healthy controls (HC, HBsAb-, HBsAg-) (n = 3) were determined and analyzed using HTS (MiSeq). Results: The overlapping rate of CDR3 clones of any two samples in AHB group was 2.00% (1.74% ~ 2.30%), CHB group was 1.77% (1.43% ~ 2.61%), and HC group was 1.82% (1.62% ~ 2.12%), and there was no significant difference among the three groups by Kruskal-Wallis H test. However, among the top 10 cumulative frequencies of clonotypes, only the frequency of clonotype (TcRBV20–1/BD1/BJ1–2) in AHB group was lower than that of HC group (P < 0.001). Moreover, exclude the 10 top clonotypes, there are 57 markedly different frequency of clones between AHB and CHB groups (18 clones up, 39 clones down), 179 (180–1) different clones between AHB and HC groups, and 134 different clones between CHB and HC groups. With regard to BV and BJ genotypes, there was no significant different frequency among the groups. Furthermore, there was no significant difference in the diversity of TcRBV CDR3 among the three groups (P > 0.05). Conclusions: Thus, there are 57 TcRBV clonotypes that may be related to HBsAg seroconversion of AHB subjects, but the diversity of TcRBV CDR3 is not significantly related to the HBsAb positive status.

Efficacy and safety of sintilimab in combination with chemotherapy in previously untreated advanced or metastatic nonsquamous or squamous NSCLC: two cohorts of an open-label, phase 1b study

Combining chemotherapy with immunotherapy improves the therapeutic outcome for first-line (1L) patients with advance nonsmall-cell lung cancer (NSCLC). Two cohorts of a phase 1b study (NCT02937116) aimed to evaluate the safety and efficacy of sintilimab, a PD-1 inhibitor, plus chemotherapy in 1L patients with nonsquamous and squamous NSCLC (nsqNSCLC/sqNSCLC); and to identify potential biomarkers for treatment response. Treatment-naïve patients with nsqNSCLC were enrolled and intravenously given sintilimab (200 mg), pemetrexed (500 mg/m2), and cisplatin (75 mg/m2), every 3 weeks (Q3W) for 4 cycles in cohort D. Treatment-naïve patients with sqNSCLC were enrolled and intravenously given sintilimab (200 mg), gemcitabine (1250 mg/m2), and cisplatin (75 mg/m2), Q3W, for 6 cycles in cohort E. The primary objective was to evaluate the safety and efficacy of the treatment. The additional objective was to explore biomarkers for the treatment efficacy. Twenty-one patients with nsqNSCLC, and 20 patients with sqNSCLC were enrolled in cohort D and cohort E, respectively. By the data cutoff (April 17, 2019), 8 (38.1%) patients in cohort D and 17 (85.0%) patients in cohort E experienced grade 3–4 adverse events. The median follow-up duration was 16.4 months (14.8–23.0) in cohort D and 15.9 months (11.7–17.7) in cohort E. The objective response rate was 68.4% (95% CI 43.4%, 87.4%) in cohort D and 64.7% (95% CI 38.3%, 85.8%) in cohort E. Neither PD-L1 expression nor tumor mutation burden value was significantly associated with an improved treatment response. Sintilimab plus chemotherapy exhibited manageable toxicity and an encouraging antitumor activity in patients with nsqNSCLC and sqNSCLC.

Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody

Sequence-defined recombinant antibodies (rAbs) have emerged as alternatives to hybridoma-secreted monoclonal antibodies (mAbs) for performing immunoassays. However, the polyploidy nature of hybridomas often leads to the coexistence of aberrant or non-specific functional variable region (VR) gene transcripts, which complicates the identification of correct VR sequences. Herein, we introduced the use of LC-MS/MS combined with next-generation sequencing to characterize VR sequences in an anti-thiacloprid mAb, which was produced by a hybridoma with genetic antibody diversity. The certainty of VR sequences was verified by the functional analysis based on the recombinant antibody (rAb) expressed by HEK293 mammalian cells. The performance of the rAb was similar to that of the parental mAb, with IC50 values of 0.73 and 0.46 μg/L as measured by ELISAs. Moreover, molecular docking analysis revealed that Ser52 (H-CDR2), Trp98, and Trp93 (L-CDR3) residues in the complementarity determining regions (CDRs) of the identified VR sequences predominantly contributed to thiacloprid-specific recognition through hydrogen bonds and the CH–π interaction. Through single-site-directed alanine mutagenesis, we found that Trp98 and Trp93 (L-CDR3) showed high affinity to thiacloprid, while Ser52 (H-CDR2) had an auxiliary effect on the specific binding. This study presents an efficient and reliable way to determine the key recognition sites of hapten-specific mAbs, facilitating the improvement of antibody properties.

A pan-cancer clinical study of personalized neoantigen vaccine monotherapy in treating patients with various types of advanced solid tumors

Purpose: Due to their high tumor specificity and immunogenicity, neoantigens have been considered as ultimate targets for cancer immunotherapy. Neoantigen-based vaccines have demonstrated promising efficacy for several cancer types. To further investigate the anti-tumor potentials for other types of solid tumors, we designed a peptide-based neoantigen vaccine, iNeo-Vac-P01, and conducted a single-arm, open-labeled, investigator-initiated clinical trial (NCT03662815). Experimental Design: Personalized neoantigen vaccines were designed and manufactured according to our bioinformatics analysis results from the whole-exome sequencing (WES) of tumor and peripheral blood cell DNAs. Patients were scheduled to be vaccinated subcutaneously (s.c.) with adjuvant on days 1, 4, 8, 15, 22 (prime phase), and days 78, 162 (boost phase). Additional immunizations were administrated every 2~3 months as per patient's potential benefit. The safety and efficacy were assessed through adverse events, progression-free survival (PFS), overall survival (OS) and other parameters. Results: Of the 22 patients enrolled with advanced malignancies, twenty had no or mild adverse events, while two had grade 3 or 4 acute allergic reactions only after their 6th boost vaccination. The disease control rate (DCR) was 71.4%. The median PFS was 4.6 months, whereas the median OS was not reached (12-month OS=55.1%). Around 80% of individual peptides or peptide pools elicited measurable specific immune response. Additionally, our findings revealed several potential biomarkers for the prediction of better response. Conclusions: iNeo-Vac-P01 as monotherapy is feasible and safe for patients with advanced solid tumors. It could elicit T cell-mediated immune response targeting tumor neoantigens, and might have promising antitumor efficacy.

Restricted TcR β chain CDR3 Clonotype Is Associated with Resolved Acute Hepatitis B (AHB) patients with HBsAb

Background: T cells play an important role in the prognosis of HBV infection, and are involved in the seroconversion of a patient from HBsAb negative to HBsAb positive. To compare the T-cell receptor beta chain variable region (TcRBV) complementarity-determining region 3 (CDR3) in subjects with or without HBsAg seroconversion to HBsAb, the TcRBV was determined using high throughput sequencing (HTS). Methods: Clonotype and diversity of CDR3 in peripheral blood mononuclear cells (PBMCs) of subjects with resolved acute hepatitis B (AHB, HBsAb+, and HBsAg-) (n = 5), chronic hepatitis B (CHB, HBsAb-, and HBsAg+) (n = 5), and healthy controls (HC, HBsAb-, and HBsAg-) (n = 3) were determined and analyzed using HTS. Findings: The overlapping rate of CDR3 clones for any two samples in the AHB, CHB, and HC groups were 2.00% (1.74–2.30%), 1.77% (1.43–2.61%), and 1.82% (1.62%–2.12%), respectively; no significant difference was found among the three groups by KruskalWallis H test. In addition, among the top 10 cumulative frequencies of clonotypes, only the frequency of clonotype (TcRBV20-1/TcRBD1/TcRBJ1-2) in the AHB group was lower than that in the HC group (P < 0.001). Additionally, excluding the 10 top clonotypes, there were 57 markedly different frequencies of clones between the AHB and CHB groups. Furthermore, there was no significant difference in the diversity of TcRBV CDR3 among the three groups (P<0.05). InterpretationL Thus, there are 57 TcRBV clonotypes that may be related to HBsAg seroconversion in AHB subjects, but the diversity of TcRBV CDR3 is not significantly related to the HBsAb positive status.

Quantitative Analysis of Thymus-Independent Donor-Derived T Cell Expansion in Transplant Patients

Although thymus-independent donor-derived T cell expansion may determine the occurrence of graft-versus-host disease (GVHD) and relapse after transplantation, the characteristics and dynamics of the expansion process remain unclear. To address this issue, we monitored T cell receptor β repertoire at day 0, day 28, and day 61 after transplantation in 30 patients with hematologic malignancies by next-generation sequencing. The clonality index showed an increasing clonality over time (P = .001). The top 200 clonotypes accounted for more than half of the total clonotypes (median frequency, 63.55%) at day 61, and there was a remarkable overlapping between the top 200 clonotypes of each repertoire and its former repertoire (>50%). A normalized index, called the T Cell Response Index (TCRI), was designed on the basis of rank-shift analysis to quantify antigen-driven expansion. The TCRI during the first month was not related to relapse or GVHD (P> .05), whereas the TCRI during the second month was related to relapse (P = .006). Recipients with a TCRI below 1.0 during the second month had a higher cumulative relapse rate (31.25% versus 0%, P = .0323) and had a lower 1-year survival rate (56.25% versus 78.57%, P = .281). The clonotypes with strong competitiveness in the second month in the nonrelapse group preferentially used TRBV2, TRBV12-3, TRBJ1-1 and TRBJ1-5 segments (P< .01). In conclusion, homeostatic expansion predominates in the first month due to nonspecific T cell proliferation, whereas antigen-driven expansion predominates in the second month and results in a graft-versus-tumor (GvT) effect. Moreover, TCRI could serve as a quantitative indicator of GvT against relapse within the first year. The difference in V and J segment usage reveals that T cells responsible for potent GvT effect are similar among patients.

Engineering Magnetosomes for High-Performance Cancer Vaccination

A novel cancer vaccine is developed by using Fe3O4 magnetic nanoclusters (MNCs) as the core and cancer cell membranes decorated with anti-CD205 as the cloak. Because of the superparamagnetism and magnetization of MNCs, it is first achieved for the magnetic retention of vaccine in the lymph nodes with a magnetic resonance imaging (MRI) guide, which opened the time window for antigen uptake by dendritic cells (DCs). Meanwhile, the camouflaged cancer cell membranes serve as a reservoir of various antigens, enabling subsequent multiantigenic response. Additionally, the decorated anti-CD205 direct more vaccine into CD8+ DCs, facilitating the major histocompatibility complex (MHC) I cross-presentation. These unique advantages together lead to a great proliferation of T cells with superior clonal diversity and cytotoxic activity. As a result, potent prophylactic and therapeutic effects with few abnormalities are observed on five different tumor models. Therefore, such a cancer-derived magnetosome with the integration of various recent nanotechnologies successfully demonstrates its promise for safe and high-performance cancer vaccination.

Quantitative characterization of T-cell repertoire alteration in Chinese patients with B-cell acute lymphocyte leukemia after CAR-T therapy

Chimeric antigen receptor (CAR) T-cell therapy has displayed potent anti-leukemia activity in acute lymphocytic leukemia (ALL), acting as a new ray of hope to refractory/relapsed patients. However, the influence of CAR-T therapy on host immune system has not been well elucidated. Thus, We applied high-throughput T cell receptor β chain sequencing to track the dynamic change of T-cell repertoire induced by CAR-T therapy in B-cell ALL patients. Six Chinese patients achieving complete remission were under observation, whose blood samples, bone marrow samples and infused CAR-T samples were collected at serial time points before and after CAR-T therapy. We observed decreased TCR diversity and increased clonality of T-cell repertoire in both peripheral blood and bone marrow after CAR-T administration. The persistent T cell clones in blood and bone marrow expanded following leukemic cell destruction and were barely detected in CAR T-cell pool. For the first time, our results demonstrated CAR-T therapy could stimulate the clonal proliferation of CAR-negative T cells in patients. Considering other groups’ animal results indicating that CAR-T therapy could facilitate the proliferation of tumor antigen-specific T cells and that the emergence of these T cell clones followed the destruction of leukemic cells, they are most likely tumor antigen-specific.

Deep sequencing of the t-cell receptors for monitoring peripheral CD8+T cells in advanced NSCLC Chinese patients treated with anti-PD-L1 antibody

Background: Atezolizumab, a high-affinity engineered human anti-PD-L1 monoclonal immunoglobulin-G1 antibody, has dramatically produced clinical benefit for patients with advanced non-small cell lung cancer (NSCLC). However, the biomarker and immunomodulatory effects of PD-L1 blockade in peripheral immunity remains to be illustrated. Methods: In a prospective cohort with twelve Chinese advanced NSCLC patients who received intravenous atezolizumab 1200 mg once every 3 weeks as second-line treatment. Blood samples were obtained prior to and 6 weeks after atezolizumab initiation, and the time when disease progression confirmed. Patients were classified into response or progression group according to their clinical efficacy to atezolizumab. TCRβ chains of CD8+ T cells were analyzed by NGS using TCR profiling system at the deep level (ImmuQuad Biotech, Hangzhou China). Briefly, a 5' RACE unbiased amplification protocol was applied. Sequencing was performed on an Illumina MiSeq system. Meanwhile, fresh PBMCs from patients were stained with anti-human CD3, CD8, and PD1 antibodies for flow cytometry analysis. Results: Clonal expansion with high PD1 expression was detected in all patient peripheral CD8+ T cells before atezolizumab therapy. Unlike progression group, diversity of TCR repertoire and singletons in TCRβ pool, apparently increased overtime with atezolizumab administration and the TCR repertoire showed dynamic changes by calculated with Baroni-Urbani & Buser similarity index in response group. Meanwhile, the frequency of CD8+PD1high terminal exhausted T cells declined in response group after treatment. Conclusions: Results from deep sequencing of the T-cell Receptors highly indicated the existing of tumor antigen-specific T cells with exhaustion phenotype caused by persistent tumor antigen stimulation in Chinese advanced NSCLC patients. Our study demonstrated that an efficient anti-PD-L1 therapy could reshape T-cell repertoire for targeting and adapting to tumor heterogeneity, and reinvigorate the progenitor exhausted T cell population which includes tumor antigen-specific T cells for fighting tumor cells.

Chimeric Antigen Receptor T-Cell Therapy Shapes T Cell Repertoire in Chinese Patients with B-Cell Acute Lymphocyte Leukemia

Introduction: Chimeric antigen receptor (CAR) T-cell therapy has displayed potent anti-leukemia activity in refractory/relapsed acute lymphocytic leukemia (ALL). However, the influence of CAR-T therapy on host systemic and local immunity has not been well examined. We therefore applied high-throughput T cell receptor β chain sequencing to track dynamic change of T-cell repertoire in vivo induced by CAR-T therapy in B-cell acute lymphocytic leukemia patients. Patients and Methods: Six patients with 45 samples were under observation. The samples obtained to be tested were the peripheral blood mononuclear cell (PBMC) samples and bone marrow mononuclear cell (BMMC) samples before and after CAR-T administration, as well as the CAR-transduced autologous T cell samples on the day when they were to be infused to patients. The information of samples and patients was summarized in Table 1. The TCR full length mRNAs of these samples were deeply sequenced using the ImmunHub® TCR profiling system (ImmunQuad Biotech). Briefly, a 5'RACE unbiased amplification protocol was used. An algorithm was applied to raw sequencing data for PCR and sequencing errors correction and V, D, J, C gene segments mapping with IMGT®. The inverse Simpson index and the clonality index was calculated to estimate TCRβ clonotype diversity and the state of clonal proliferation of T cells. The donut chart and clone tracking heat map were generated by R.