Interferon-γ Enzyme-linked Immunospot Assay Research Articles

Irreversible electroporation of localised prostate cancerdownregulates immune suppression andinduces systemic anti-tumour T-cell activation - IRE-IMMUNO study.

To prospectively compare systemic anti-tumour immune responses induced by irreversible electroporation (IRE) and robot-assisted radical prostatectomy (RARP) in patients with localised intermediate-risk prostate cancer (PCa). Between February 2021 and June 2022, before and after treatment (at 5, 14 and 30 days) peripheral blood samples of 30 patients with localised PCa were prospectively collected. Patient inclusion criteria were: International Society of Urological Pathologists Grade 2-3, clinical cancer stage ≤T2c, prostate-specific antigen level <20 ng/mL). Patients were treated with IRE (n = 20) or RARP (n = 10). Frequency and activation status of lymphocytic and myeloid immune cell subsets were determined using flow cytometry. PCa-specific T-cell responses to prostatic acid phosphatase(PSAP) and cancer testis antigen (New York oesophageal squamous cell carcinoma 1 [NY-ESO-1]) were determined by interferon-γ enzyme-linked immunospot assay (ELISpot). Repeated-measures analysis of variance and two-sided Student's t-tests were used to compare immune responses over time and between treatment cohorts. Patient and tumour characteristics were similar between the cohorts except for age (median 68 years [IRE] and 62 years [RARP], P = 0.01). IRE induced depletion of systemic regulatory Tcells (P = 0.0001) and a simultaneous increase in activated cytotoxic T-lymphocyte antigen 4 (CTLA-4)+ cluster of differentiation (CD)4+ (P < 0.001) and CD8+ (P = 0.032) Tcells, consistent with reduction of systemic immune suppression allowing for effector T-cell activation, peaking 14 days after IRE. Effects were positively correlated with tumour volume/ablation size. Accordingly, IRE induced expansion of PSAP and/or NY-ESO-1 specific T-cell responses in four of the eight immune competent patients. Temporarily increased activated myeloid derived suppressor cell frequencies (P = 0.047) were consistent with transient immunosuppression after RARP. Irreversible electroporation induces a PCa-specific systemic immune response in patients with localised PCa, aiding conversion of the tumour microenvironment into a more immune permissive state. Therapeutic efficacy might be further enhanced by combination with CTLA-4 checkpoint inhibition, potentially opening up a new synergistic treatment paradigm for high-risk localised or (oligo)metastatic disease.

Development and evaluation of a Mycobacterium bovis interferon-γ enzyme-linked immunospot (ELISpot) assay for detection of bovine tuberculosis

Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important zoonotic disease. This infection is difficult to control because of the limited ability of the tuberculin skin test (TST) and ancillary IFN-γ release assay to detect all infected animals. In this study, we aimed to develop an efficient assay based on the enzyme-linked immunospot (ELISpot) technique for the diagnosis of bTB, with IFN-γ monoclonal antibodies 3E9 and Bio-labeled 6F8 used as capture and detection antibodies, respectively. As expected, there were significantly more M. bovis-specific spot-forming units (SFU) in bTB-infected cattle than in healthy cattle when an M. bovis-specific antigen, CFP-10-ESAT-6 fusion protein (CE protein), was used. The M. bovis IFN-γ ELISpot assay demonstrated a high level of agreement (90.83%) with the BOVIGAM ELISA test (Thermo Fisher Scientific) for detecting bTB. Furthermore, 3 of 109 cattle tested negative by both the TST and the BOVIGAM ELISA tests, but positive by the ELISpot assay (TST- ELISA- ELISpot+). During subsequent long-term monitoring, these 3 cattle became TST+ ELISA+ ELISpot+. These results suggest that the M. bovis IFN-γ ELISpot assay we established could detect infected cattle earlier than the BOVIGAM ELISA test.

Immunogenicity and Reactogenicity of Ad26.COV2.S in Korean Adults: A Prospective Cohort Study.

BackgroundAs the coronavirus disease 2019 (COVID-19) pandemic continues, there are concerns regarding waning immunity and the emergence of viral variants. The immunogenicity of Ad26.COV2.S against wild-type (WT) and variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) needs to be evaluated.MethodThis prospective cohort study was conducted between June 2021 and January 2022 at two university hospitals in South Korea. Healthy adults who were scheduled to be vaccinated with Ad26.COV2.S were enrolled in this study. The main outcomes included anti-spike (S) IgG antibody and neutralizing antibody responses, S-specific T-cell responses (interferon-γ enzyme-linked immunospot assay), solicited adverse events (AEs), and serious AEs.ResultsFifty participants aged ≥ 19 years were included in the study. Geometric mean titers (GMTs) of anti-S IgG were 0.4 U/mL at baseline, 5.2 ± 3.0 U/mL at 3–4 weeks, 55.7 ± 2.4 U/mL at 5–8 weeks, and 81.3 ± 2.5 U/mL at 10–12 weeks after vaccination. GMTs of 50% neutralizing dilution (ND50) against WT SARS-CoV-2 were 164.6 ± 4.6 at 3-4 weeks, 313.9 ± 3.6 at 5–8 weeks, and 124.4 ± 2.6 at 10–12 weeks after vaccination. As for the S-specific T-cell responses, the median number of spot-forming units/106 peripheral blood mononuclear cell was 25.0 (5.0–29.2) at baseline, 60.0 (23.3–178.3) at 5-8 weeks, and 35.0 (13.3–71.7) at 10–12 weeks after vaccination. Compared to WT SARS-CoV-2, ND50 against Delta and Omicron variants was attenuated by 3.6-fold and 8.2-fold, respectively. The most frequent AE was injection site pain (82%), followed by myalgia (80%), fatigue (70%), and fever (50%). Most AEs were grade 1–2, and resolved within two days.ConclusionSingle-dose Ad26.COV2.S was safe and immunogenic. NAb titer and S-specific T-cell immunity peak at 5–8 weeks and rather decrease at 10–12 weeks after vaccination. Cross-reactive neutralizing activity against the Omicron variant was negligible.

T-cell and antibody responses to first BNT162b2 vaccine dose in previously infected and SARS-CoV-2-naive UK health-care workers: a multicentre prospective cohort study

SummaryBackgroundPrevious infection with SARS-CoV-2 affects the immune response to the first dose of the SARS-CoV-2 vaccine. We aimed to compare SARS-CoV-2-specific T-cell and antibody responses in health-care workers with and without previous SARS-CoV-2 infection following a single dose of the BNT162b2 (tozinameran; Pfizer–BioNTech) mRNA vaccine.MethodsWe sampled health-care workers enrolled in the PITCH study across four hospital sites in the UK (Oxford, Liverpool, Newcastle, and Sheffield). All health-care workers aged 18 years or older consenting to participate in this prospective cohort study were included, with no exclusion criteria applied. Blood samples were collected where possible before vaccination and 28 (±7) days following one or two doses (given 3–4 weeks apart) of the BNT162b2 vaccine. Previous infection was determined by a documented SARS-CoV-2-positive RT-PCR result or the presence of positive anti-SARS-CoV-2 nucleocapsid antibodies. We measured spike-specific IgG antibodies and quantified T-cell responses by interferon-γ enzyme-linked immunospot assay in all participants where samples were available at the time of analysis, comparing SARS-CoV-2-naive individuals to those with previous infection.FindingsBetween Dec 9, 2020, and Feb 9, 2021, 119 SARS-CoV-2-naive and 145 previously infected health-care workers received one dose, and 25 SARS-CoV-2-naive health-care workers received two doses, of the BNT162b2 vaccine. In previously infected health-care workers, the median time from previous infection to vaccination was 268 days (IQR 232–285). At 28 days (IQR 27–33) after a single dose, the spike-specific T-cell response measured in fresh peripheral blood mononuclear cells (PBMCs) was higher in previously infected (n=76) than in infection-naive (n=45) health-care workers (median 284 [IQR 150–461] vs 55 [IQR 24–132] spot-forming units [SFUs] per 106 PBMCs; p<0·0001). With cryopreserved PBMCs, the T-cell response in previously infected individuals (n=52) after one vaccine dose was equivalent to that of infection-naive individuals (n=19) after receiving two vaccine doses (median 152 [IQR 119–275] vs 162 [104–258] SFUs/106 PBMCs; p=1·00). Anti-spike IgG antibody responses following a single dose in 142 previously infected health-care workers (median 270 373 [IQR 203 461–535 188] antibody units [AU] per mL) were higher than in 111 infection-naive health-care workers following one dose (35 001 [17 099–55 341] AU/mL; p<0·0001) and higher than in 25 infection-naive individuals given two doses (180 904 [108 221–242 467] AU/mL; p<0·0001).InterpretationA single dose of the BNT162b2 vaccine is likely to provide greater protection against SARS-CoV-2 infection in individuals with previous SARS-CoV-2 infection, than in SARS-CoV-2-naive individuals, including against variants of concern. Future studies should determine the additional benefit of a second dose on the magnitude and durability of immune responses in individuals vaccinated following infection, alongside evaluation of the impact of extending the interval between vaccine doses.FundingUK Department of Health and Social Care, and UK Coronavirus Immunology Consortium.

Protective heterologous T cell immunity in COVID-19 induced by the trivalent MMR and Tdap vaccine antigens

BackgroundT cells control viral infection, promote vaccine durability, and in coronavirus disease 2019 (COVID-19) associate with mild disease. We investigated whether prior measles-mumps-rubella (MMR) or tetanus-diphtheria-pertussis (Tdap) vaccination elicits cross-reactive T cells that mitigate COVID-19.MethodsAntigen-presenting cells (APC) loaded ex vivo with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), MMR, or Tdap antigens and autologous T cells from COVID-19-convalescent participants, uninfected individuals, and COVID-19 mRNA-vaccinated donors were co-cultured. T cell activation and phenotype were detected by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assays and flow cytometry. ELISAs (enzyme-linked immunosorbant assays) and validation studies identified the APC-derived cytokine(s) driving T cell activation. TCR clonotyping and single-cell RNA sequencing (scRNA-seq) identified cross-reactive T cells and their transcriptional profile. A propensity-weighted analysis of COVID-19 patients estimated the effects of MMR and Tdap vaccination on COVID-19 outcomes.FindingsHigh correlation was observed between T cell responses to SARS-CoV-2 (spike-S1 and nucleocapsid) and MMR and Tdap proteins in COVID-19-convalescent and -vaccinated individuals. The overlapping T cell population contained an effector memory T cell subset (effector memory re-expressing CD45RA on T cells [TEMRA]) implicated in protective, anti-viral immunity, and their detection required APC-derived IL-15, known to sensitize T cells to activation. Cross-reactive TCR repertoires detected in antigen-experienced T cells recognizing SARS-CoV-2, MMR, and Tdap epitopes had TEMRA features. Indices of disease severity were reduced in MMR- or Tdap-vaccinated individuals by 32%–38% and 20%–23%, respectively, among COVID-19 patients.ConclusionsTdap and MMR memory T cells reactivated by SARS-CoV-2 may provide protection against severe COVID-19.FundingThis study was supported by a National Institutes of Health (R01HL065095, R01AI152522, R01NS097719) donation from Barbara and Amos Hostetter and the Chleck Foundation.

Spk-9001: Adeno-Associated Virus Mediated Gene Transfer for Hemophilia B Achieves Sustained Factor IX with Minimal Immune Response

▪Background: Data from earlier hemophilia B (HB) AAV-mediated liver gene transfer trials demonstrated a dose-dependent, capsid-specific immune response that may result in clearance of transduced hepatocytes and loss of transgene expression (Manno et al. 2006, Mingozzi et al. 2007). This has not posed major safety concerns, but may limit efficacy. Prior work incorporated the use of steroids to abort this immune response and maintain factor IX (FIX) expression (Nathwani et al 2014). The percent of transgene expression lost increased with number of elapsed days from transaminase elevation to steroid initiation. Once vector responsiveness to steroids is established, the need for steroids is not a limitation in and of itself but highlights the requirement to reliably and expeditiously recognize an immune response and initiate steroids. Here we present our immunomonitoring data following infusion of SPK-9001, an AAV vector designed to achieve therapeutic FIX:C at a low vector dose to minimize capsid-specific T cell responses.Method: Ten HB subjects were infused with 5 x 1011 vg/kg SPK-9001, containing a transgene cassette encoding FIX-Padua that confers ~8-12 - fold higher specific activity than wild-type factor IX (Simioni et al. 2009, Crudele et al. 2016). T cell responses to the SPK-9001 AAV capsid and transgene product were monitored post-infusion using a validated interferon-γ enzyme-linked immunospot (ELISpot) assay, with a threshold for positivity of > 50 spot forming units (SFU) per 106 PBMC.Results: As of 7/26/17, 10 subjects are 26 -78 weeks post SPK-9001 infusion with mean sustained FIX:C of 33.8±18.1% (mean±SD). There were no vector or procedure-related adverse events. All subjects discontinued prophylaxis and had a significant reduction in the annualized bleeding rate (p<0.0001). No patients exhibited a significant T cell response to the transgene product, including the Padua mutation site. T cell responses against the viral capsid generally fell into 3 categories: 5 subjects had no detectable response to capsid; 3 subjects had a transient T cell response that peaked at week 2 that quickly resolved without intervention; 2 subjects demonstrated a T cell response that persisted for longer than 3 weeks. The persistent T cell responses observed in the 2 subjects correlated with an asymptomatic increase of liver transaminases. One subject was administered a course of prednisone starting on day 56, after which FIX:C levels stabilized and T cell reactivity quickly returned to baseline levels. An orthogonal peptide matrix approach identified 1 candidate AAV epitope, SEYQLPYVL, which is a MHC Class I predicted binder for the subject's HLA-B*18 haplotype according to bioinformatics prediction algorithms. This epitope is highly conserved across AAV serotypes. In response to grade I transaminase toxicity, the second subject was administered steroids starting on day 36. Concurrent ELISPOT values were elevated (1260 SFU/106 PBMC), but not associated with a decline in FIX:C and the subject maintained FIX:C in the normal range. Like the subject's LFTs, the ELISPOT response markedly decreased with steroids while his FIX:C remained stable at ~80%. This subject continues to demonstrate a low-positivity to AAV capsid peptides through 32 weeks and off steroids, but the persistent T cell response has not been associated with a decline in FIX:C or impact on overall efficacy. Further ELISPOT analysis on this subject identified 4 AAV T cell epitopes that are predicted binders for 2 of this subject's alleles, HLA-A*03 and HLA-B*07.Conclusion:We report consistent levels of sustained plasma FIX:C of 33.8±18.1% (mean±SD) in 10 subjects to date. Observed T cell responses to the AAV capsid occurred in 2/10 subjects that required steroid intervention. The early administration of immunosuppression and subsequent decrease in T cell response in the absence of a corresponding loss in FIX:C suggests that prompt recognition of a capsid immune response and early steroid intervention is critical. Furthermore, persistence of detectable T cell responses late in the time-course, with no association with a diminution in FIX:C, may suggest that circulating T cells may be present long after transduced hepatocytes cease presenting capsid peptides. Preliminary data suggest that our validated method for assessing T cell responses can detect responses that require management with steroids to ensure continuous FIX expression. DisclosuresHui:Spark Therapeutics: Employment, Equity Ownership. Liu:Spark Therapeutics: Employment. Patel:Spark Therapeutics: Employment. Chen:Spark Therapeutics: Employment. George:Pfizer: Consultancy; Spark Therapeutics: Other: Principal Investigator of Ongoing Phase I/II Gene Therapy Trials for Hemophilia A and B. Rasko:Genea: Equity Ownership; Novartis: Other: Clinical trials, Speakers Bureau; Spark: Equity Ownership, Other: clinical trials, Speakers Bureau; Rarecyte: Consultancy, Equity Ownership; President-Elect International Society for Cellular Therapy: Membership on an entity's Board of Directors or advisory committees; IMAGO Biosciences: Consultancy, Equity Ownership. Ducore:Octapharma: Research Funding; Bayer, Shire, HemaBiologics, Bioverativ, Octapharma, Spark Therapeutics: Other: Advisory board. Dasen:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Anguela:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties.

SARS-CoV-2-Specific Antibody and T Cell Response Kinetics According to Symptom Severity.

ABSTRACT.Data on the longevity of humoral and cell-mediated immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with coronavirus disease 2019 (COVID-19) are limited. We evaluated the detailed kinetics of antibody and T-cell responses at the acute, convalescent, and post-convalescent phases in COVID-19 patients with a wide range of severity. We enrolled patients with COVID-19 prospectively from four hospitals and one community treatment center between February 2020 and January 2021. symptom severity was classified as mild, moderate, or severe/critical. Patient blood samples were collected at 1 week (acute), 1 month (convalescent), and 2 months after symptom onset (post-convalescent). Human SARS-CoV-2 IgG and IgM antibodies were measured using in-house-developed ELISA. The SARS-CoV-2-specific T-cell responses against overlapping peptides of spike proteins and nucleoprotein were measured by interferon-γ enzyme-linked immunospot assays. Twenty-five COVID-19 patients were analyzed (mild, n = 5; moderate, n = 9; severe/critical, n = 11). IgM and IgG antibody responses peaked at 1 month after symptom onset and decreased at 2 months. IgG response levels were significantly greater in the severe/critical group compared with other groups. Interferon-γ-producing T-cell responses increased between 1 week and 1 month after symptom onset, and had a trend toward decreasing at 2 months, but did not show significant differences according to severity. Our data indicate that SARS-CoV-2-specific antibody responses were greater in those with severe symptoms and waned after reaching a peak around 1 month after symptom onset. However, SARS-CoV-2-specific T-cell responses were not significantly different according to symptom severity, and decreased slowly during the post-convalescent phase.

Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials.

Effect of epitope variant co-delivery on the depth of CD8 T cell responses induced by HIV-1 conserved mosaic vaccines

To stop the HIV-1 pandemic, vaccines must induce responses capable of controlling vast HIV-1 variants circulating in the population as well as those evolved in each individual following transmission. Numerous strategies have been proposed, of which the most promising include focusing responses on the vulnerable sites of HIV-1 displaying the least entropy among global isolates and using algorithms that maximize vaccine match to circulating HIV-1 variants by vaccine cocktails of optimized complementing sequences. In this study, we investigated CD8 T cell responses induced by a bi-valent mosaic of highly conserved HIVconsvX regions delivered by a combination of simian adenovirus ChAdOx1 and poxvirus MVA. We compared partially and fully mono- and bi-valent prime-boost regimens and their ability to elicit T cells recognizing natural epitope variants using an interferon-γ enzyme-linked immunospot (ELISPOT) assay. We used 11 well-defined CD8 T cell epitopes in two mouse haplotypes and, for each epitope, assessed recognition of the two vaccine forms together with the other most frequent epitope variants in the HIV-1 database. We conclude that for the magnitude and depth of epitope recognition, CD8 T cell responses benefitted in most comparisons from the combined bi-valent mosaic and envisage the main advantage of the bi-valent vaccine during its deployment to diverse populations.

Branched Multipeptide-combined Adjuvants Potentially Improve the Antitumor Effects on Glioblastoma.

The promising immunotherapy effects of a multiple antigenic peptide on glioblastoma (GBM) in a previous study encourage the use of adjuvants to enhance its therapeutic efficacy. Among adjuvants, pan HLA-DR-binding epitope (PADRE) and anti-programmed cell death protein 1 (anti-PD1) have potentially been tested for cancer immunotherapy. Therefore, here we evaluated the ability of PADRE and anti-PD1 to enhance the function of the branched multipeptide against GBM. The potential utility of tumor-associated antigens (ErbB-2 and WT-1) targeting GBM with HLA-A24 was confirmed and a branched multipeptide was constructed from these antigens. The effects of the branched multipeptide and PADRE on immunophenotyping and polarized Th cytokine production in dendritic cells were clarified. The expression of PD1 on T cells and PDL1 on GBM cells was also investigated. The interferon-γ enzyme-linked immunospot and lactate dehydrogenase release assays were performed to determine the function of GBM peptide antigen-specific cytotoxic T cells against GBM cells. Overall, this study showed that both ErbB-2 and WT-1 are potential candidates for branched multipeptide construction. The branched multipeptide and PADRE enhanced the expression of major histocompatibility complex and co-stimulatory molecules and the production of polarized Th1 cytokines in dendritic cells. The increase in the number of interferon-γ+ effector T cells was consistent with the increase in the percentage specific lysis of GBM target cells by GBM peptide antigen-specific cytotoxic T cells in the presence of the branched multipeptide, PADRE, and anti-PD1. Our study suggests the combination of branched multipeptide and adjuvants such as PADRE and anti-PD1 can potentially enhance the effects of immunotherapy for GBM treatment.

Safety and Immunogenicity of the ChAdox1 nCoV-19 (AZD1222) Vaccine Against SARS-CoV-2 in HIV Infection

Background: The ChAdOx1 nCoV-19 (AZD1222) vaccine is immunogenic and protects against COVID-19. However, data on vaccine immunogenicity are needed for the 40 million people living with HIV (PWH), who may have less functional immunity and more associated co-morbidities than the general population. Methods: Between the 5th and 24th November 2020, 54 adults with HIV, aged 18-55 years, were enrolled into a single arm open label vaccination study within the protocol of the larger phase 2/3 COV002 trial. A prime-boost regimen of ChAdOx1 nCoV-19, with two doses (5 × 1010 vp) was given 4-6 weeks apart. All participants were on antiretroviral therapy (ART) with undetectable plasma HIV viral loads and CD4+ T cell counts >350 cells/µl at enrolment. Data were captured on adverse events. Humoral responses were measured by anti-spike IgG ELISA and antibody-mediated live virus neutralisation. Cell-mediated immune responses were measured by ex-vivo interferon-γ enzyme-linked immunospot assay (ELISpot) and T cell proliferation. All outcomes were compared with a HIV uninfected group from the main COV002 study.Findings: 54 participants with HIV (median age 42.5 years (IQR 37.2-49.8)) received two doses of ChAdOx1 nCoV-19. Median CD4+ T cell count at enrolment was 694 cells/µl (IQR 562-864). Results are reported for 56 days of follow-up. Local and systemic reactions occurring during the first 7 days after prime vaccination included pain at the injection site (49%), fatigue (47%), headache (47%), malaise (34%), chills (23%), and muscle or (36%) joint pain (9%), the frequencies of which were similar to the HIV-negative participants. There were no serious adverse events. Anti-spike IgG responses by ELISA peaked at Day 42 (median 1440 ELISA units, IQR 704-2728) and were sustained out to Day 56. There was no correlation with CD4+ T cell count or age and the magnitude of the anti-spike IgG response at Day 56 (P>0.05 for both). ELISpot and T cell proliferative responses peaked between Day 14 and 28 after prime and were sustained through to Day 56. When compared to participants without HIV there was no statistical difference in magnitude or persistence of SARS-CoV-2 spike-specific humoral or cellular responses (P>0.05 for all analyses).Interpretation: In this study of PWH, vaccination with ChAdOx1 nCoV-19 was well tolerated and there was no difference in humoral and cell-mediated immune responses compared to an adult cohort without HIV who received the same vaccination regime. Trial Registration: Trial Registration number is NCT04400838. Funding: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.Declaration of Interest: Oxford University has entered into a partnership with AstraZeneca for further development of ChAdOx1 nCoV-19 (AZD1222). AstraZeneca reviewed the data from the study and the final manuscript before 474 submission, but the authors retained editorial control. SCG is cofounder of Vaccitech (a collaborator in the early development of this vaccine candidate) and named as an inventor on a patent covering use of ChAdOx1-vectored vaccines (PCT/GB2012/000467) and a patent application covering this SARS-CoV-2 vaccine. TL is named as an inventor on a patent application covering this SARS-CoV-2 vaccine and was consultant to Vaccitech. PMF is a consultant to Vaccitech. AJP is Chair of the UK Department of Health and Social Care’s JCVI, but does not participate in policy advice on coronavirus vaccines, and is a member of the WHO Strategic Advisory Group of Experts (SAGE). AVSH is a cofounder of and consultant to Vaccitech and is named as an inventor on a patent covering design and use of ChAdOx1-vectored vaccines (PCT/GB2012/000467).Ethical Approval: Written informed consent was obtained from all participants, and the trial was done in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. This study was approved in the UK by the Medicines and Healthcare products Regulatory Agency (reference 21584/0424/001-0001) and the South Central Berkshire Research Ethics Committee (reference 20/SC/0145). Vaccine use was authorised by Genetically Modified Organisms Safety Committees at each participating site.

Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial

BackgroundThe pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2.MethodsWe did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18–55 years with no history of laboratory confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 × 1010 viral particles or MenACWY as a single intramuscular injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque reduction neutralisation assay [PRNT50]; a microneutralisation assay [MNA50, MNA80, and MNA90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606.FindingsBetween April 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p<0·05). There were no serious adverse events related to ChAdOx1 nCoV-19. In the ChAdOx1 nCoV-19 group, spike-specific T-cell responses peaked on day 14 (median 856 spot-forming cells per million peripheral blood mononuclear cells, IQR 493–1802; n=43). Anti-spike IgG responses rose by day 28 (median 157 ELISA units [EU], 96–317; n=127), and were boosted following a second dose (639 EU, 360–792; n=10). Neutralising antibody responses against SARS-CoV-2 were detected in 32 (91%) of 35 participants after a single dose when measured in MNA80 and in 35 (100%) participants when measured in PRNT50. After a booster dose, all participants had neutralising activity (nine of nine in MNA80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R2=0·67 by Marburg VN; p<0·001).InterpretationChAdOx1 nCoV-19 showed an acceptable safety profile, and homologous boosting increased antibody responses. These results, together with the induction of both humoral and cellular immune responses, support large-scale evaluation of this candidate vaccine in an ongoing phase 3 programme.FundingUK Research and Innovation, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research (NIHR), NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and the German Center for Infection Research (DZIF), Partner site Gießen-Marburg-Langen.

Efficacy of immunotherapy targeting the neoantigen derived from epidermal growth factor receptor T790M/C797S mutation in non-small cell lung cancer.

Lung cancer is the leading cause of cancer‐related deaths worldwide. Epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKI) often have good clinical activity against non–small cell lung cancer (NSCLC) with activating EGFR mutations. Osimertinib, which is a third‐generation EGFR‐TKI, has a clinical effect even on NSCLC harboring the threonine to methionine change at codon 790 of EGFR (EGFR T790M) mutation that causes TKI resistance. However, most NSCLC patients develop acquired resistance to osimertinib within approximately 1 year, and 40% of these patients have the EGFR T790M and cysteine to serine change at codon 797 (C797S) mutations. Therefore, there is an urgent need for the development of novel treatment strategies for NSCLC patients with the EGFR T790M/C797S mutation. In this study, we identified the EGFR T790M/C797S mutation‐derived peptide (790‐799) (MQLMPFGSLL) that binds the human leukocyte antigen (HLA)‐A*02:01, and successfully established EGFR T790M/C797S‐peptide‐specific CTL clones from human PBMC of HLA‐A2 healthy donors. One established CTL clone demonstrated adequate cytotoxicity against T2 cells pulsed with the EGFR T790M/C797S peptide. This CTL clone also had high reactivity against cancer cells that expressed an endogenous EGFR T790M/C797S peptide using an interferon‐γ (IFN‐γ) enzyme‐linked immunospot (ELISPOT) assay. In addition, we demonstrated using a mouse model that EGFR T790M/C797S peptide‐specific CTL were induced by EGFR T790M/C797S peptide vaccine in vivo. These findings suggest that an immunotherapy targeting a neoantigen derived from EGFR T790M/C797S mutation could be a useful novel therapeutic strategy for NSCLC patients with EGFR‐TKI resistance, especially those resistant to osimertinib.

HBV X protein-based therapeutic vaccine accelerates viral antigen clearance by mobilizing monocyte infiltration into the liver in HBV carrier mice

BackgroundHepatitis B virus (HBV) persistently infected about 250 million people worldwide, and a curative treatment remains an unmet medical need. Among many approaches to treat chronic hepatitis B (CHB), therapeutic vaccines have been developed for two decades, but none have yielded promising results in clinical trials. Therefore, dissection of HBV clearance mechanisms during therapeutic vaccination in appropriate models, which could give rise to new curative therapies, is urgently needed. Growing evidence indicates that prolonged and intensive exposure of antigen-specific T cells to viral antigens is a major cause of T cell exhaustion, and decreases anti-HBV immunity efficacy of therapeutic vaccination. HBV X protein (HBx) is expressed at low levels, and the understanding of its immunogenicity and potential in therapeutic CHB vaccines is limited.MethodsHBV genome sequences from CHB patients were cloned into a pAAV plasmid backbone and transfected into immunocompetent mouse hepatocytes through hydrodynamic injection. Mice carrying > 500 IU/mL serum HBV surface antigen (HBs) for more than 4 weeks were considered HBV carriers mimicking human CHB and received 3 doses of weekly HBx vaccine by subcutaneous immunization. Serum HBV clearance was evaluated by monitoring serum HBs and HBV-DNA titers. Residual HBV in the liver was evaluated by western blotting for HBV core antigen. The splenic antigen-specific T cell response was quantified by a 15-mer overlapping peptide-stimulated interferon-γ enzyme-linked immunospot assay. Blood and hepatic immune cells were quantified by flow cytometric analysis.ResultsOur HBx-based vaccine induced systemic HBx-specific CD4+ and CD8+ T cell responses in HBV carrier mice and demonstrated significant HBs and HBV-DNA elimination. The protective effect persisted for at least 30 days without additional booster immunization. Different infiltrating myeloid cell subsets, each with distinctive roles during immune-mediated HBV clearance, were found in the liver of vaccinated mice. During vaccine therapy, inflammatory monocyte depletion resulted in sustained HBV clearance inhibition, whereas phagocytic monocyte-derived macrophage and Kupffer cell elimination resulted in only transient inhibition of vaccine-induced HBV clearance.ConclusionsWe report the potential role of HBx as a major immunogen in an HBV therapeutic vaccine and the significance of a liver-infiltrating monocyte subset during hepatic viral clearance.

Usefulness of BK virus-specific interferon-γ enzyme-linked immunospot assay for predicting the outcome of BK virus infection in kidney transplant recipients.

Background/AimsTo investigate if BK virus (BKV)-specific T cell immunity measured by an interferon-γ enzyme-linked immunospot (ELISPOT) assay can predict the outcome of BK virus infection in kidney transplant recipients (KTRs). MethodsWe included 68 KTRs with different viremia status (no viremia [n = 17], BK viremia [n = 27], and cleared viremia [n = 24]) and 44 healthy controls (HCs). The BK viremia group was divided into controller (< 3 months) and noncontroller (> 3 months) according to sustained duration of BKV infection. We compared BKV-ELISPOT results against five BKV peptides (large tumor antigen [LT], St, VP1-3). ResultsBKV-ELISPOT results were higher in three KTRs groups with different BKV infection status than the HCs group (p < 0.05). In KTR groups, they were higher in cleared viremia group than no viremia or BK viremia group. Within the BK viremia group, controller group had higher LT-ELISPOT results compared to noncontroller group (p = 0.032). Also, KTRs without BK virus-associated nephropathy (BKVN) had higher LT, St, VP1, and VP2-ELISPOT results than those with BKVN (p < 0.05). ConclusionsBKV-ELISPOT assay may be effective in predicting clinical outcomes of BKV infection in terms of clearance of BK virus and development of BKVN.

Safety and immunogenicity of VGX-3100 formulations in a healthy young adult population

ABSTRACT HPV remains the most common sexually transmitted disease worldwide, despite improvements in awareness, screening, prophylactic vaccination uptake, and surgical treatment. VGX-3100 is an immunotherapy that uses electroporation to introduce DNA encoding for modified HPV-16 and HPV-18, E6-and E7 proteins into myocytes to stimulate an effector T cell response. We now report immunogenicity and safety of VGX-3100 for a refrigeration-stable formulation, which improves patient-care setting usability. This multi-arm, double-blinded, randomized trial enrolled 235 healthy men and women to receive either a refrigerated (RF) or frozen formulation (FF) of VGX-3100. Three doses were administered intramuscularly with electroporation at 0, 4, and 12 weeks. Non-inferiority of RF to FF was assessed by comparing the proportion of subjects who achieved a ≥2-fold increase from baseline to Week 14 in Spot Forming Units/106 PMBCs using an interferon-γ enzyme-linked immunospot assay. There were no related SAEs. Injection site reactions were the most common adverse event (54%, RF; 66%, FF) the majority of which resolved within a few minutes following administration. The primary endpoint was met with 89.9% of RF recipients and 97.2% of FF recipients reaching a ≥2-fold rise in SFU/106 PBMC, 2 weeks following the last dose; RF was statistically non-inferior to FF (p = .022). A systemic, immunologic approach has the potential to fill a critical gap in the ability to treat men and women with high grade HPV diseases. These safety and immunogenicity data are supportive of the continued development of a refrigerated formulation of VGX-3100.

Maintaining the Cellular Anti-Viral and Anti-Leukemic Activities in GvHD Patients Undergoing Extracorporeal Photophoresis Therapy

Introduction: Systemic steroids with their strong immunosuppressive and anti-inflammatory effects are the current gold standard for initial treatment of the graft-versus-host disease (GvHD). But, the effectiveness of high-dose steroids comes along with an increased susceptibility to infections and other secondary malignancies. Extracorporeal photophoresis (ECP), an immunotherapeutic therapy, is currently being used as a clinically well-established second-line treatment for GvHD. Contrary to steroid treatment, there is no clinical data showing that ECP is associated with an increased risk of infection. However, the exact mechanism of action how ECP preserves the anti-viral and anti-leukemic effects on the cellular level has not been discovered yet. Materials and methods: Thirty-four patients with steroid-refractory/resistant aGvHD ≥ II and moderate to severe cGvHD were treated with ECP at the University Hospitals in Heidelberg, Greifswald in Germany and Chaim Sheba Medical Center in Israel. A comprehensive analysis of cell subsets was performed using multi-parametric flow cytometry. Additionally, the quantity and quality of CMV-specific T cells was determined by tetramer staining and interferon-γ enzyme-linked immunospot assay. The NK activity in terms of killing functionality and cytokine release was analyzed by intracellular cytokine staining and chromium-51 release assay, respectively. The proliferative capacity of effector cells was determined by carboxyfluorescein succinimidyl ester (CFSE) staining. Results: ECP therapy was effective with an overall response rate of 75% for patients with aGvHD (x/y) and 78% (x/y) for patients with cGvHD. Of note, all patients showed neither the increased susceptibility to infections nor the reactivation of CMV nor the tumor relapse. Overall, the frequency of well-established protective cell subsets in terms of cytotoxic CD8+ T cells, CD4+CD8+ T cells, γδ T cells, NK cells and NKT cells remained constant under the ECP therapy. Specifically, no significant influence of ECP therapy on the CD56dimCD57+NKG2C+ NK cells, a specialized anti-viral/tumor population, and the CMV+CD8+ T cells could be observed. The further phenotyping of CMV+CD8+ T cells showed that CD45RA+CCR7-CMV+CD8+ TE cells and CD45RA-CCR7-CMV+CD8+ TEM cells could keep stable under the ECP therapy. Besides these, priming, differentiation and maturation of NK cells through upregulation of CD57 by ECP therapy bridged the T-cell-deficient period after HSCT in order to control the viral infections and eliminate the residual malignant cells. Moreover, ECP could reduce the CD62L expression on TE population which not only facilitates the hematopoietic engraftment and contributes to the phenotypic and functional T cell reconstitution after transplantation without causing GvHD, but also enhances the functional immune reconstitution against tumor and viral antigens. Functionally, neither IFN-g released by virus specific T cells nor production of TNF-a and IFN-g by NK cells in response to K562 cells was hampered by ECP. Of note, the functions of NK cells in terms of the MFI of cytokines and the polyfunctionality of NK cells were stable under ECP treatment. Additionally, the proliferation of NK cells, CD4+ T cells and CD8+ T cells providing an expanded pool of effector cells against the pathogens was not hampered by ECP therapy as well. Conclusions: ECP therapy constitutes an effective strategy to treat GvHD. In addition, ECP appears to be a safe therapy compared to continued steroid use. Our current results suggest that ECP allows to maintain immunity against infections and tumors. Disclosures Schönland: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Other: Travel Grant. Dreger:Neovii, Riemser: Research Funding; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia. Schmitt:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Therakos Mallinckrodt: Other: Financial Support. Schmitt:Therakos Mallinckrodt: Other: Financial Support .

Co-Prevalence of Pre-Existing Immunity to Different Serotypes of Adeno-Associated Virus (AAV) in Adults with Hemophilia

Background: Recombinant and synthetic adeno-associated virus (AAV) vectors are in development for gene transfer in patients with hemophilia A (HA) or hemophilia B (HB). These include liver-directed recombinant AAV8 vectors BAX 888/SHP654/TAK-754 factor VIII (FVIII) gene therapy (GT) for severe HA, and SHP648/TAK-748 factor IX (FIX) GT for HB (Baxalta US Inc., a Takeda company, Lexington, MA, USA). However, environmental exposure to wild-type AAVs can result in individuals developing antibodies and cell-mediated immune responses to the naturally occurring AAV. While natural exposure to AAV does not result in any known disease in humans, presence of preexisting immunity can block delivery and prevent sustained expression of the transgene by an AAV-based vector in a gene therapy setting. Of the AAV serotypes, AAV2 is the most frequently encountered natural human infection and AAV5 and AAV8 have been the most commonly used vectors for hemophilia GT. Therefore, it is important to assess the prevalence and co-prevalence of antibody and T cell-mediated responses against each of these AAV serotypes and to better characterize the association between humoral and cellular immunity in people with hemophilia. Aims: To determine the prevalence of preexisting antibody-mediated immunity against AAV2, AAV5 and AAV8 and the association between AAV8-specific humoral and cell-mediated responses in adult patients with HA and HB in an international prospective, epidemiological study. Methods: This ongoing seroprevalence study involved adult male patients (18-75 years of age) with severe HA (&lt;1% plasma FVIII activity) or severe/moderate HB (≤2% plasma FIX activity) recruited from hemophilia treatment centers in the United States and Europe (NCT03185897). Participants consented to collection of peripheral blood at either a single or multiple annual outpatient study visits, in order to explore fluctuations of the immune response over time. Local ethics committee approval was obtained. Titers for anti-AAV2, anti-AAV5 and anti-AAV8 neutralizing antibodies (NAbs) were determined using a cell-based transduction inhibition assay, with seropositivity defined as a titer ≥1:5. Titers for anti-AAV2, anti-AAV5 and anti-AAV8 binding antibodies (BAbs) were quantitated by indirect enzyme-linked immunosorbent assay (ELISA), with seropositivity defined as a titer ≥1:80. Cell-mediated immune responses to AAV8 peptide antigens were measured in peripheral blood mononuclear cells using an interferon-γ enzyme-linked immunospot (ELISpot) assay. Samples with a signal ≥3 times background and &gt;60 spots per million cells were defined as positive. Results: Here we present data from patients who completed at least a single visit at the time of the interim, one year data cut (November 9, 2018). Of 242 patients enrolled (mean ± SD age: 35.3 ± 11.4 years), 194 patients had HA and 48 patients had HB. The overall co-prevalence of NAbs and BAbs to AAV2, AAV5 and AAV8 was 39.7% (HA: 38.1%, 72/189; HB: 45.8%, 22/48) and 16.1% (HA: 16.5%, 31/188; HB: 14.6%, 7/48) respectively, with further details shown in Table 1. Overall, 38.3% of patients (82/214) exhibited a T cell-mediated immune response to AAV8 peptide antigens (HA: 35.9%, 61/170; HB: 47.7%, 21/44). Among patients with AAV-8-specific NAbs, 37.9% (39/103) demonstrated positive AAV8-specific ELISPOT results. (HA: 35.7%, 30/84; HB: 47.4%, 9/19). Conclusion: The findings from this ongoing study demonstrate that approximately 50% of patients with hemophilia have preexistent NAb responses to AAV2, AAV5 or AAV8 with 40% demonstrating co-prevalence to all 3 evaluated AAV serotypes. Similar percentages of patients exhibited a positive cellular response to AAV8 antigens. Further, patients with HB demonstrated a slightly higher co-prevalence and a higher cellular response than patients with HA. In the combined HA and HB cohorts, co-prevalence was almost 40% for AAV8-specific humoral and T-cell mediated immunity. These data will add to our appreciation of preexisting AAV immunity that prevent patient participation in gene therapy trials. Disclosures Rajavel: Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Ayash-Rashkovsky:Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Tang:Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Gangadharan:Baxalta Innovations GmbH, a Takeda company: Employment. de la Rosa:Baxalta Innovations GmbH, a Takeda company: Employment. Ewenstein:Baxalta US Inc., a Takeda company: Employment, Equity Ownership, Other: a Takeda stock owner.

Efficacy and Safety in 15 Hemophilia B Patients Treated with the AAV Gene Therapy Vector Fidanacogene Elaparvovec and Followed for at Least 1 Year

Background: Adeno-Associated Virus (AAV) based liver transduction has emerged as a potentially viable gene therapy approach for the treatment of hemophilia patients. Fidanacogene elaparvovec (previously SPK-9001) is a hepatotropic bioengineered AAV based vector that delivers a high activity factor IX (FIX) transgene driven by a liver specific promoter. The Phase 1/2a development consists of a dosing study where patients are followed for 52 weeks post vector infusion followed by a long-term follow-up study for an additional 5 years. Data on the first 10 patients were previously published and demonstrated safe and sustained expression of a high activity FIX protein with an associated decreased requirement for exogenous factor administration and markedly reduced annualized bleeding rate. Here we present data on 15 patients infused with fidanacogene elaparvovec with ≥ 1 year of follow-up, which represents the largest cohort of Hemophilia B (HB) patients treated with the same vector at the same dose. Methods: Fifteen (15) adult HB patients were infused with 5 x 1011 vg/kg of fidanacogene elaparvovec and followed for at least 1 year as part of the Phase 1/2a dosing study. FIX activity (FIX:C) levels were measured using a one-stage assay. Endpoints include: Safety and tolerability, steady-state activity calculated as the geometric mean of all observed FIX:C activity levels from week 12 through week 52; annualized bleeding rate (ABR) prior to and 52 weeks after vector infusion; T cell response to fidanacogene elaparvovec capsid and transgene monitored post-infusion using an interferon-γ enzyme-linked immunospot (ELISpot) assay. Results: Three of fifteen patients were treated with corticosteroids for elevations in hepatic transaminases of which 2 were positive for capsid reactive T cells by interferon-γ ELISpot. There were otherwise no treatment related adverse events. The mean post-infusion steady-state FIX:C was 22.9%±9.9% at 1 year post vector infusion as measured in a central laboratory by one-stage assay utilizing Actin-FSL. Mean ABR during the first 52 weeks following fidanacogene elaparvovec infusion was 0.4±1.1 compared to 8.9±14.0 in the 52 weeks preceding infusion (p&lt;0.001). Twelve (12) out of 15 patients reported zero bleeds in the 52 weeks post-vector infusion. Five of 15 subjects infused factor for a total of 20 infusions. Additional follow-up data will be presented for all patients enrolled in the long-term follow-up study. Conclusions: Fidanacogene elaparvovec was well tolerated in 15 patients with no serious adverse events. Data for all patients at 52 weeks post-infusion demonstrated a marked reduction in bleeding frequency and exogenous FIX use. All hepatic transaminase elevations responded to treatment with corticosteroids. Collectively, to date, this represents the largest cohort of HB patients treated with the same AAV based gene therapy and at the lowest dose. Treatment has been efficacious for all patients with manageable immune responses when present. These data support progression to a pivotal Phase 3 study at the dose evaluated. Disclosures George: University of Pennyslvania: Employment; Avrobio: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy. Sullivan:Octapharma: Consultancy, Other: Advisory Board. Rasko:bluebird bio: Honoraria; Celgene: Honoraria; Novartis: Honoraria; FSHD Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Rarecyte: Consultancy, Equity Ownership; Gene Technology Technical Advisory, Australian Government: Other: Advisory committee; GSK: Honoraria; Takeda: Honoraria; Cynata: Honoraria; Genea: Equity Ownership; Cure The Future Foundation: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Pfizer: Honoraria; Spark: Honoraria; Imago: Consultancy; Advisory Committee on Biologics, Australian Government: Other: Advisory Committee; NHMRC Mitochondrial Donation Expert Working Committee: Other: Advisory Committee; Australian Cancer Research Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees. Giermasz:Genentech/Roche: Consultancy, Other: Research, Speakers Bureau; uniQure: Consultancy, Other: Research; Bioverativ/Sanofi: Consultancy, Speakers Bureau; BioMarin: Consultancy, Other: Research; Sangamo: Other: Research. Samelson-Jones:The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania: Employment. Ducore:Bayer: Consultancy, Honoraria, Other: speaker (not bureau); Spark Therapeutics: Research Funding; Shire: Consultancy, Honoraria; Octapharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; HEMA Biologics: Consultancy, Honoraria; BioMarin: Research Funding; Bioverativ: Research Funding. Teitel:BioMarin: Consultancy; Bayer: Consultancy, Research Funding; Shire: Consultancy; Pfizer: Consultancy, Research Funding; Novo Nordisk: Consultancy; Octapharma: Consultancy; CSL Behring: Consultancy. McGuinn:Biogen: Research Funding; Roche/Genetech: Research Funding; Spark: Research Funding; Shire/Baxalta: Consultancy, Research Funding. Wright:Solid Biosciences: Consultancy; Yposkesi: Other: Senior Advisor, SAB; LogicBio Therapeutics: Other: Member, SAB; Memorial Sloan Kettering Cancer Center: Consultancy; Agilis Biotherapeutics: Consultancy; Axovant Sciences: Other: Chief Technology Officer, Gene Therapies; Akous Therapeutics: Consultancy; National Institutes of Health: Consultancy; Leland Stanford Junior University: Consultancy; Wright Biologics: Other; Sanofi Genzyme: Consultancy; Spark Therapeutics: Consultancy, Other: co-founder, Chief Technology Advisor/Officer, Member, SAB; Adrenas Therapeutics: Other: Member, SAB; Ambys Medicines: Consultancy; CEVEC Pharmaceuticl: Other: Member, SAB. Anguela:Spark Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties. High:Spark Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties. Rybin:Pfizer: Employment. Murphy:Pfizer Inc.: Employment. Rupon:Pfizer: Employment.

Expression of Lymphocyte-Activation Gene 3 (LAG-3) Immune Checkpoint Receptor Identifies a Tumor-Reactive T Cell Population in the Peripheral Blood of Patients with Colorectal Cancer.

BackgroundThe use of adoptive T cell therapy has proven to be effective in some advanced malignancies. This study aimed to investigate the effects of lymphocyte-activation gene 3 (LAG-3) immune checkpoint receptor in the enrichment of tumor antigen-specific CD8+ T lymphocytes derived from peripheral blood mononuclear cells (PBMCs) in patients with colorectal cancer.Material/MethodsPeripheral blood samples were obtained from 20 patients with colorectal cancer and apheresis was performed with enrichment and cell sorting to obtain CD8+LAG-3+ T cells, which were expanded using high-dose interleukin-2 (IL-2). T cell subsets were detected using fluorescence-activated cell sorting (FACS), and T cell receptor (TCR) sequencing was used to determine specific clone types. Interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) and cell counting kit-8 (CCK-8) assays were used to measure cell avidity and cytotoxicity.ResultsThe cultured cells increased in number over time and had the greatest proliferative activity at 15 days, at which time the percentage of CD3+, CD3+CD8+, and CD8+CD28+ reached maximal levels. High purity CD8+LAG-3+ T cells were isolated by FACS and at 15 days. TCR sequencing showed that CD8+LAG-3+ T cells were oligoclonal, ELISpot identified increased production of tumor-specific IFN-γ, and the CCK-8 assay showed increased cytotoxicity when compared with pre-cultured CD8+LAG-3− T cells.ConclusionsIn patients with colorectal cancer, CD8+LAG-3+ T cells showed more specific anti-tumor activity following cell culture in vitro, which supported the potential role for the LAG-3 immune checkpoint receptor in enriching tumor-specific T cells in patients with cancer.