Idiotypic DNA vaccination for the treatment of multiple myeloma: safety and immunogenicity in a phase I clinical study

We report on the safety and immunogenicity of idiotypic DNA vaccination in a phase I, non-randomised, open-label study in patients with multiple myeloma. The study used DNA fusion gene vaccines encoding patient-specific single chain variable fragment, or idiotype (Id), linked to fragment C (FrC) of tetanus toxin. Patients in complete or partial response following high-dose chemotherapy and autologous stem cell transplant were vaccinated intramuscularly with 1 mg DNA on six occasions, beginning at least 6 months post-transplant; follow-up was to week 52. Fourteen patients were enrolled on study and completed vaccinations. Idiotypic DNA vaccines were well tolerated with vaccine-related adverse events limited to low-grade constitutional symptoms. FrC- and Id-specific T-cell responses were detected by ex vivo ELISPOT in 9/14 and 3/14 patients, respectively. A boost of pre-existing anti-FrC antibody (Ab) was detected by ELISA in 8/14 patients, whilst anti-Id Ab was generated in 1/13 patients. Overall, four patients (29 %) made an immune response to FrC and Id, with six patients (43 %) responding to FrC alone. Over the 52-week study period, serum paraprotein was undetectable, decreased or remained stable for ten patients (71 %), whilst ongoing CR/PR was maintained for 11 patients (79 %). The median time to progression was 38.0 months for 13/14 patients. Overall survival was 64 % after a median follow-up of 85.6 months

Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8+ T cells with detection by ELISPOT and HLA-multimer staining

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R(2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00262-014-1593-0) contains supplementary material, which is available to authorized users

Correlation of HPV16 Gene Status and Gene Expression With Antibody Seropositivity and TIL Status in OPSCC.

IntroductionHuman papillomavirus 16 (HPV16) is the main cause of oropharyngeal squamous cell carcinoma (OPSCC). To date, the links between HPV16 gene expression and adaptive immune responses have not been investigated. We evaluated the correlation of HPV16 DNA, RNA transcripts and features of adaptive immune response by evaluating antibody isotypes against E2, E7 antigens and density of tumor-infiltrating lymphocytes (TIL).Material and methodsFFPE-tissue from 27/77 p16-positive OPSCC patients was available. DNA and RNA were extracted and quantified using qPCR for all HPV16 genes. The TIL status was assessed. Immune responses against E2 and E7 were quantified by ELISA (IgG, IgA, and IgM; 77 serum samples pre-treatment, 36 matched post-treatment).ResultsAmounts of HPV16 genes were highly correlated at DNA and RNA levels. RNA co-expression of all genes was detected in 37% (7/19). E7 qPCR results were correlated with higher anti-E7 antibody (IgG, IgA) level in the blood. Patients with high anti-E2 IgG antibody (>median) had better overall survival (p=0.0311); anti-E2 and anti-E7 IgA levels had no detectable effect. During the first 6 months after treatment, IgA but not IgG increased significantly, and >6 months both antibody classes declined over time. Patients with immune cell-rich tumors had higher levels of circulating antibodies against HPV antigens.ConclusionWe describe an HPV16 qPCR assay to quantify genomic and transcriptomic expression and correlate this with serum antibody levels against HPV16 oncoproteins. Understanding DNA/RNA expression, relationship to the antibody response in patients regarding treatment and outcome offers an attractive tool to improve patient care

Intermittent PI3Kδ inhibition sustains anti-tumour immunity and curbs irAEs

Phosphoinositide 3-kinase δ (PI3Kδ) has a key role in lymphocytes, and inhibitors that target this PI3K have been approved for treatment of B cell malignancies1-3. Although studies in mouse models of solid tumours have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumour immunity4,5, its effect on solid tumours in humans remains unclear. Here we assessed the effects of the PI3Kδi AMG319 in human patients with head and neck cancer in a neoadjuvant, double-blind, placebo-controlled randomized phase II trial (EudraCT no. 2014-004388-20). PI3Kδ inhibition decreased the number of tumour-infiltrating regulatory T (Treg) cells and enhanced the cytotoxic potential of tumour-infiltrating T cells. At the tested doses of AMG319, immune-related adverse events (irAEs) required treatment to be discontinued in 12 out of 21 of patients treated with AMG319, suggestive of systemic effects on Treg cells. Accordingly, in mouse models, PI3Kδi decreased the number of Treg cells systemically and caused colitis. Single-cell RNA-sequencing analysis revealed a PI3Kδi-driven loss of tissue-resident colonic ST2 Treg cells, accompanied by expansion of pathogenic T helper 17 (TH17) and type 17 CD8+ T (TC17) cells, which probably contributed to toxicity; this points towards a specific mode of action for the emergence of irAEs. A modified treatment regimen with intermittent dosing of PI3Kδi in mouse models led to a significant decrease in tumour growth without inducing pathogenic T cells in colonic tissue, indicating that alternative dosing regimens might limit toxicity

DNA fusion vaccines enter the clinic

Induction of effective immune attack on cancer cells in patients requires conversion of weak tumor antigens into strong immunogens. Our strategy employs genetic technology to create DNA vaccines containing tumor antigen sequences fused to microbial genes. The fused microbial protein engages local CD4+ T cells to provide help for anti-tumor immunity, and to reverse potential regulation. In this review, we focus on induction of CD8+ T cells able to kill target tumor cells. The DNA vaccines incorporate tumor-derived peptide sequences fused to an engineered domain of tetanus toxin. In multiple models, this design induces strong CD8+ T-cell responses, able to suppress tumor growth. For clinical relevance, we have used "humanized" mice expressing HLA-A2, successfully inducing cytolytic T-cell responses against a range of candidate human peptides. To overcome physical restriction in translating to patients, we have used electroporation. Clinical trials of patients with cancer are showing induction of responses, with preliminary indications of suppression of tumor growth and evidence for clinically manageable concomitant autoimmunity

Peripheral immunophenotype in Dementia with Lewy bodies and Alzheimer’s disease: an observational clinical study

Background: Inflammation plays a key role in the aetiology and progression of Alzheimer’s disease (AD). However, the immunophenotype of the second most common neurodegenerative cause of dementia, dementia with Lewy bodies (DLB), remains unclear. To date there have been no studies examining peripheral inflammation in DLB using multiplex immunoassay and flow cytometry concomitantly. We hypothesised that, using blood biomarkers, DLB would show an increased proinflammatory profile compared with controls, and that there would be a distinct profile compared with AD.Methods: 93 participants (31 with DLB, 31 with AD and 31 healthy older controls) completed a single study visit for neuropsychiatric testing and phlebotomy. Peripheral blood mononuclear cells were quantified for T and B cell subsets using flow cytometry, and serum cytokine concentrations were measured using multiplex immunoassay.Results: We detected reduced relative numbers of helper T cells and reduced activation of B cells in DLB compared with AD. Additionally, interleukin (IL)-1β was detected more frequently in DLB and the serum concentration of IL-6 was increased compared with controls.Conclusions: Peripheral inflammation is altered in DLB compared with AD, with T cell subset analysis supporting a possible shift towards senescence of the adaptive immune system in DLB. Furthermore, there is a proinflammatory signature of serum cytokines in DLB. Identification of this unique peripheral immunophenotype in DLB could guide development of an immune-based biomarker and direct future work exploring potential immune modulation as a novel treatment

Flow cytometric analysis of vaccine-specific and bystander CD4⁺ T cell responses to TT recall vaccination detected by combined CD40L and cytokine intracellular staining.

<p>After a short term (6h) <i>in vitro</i> culture in the absence (CTRL) or in the presence of either TT (10μg/ml), PPD (15μg/ml) or <i>C</i>.<i>Alb</i> (10μg/ml), PBMNC were first stained for surface CD3 and CD4, then permeabilized and stained intracellularly with fluorescent antibodies specific for CD40L and the cytokines IL-2 and IFN-γ. (A) Data from one of the individuals (subject 1) show the gating strategy (top three dot plots) and the detection of vaccine-specific (TT) and bystander (PPD and <i>C</i>.<i>Alb</i>) CD4⁺ T cell responses before (pre-vaccination) and one week after (week 1) a booster injection of TT. Percentages indicate the frequency of positive events within the CD3⁺CD4⁺ lymphocyte population. (B) Kinetics of vaccine-specific and bystander CD4⁺ T cell responses detected by intracellular CD40L and cytokine staining in the same individual who received a booster vaccination (Wk 0) with TT. Data indicate the frequency of positive cells within the CD3⁺CD4⁺ population, obtained from the antigen-stimulated samples after subtracting the frequency of events in the control cultures. Responses were considered positive if they met the criteria described in Materials and Methods. The dotted line shows the cut off value of 0.01%.</p

Analyis of activation (CD38, HLA-DR) and proliferation (Ki-67) markers on vaccine-specific (TT) and bystander (PPD and <i>C</i>.<i>Alb</i>) CD4⁺CD40L⁺ T cells.

<p>After a short term (6h) <i>in vitro</i> culture in the absence (control) or in the presence of either TT (10μg/ml), PPD (15μg/ml) or <i>C</i>.<i>Alb</i> (10μg/ml), PBMNC were first stained for surface CD3, CD4, CD38 and HLA-DR, then permeabilized and stained intracellularly for CD40L and Ki-67. (A) Data indicate the percentage of CD38, HLA-DR, Ki-67 single positive and CD38⁺Ki-67⁺ double positive cells within the CD3⁺CD4⁺CD40L⁺ population in five healthy subjects one week after receiving a booster vaccination with TT. Median values are indicated by a bar. Significance of the difference between vaccine specific and bystander populations are indicated (NS—non-significant, * = P<0.01, ** = P<0.001), as analysed using non-parametric Mann-Whitney U-test. (B) Representative dot plots from subject 5, showing how at week 1 activated and proliferating (CD38⁺Ki-67⁺) cells are only found among the TT-specific cells, but not among the bystander PPD- and <i>C</i>.<i>Alb</i>-specific cells. Percentages of positive cells in the CD3⁺CD4⁺CD40L⁺ gated population are indicated in each quadrant. Gates in dot plots were set using the appropriate isotype-matched controls. (C) Analysis of expression (%) of CD38, HLA-DR, Ki-67 single positive and CD38⁺Ki-67⁺ double positive cells within the TT- and PPD-specific CD4⁺CD40L⁺ T cell populations before (Wk 0) and at various time points after TT booster vaccination. Data indicate the mean ± standard deviation calculated from the individuals showing detectable responses. P-values indicate the significant changes in expression over baseline for all time points, calculated using paired T-test and confirmed using Friedman’s test; non-significant changes are unlabeled. At week 0, responses to TT and PPD were detected in subjects 4 and 5; at week 2, responses to PPD were detected in subjects 2 and 4; for all the remaining time points, responses to TT and PPD were detected in all subjects.</p

Total and differential white blood cell (WBC) counts (N x 10⁹/L) before (Week 0) and at various time points after TT booster vaccination in three healthy subjects.

<p>Full blood counts were carried out at each study visit by the routine NHS laboratory.</p