Targeting Carcinoembryonic Antigen with DNA Vaccination: On-Target Adverse Events Link with Immunologic and Clinical Outcomes.

PURPOSE: We have clinically evaluated a DNA fusion vaccine to target the HLA-A*0201-binding peptide CAP-1 from carcinoembryonic antigen (CEA605-613) linked to an immunostimulatory domain (DOM) from fragment C of tetanus toxin. EXPERIMENTAL DESIGN: Twenty-seven patients with CEA-expressing carcinomas were recruited: 15 patients with measurable disease (arm-I) and 12 patients without radiological evidence of disease (arm-II). Six intramuscular vaccinations of naked DNA (1 mg/dose) were administered up to week 12. Clinical and immunologic follow-up was up to week 64 or clinical/radiological disease. RESULTS: DOM-specific immune responses demonstrated successful vaccine delivery. All patients without measurable disease compared with 60% with advanced disease responded immunologically, while 58% and 20% expanded anti-CAP-1 CD8+ T cells, respectively. CAP-1-specific T cells were only detectable in the blood postvaccination but could also be identified in previously resected cancer tissue. The gastrointestinal adverse event diarrhea was reported by 48% of patients and linked to more frequent decreases in CEA (P < 0.001) and improved global immunologic responses [anti-DOM responses of greater magnitude (P < 0.001), frequency (P = 0.004), and duration] compared with patients without diarrhea. In advanced disease patients, decreases in CEA were associated with better overall survival (HR = 0.14, P = 0.017). CAP-1 peptide was detectable on MHC class I of normal bowel mucosa and primary colorectal cancer tissue by mass spectrometry, offering a mechanistic explanation for diarrhea through CD8+ T-cell attack. CONCLUSIONS: Our data suggest that DNA vaccination is able to overcome peripheral tolerance in normal and tumor tissue and warrants testing in combination studies, for example, by vaccinating in parallel to treatment with an anti-PD1 antibody. Clin Cancer Res; 22(19); 4827-36. ©2016 AACR.

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.

DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8(+) T-cell responses and increases PSA doubling time.

We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA(27-35). We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2(+) patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2(-) control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4(+) and PSMA(27)-specific CD8(+) T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.00248, respectively). Of 30 patients, 29 had a measurable CD4(+) T-cell response and PSMA(27)-specific CD8(+) T cells were detected in 16/30 patients, with or without EP. At week 24, before cross-over, both delivery methods led to increased CD4(+) and CD8(+) vaccine-specific T cells with a trend to a greater effect with EP. PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP. The high frequency of immunological responses to DOM-PSMA(27) vaccination and the clinical effects are sufficiently promising to warrant further, randomized testing.

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.

DNA vaccination with electroporation induces increased antibody responses in patients with prostate cancer.

We are evaluating the use of electroporation (EP) to deliver a novel DNA vaccine, p.DOM-PSMA(27). This vaccine encodes a domain (DOM) of fragment C of tetanus toxin to induce CD4(+) T cell help, fused to a tumor-derived epitope from prostate-specific membrane antigen (PSMA) for use in HLA-A2(+) patients with recurrent prostate cancer. We report on safety and tolerability and on antibody response to DOM as a first indication of the effect of EP in patients. In this open label phase I/II, two-arm, dose escalation trial DNA was delivered either by intramuscular injection or by intramuscular injection followed by EP (DNA+EP), with five patients per dose level. Three vaccinations were given at 0, 4, and 8 weeks,with booster doses at 24 and 48 weeks; here we allowed crossover between study arms if supported by the safety and immunological data. In the 20 patients in the first two dose cohorts we observed that beyond brief and acceptable pain at the injection site, EP did not appear to add toxicity to the vaccination. We evaluated humoral responses to DOM. Low anti-DOM IgG antibody responses were observed after intramuscular injection of DNA without EP (at week 12: mean 1.7- vs. 24.5-fold increase over baseline with DNA+EP). These could be boosted by delivery of DNA+EP at later time points. Delivery of DNA+EP at all five vaccinations yielded the highest levels of anti-DOM antibody. Responses persisted to 18 months of follow-up. These data establish EP as a potent method for stimulating humoral responses induced by DNA vaccination in humans.

Fit for purpose? A case study: validation of immunological endpoint assays for the detection of cellular and humoral responses to anti-tumour DNA fusion vaccines.

Clinical trials are governed by an increasingly stringent regulatory framework, which applies to all levels of trial conduct. Study critical immunological endpoints, which define success or failure in early phase clinical immunological trials, require formal pre-trial validation. In this case study, we describe the assay validation process, during which the sensitivity, and precision of immunological endpoint assays were defined. The purpose was the evaluation of two multicentre phase I/II clinical trials from our unit in Southampton, UK, which assess the effects of DNA fusion vaccines on immune responses in HLA-A2+ patients with carcinoembryonic antigen (CEA)-expressing malignancies and prostate cancer. Validated immunomonitoring is being performed using ELISA and IFNgamma ELISPOTs to assess humoral and cellular responses to the vaccines over time. The validated primary endpoint assay, a peptide-specific CD8+ IFNgamma ELISPOT, was tested in a pre-trial study and found to be suitable for the detection of low frequency naturally occurring CEA- and prostate-derived tumour-antigen-specific T cells in patients with CEA-expressing malignancies and prostate cancer.

Knock-down of LAR protein tyrosine phosphatase induces insulin resistance.

To test the role of the leukocyte common antigen-related protein tyrosine phosphatase (LAR) as a regulator of insulin receptor (IR) signalling, an siRNA probe against LAR was developed. Knock-down of LAR induced post-receptor insulin resistance with the insulin-induced activation of PKB/Akt and MAP kinases markedly inhibited. The phosphorylation and dephosphorylation of the IR and insulin receptor substrate (IRS) proteins were unaffected by LAR knock-down. These results identify LAR as a crucial regulator of the sensitivity of two key insulin signalling pathways to insulin. Moreover, the siRNA probe provides a molecular tool of general applicability for further dissecting the precise targets and roles of LAR.

Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking.

PKCzeta (protein kinase Czeta) is a serine/threonine protein kinase controlled by insulin, various growth factors and phosphoinositide 3-kinase. It has been implicated in controlling glucose transport in response to insulin by the translocation of GLUT4-(glucose transporter 4) containing vesicles to the plasma membrane in stimulated cells. How PKCzeta modulates GLUT4 vesicle trafficking remains unknown. A yeast two-hybrid screen using full-length human PKCzeta identified 80K-H protein as an interactor with PKCzeta. GST (glutathione S-transferase) pull-down assays with GST-tagged 80K-H constructs confirmed the interaction and showed that the N-terminal portion of 80K-H was not required for the interaction. Immunoprecipitates of endogenous PKCzeta from Cho cells, 3T3-L1 adipocytes or L6 myotubes contained endogenous 80K-H, demonstrating a physiological interaction. Insulin stimulation enhanced the association 3-5-fold. Immunoprecipitates of endogenous 80K-H contained endogenous munc18c and immunoprecipitates of endogenous munc18c contained endogenous PKCzeta, with insulin markedly increasing the amount of co-immunoprecipitated protein in each case. These results show that insulin triggers interactions in vivo between PKCzeta, 80K-H and munc18c. Overexpression of 80K-H constructs mimicked the action of insulin in stimulating both glucose uptake and translocation of Myc-tagged GLUT4 in Cho cells, with the level of effect proportional to the ability of the constructs to associate with munc18c. These results identify 80K-H as a new player involved in GLUT4 vesicle transport and identify a link between a kinase involved in the insulin signalling cascade, PKCzeta, and a known component of the GLUT4 vesicle trafficking pathway, munc18c. The results suggest a model whereby insulin triggers the formation of a PKCzeta-80K-H-munc18c complex that enhances GLUT4 translocation to the plasma membrane.

Altered phospholipid composition and aggregate structure of lung surfactant is associated with impaired lung function in young children with respiratory infections.

Alterations to pulmonary surfactant structure, composition, and function contribute to the severity of respiratory infections. Analysis of bronchoalveolar lavage fluid (BALF) from children undergoing diagnostic bronchoscopy for structural abnormalities (control group, n = 24), asthma (n = 18), lung infection (n = 30), and cystic fibrosis (CF, n = 15) showed that BALF phospholipid concentration decreased with age for the control group and was elevated in all disease groups. The fractional concentration of the major surface active component, dipalmitoyl phosphatidylcholine (PC16:0/16:0), correlated (r(2) = 0.608, P < 0.01) with airway resistance (FEV(1%) predicted), and decreased PC16:0/16:0 was accompanied by increased concentrations of phospholipid components characteristic of cell membranes (PC16:0/18:1 and PI18:0/20:4). Median minimal surface tension, measured by pulsating bubble surfactometer, was elevated (P < 0.01) in both infection (17.5 mN/m) and CF (17.1 mN/m) compared with the control group (1.5 mN/m). Centrifugation (60,000 x g, 40 min) of BALF indicated that infection was accompanied by accumulation of large aggregate forms of surfactant, in contrast to previous reports of increased conversion to inactive small aggregate surfactant particles in ventilated patients with respiratory failure. This accumulation of surface-inactive, large aggregate forms of surfactant, possibly due to mixing with membrane material from inflammatory cells, may contribute to severity of lung disease in children with respiratory infections.