Vaccine against gastrin, a polyclonal antibody stimulator, decreases pancreatic cancer metastases.

Growth of pancreatic cancer is stimulated by gastrin in both a paracrine and an autocrine fashion. Traditional therapies have not significantly improved survival, and recently pancreatic cancer has been deemed a "cold" tumor due to its poor response to immunotherapy. Strategies to improve survival of pancreatic cancer are desperately needed. In the current investigation, we studied the effects of an anti-gastrin cancer vaccine, polyclonal antibody stimulator (PAS; formerly called G17DT and Gastrimmune), used alone or in combination with a programmed cell death receptor (PD)-1 immune checkpoint antibody on pancreatic cancer growth, metastases, and the tumor microenvironment (TME). Immune-competent female C57BL/6 mice bearing syngeneic orthotopic murine pancreatic cancer treated with PAS had significantly smaller tumors and fewer metastases. Examination of the TME demonstrated decreased fibrosis with fewer M2 and more M1 tumor-associated macrophages. Expression of the E-cadherin gene was significantly increased and expression of the TGFßR2 gene was decreased compared with controls. Mice treated with PAS or the combination of PAS and PD-1 antibody exhibited significantly less tumor expression of phospho-paxillin, the focal adhesion protein ß-catenin, and matrix metalloproteinase-7. This study suggests that inhibition of the cancer-promoting effects of gastrin in pancreatic cancer can decrease metastases by altering the TME and decreasing pathways that activate the epithelial mesenchymal transition. The PAS vaccine appears to change the TME, making it more susceptible to therapy with an immune checkpoint antibody. This novel combination of two immunotherapies may improve survival of pancreatic cancer by decreasing both tumor growth and metastasis formation.NEW & NOTEWORTHY Survival from advanced pancreatic cancer is poor, in part due to dense fibrosis of the tumor microenvironment, increased number of M2-polarized macrophages that promote angiogenesis and invasion, and lack of "target-specific" therapy. Herein, we report that a tumor vaccine that selectively targets gastrin decreases pancreatic cancer growth and metastases. Furthermore, the gastrin vaccine polyclonal antibody stimulator alters the tumor microenvironment rendering it more responsive to immunotherapy with a programmed cell death receptor-1 immune checkpoint antibody.

Gastrin vaccine improves response to immune checkpoint antibody in murine pancreatic cancer by altering the tumor microenvironment.

Pancreatic cancer has been termed a 'recalcitrant cancer' due to its relative resistance to chemotherapy and immunotherapy. This resistance is thought to be due in part to the dense fibrotic tumor microenvironment and lack of tumor infiltrating CD8 + T cells. The gastrointestinal peptide, gastrin, has been shown to stimulate growth of pancreatic cancer by both a paracrine and autocrine mechanism. Interruption of gastrin at the CCK receptor may reduce tumor-associated fibrosis and alter tumor immune cells. Polyclonal Ab Stimulator (PAS) is a vaccine that targets gastrin and has been shown to prolong survival of patients with pancreatic cancer. Here, we report that PAS vaccination monotherapy elicits both a humoral and cellular immune response when used in immune competent mice-bearing pancreatic tumors and that PAS monotherapy produced a marked T-cell activation and influx of CD8 + lymphocytes into pancreatic tumors. Isolated peripheral lymphocytes elicited cytokine release upon re-stimulation with gastrin in vitro demonstrating specificity of immune activation for the target peptide. Combination therapy with PAS and PD-1 Ab activated CD4 -/CD8 - TEMRA cells important in T-cell-mediated tumor death and memory. Tumors of mice treated with PAS (250 µg) or PAS (100 and 250 µg) in combination with a PD-1 Ab were significantly smaller compared to tumors from PBS or PD-1 Ab-treated mice. When PAS was given in combination with PD-1 Ab, tumors had less fibrosis, fewer inhibitory Treg lymphocytes, and fewer tumor-associated macrophages. These findings reveal a novel approach to improve treatment strategies for pancreatic cancer.

Pitfalls in diagnostic gastrin measurements.

BACKGROUND: Gastrin measurements are performed primarily for the diagnosis of gastrin-producing tumors, gastrinomas, which cause the Zollinger-Ellison syndrome (ZES). Gastrin circulates as several bioactive peptides, however, and the peptide pattern in gastrinoma patients often deviates from normal. Therefore, it is necessary to measure all forms of gastrin. CONTENT: Only immunoassays are useful for measurement of gastrin in plasma. The original assays were RIAs developed in research laboratories that used antibodies directed against the C terminus of gastrin peptides. Because the C-terminal tetrapeptide amide sequence constitutes the active site of gastrin peptides, these assays were well suited for gastrinoma diagnosis. More recently, however, most clinical chemistry laboratories have switched to commercial kits. Because of recent cases of kit-measured normogastrinemia in patients with ZES symptoms, the diagnostic sensitivity and analytical specificity of the available kits have been examined. The results show that gastrin kits frequently measure falsely low concentrations because they measure only a single gastrin form. Falsely high concentrations were also encountered, owing to overreactivity with O-sulfated gastrins or plasma proteins. Thus, more than half of the gastrin kits on the market are unsuited for diagnostics. SUMMARY: Gastrinomas are neuroendocrine tumors, some of which become malignant. A delay in diagnosis leads to fulminant ZES, with major, even lethal, complications. Consequently, it is necessary that the diagnostic sensitivity of gastrin kits be adequate. This diagnostic sensitivity requires antibodies that bind the C-terminal epitope of bioactive gastrins without the influence of O-sulfation.

Clinical usefulness of the serological gastric biopsy for the diagnosis of chronic autoimmune gastritis.

AIM: To assess the predictive value for chronic autoimmune gastritis (AIG) of the combined assay of anti-parietal-cell antibodies (PCA), anti-intrinsic-factor antibodies (IFA), anti-Helicobacter pylori (Hp) antibodies, and measurement of blood gastrin. METHODS: We studied 181 consecutive patients with anemia, due to iron deficiency resistant to oral replacement therapy or to vitamin B12 deficiency. RESULTS: 83 patients (45.8%) tested positive for PCA and underwent gastroscopy with multiple gastric biopsies. On the basis of the histological diagnosis, PCA-positive patients were divided into 4 groups: (1) 30 patients with chronic atrophic gastritis; they had high concentrations of PCA and gastrin and no detectable IFA; (2) 14 subjects with metaplastic gastric atrophy; they had high PCA, IFA, and gastrin; (3) 18 patients with nonspecific lymphocytic inflammation with increased PCA, normal gastrin levels, and absence of IFA; (4) 21 patients with multifocal atrophic gastritis with "borderline" PCA, normal gastrin, absence of IFA and presence of anti-Hp in 100% of the cases. CONCLUSIONS: The assay of four serological markers proved particularly effective in the diagnostic classification of gastritis and highly correlated with the histological profile. As such, this laboratory diagnostic profile may be considered an authentic "serological biopsy."

Supersensitive gastrin assay using antibodies raised against a cholecystokinin homolog.

Peptide hormones may occur in particularly low amounts in samples from small animals. Hence, in a rat microdialysis study conventional immunoassays were not sufficiently sensitive to measure gastrin in the dialysis samples. We therefore exploited the observation that antibodies raised against the homologous hormone cholecystokinin (CCK) occasionally bind gastrin peptides with significantly higher affinity than the proper ligand. The immunoassay thus established could detect 1.0 pmol/l in 15 µl microdialysate, which corresponds to 23 attomol gastrin. Such detection limit is five-fold lower than that obtained with the most avid conventional gastrin antibodies. The results may encourage similar approaches for other peptides using homologue-raised antibodies when supersensitivity is required.

Immunohistochemical identification and localisation of gastrin and somatostatin in endocrine cells of human pyloric gastric mucosa.

The detailed description of the distribution of endocrine cells G and D producing important hormones that regulate activation of other cells in the human stomach may be a valuable source of information for opinions about mucosa changes in different diseases of the alimentary tract. The density and distribution of immunoreactive G and D cells in the pylorus of humans (donors of organs) were evaluated. The pylorus samples were collected after other organs were harvested for transplantation. The number of G cells in the pyloric mucosa of healthy people was higher than the number of D cells. G and D cells were distributed between columnar cells of epithelium mucosa. Multiform endocrine cells generally occurred: gastrin in the middle third of the mucosa and somatostatin cells in the basal half of the pyloric mucosa. The investigation of the pyloric part of the healthy human stomach showed a characteristic distribution of cells that reacted with antisera against gastrin and somatostatin.

Determination of trace gastrin and diagnosis of human diseases using CdTe quantum dots labelled gastrin antibodies as phosphorescence sensors.

CdTe quantum dots (CdTe-QDs) can emit strong and stable room temperature phosphorescence (RTP) via the perturbation effect of a Pb(2+) ion on the surface of a nitrocellulose membrane (NCM). CdTe-QDs-Ab(GAS), the product of CdTe-QDs labelled gastrin antibodies (Ab(GAS)), can not only maintain good RTP characteristics, but can also be used as a RTP sensor and carry out highly specific immunoreactions with gastrin (GAS) to form GAS-Ab(GAS)-CdTe-QDs causing the ΔI(p) of the system to sharply enhance. Thus, a new solid substrate room temperature phosphorescence immunoassay (SSRTPIA) for the determination of GAS was established based on the linear relativity between the ΔI(p) of the system and the content of GAS. The limit of quantification (LOQ) of this method was 0.43 fg spot(-1) with the corresponding concentration being 1.1 × 10(-12) g mL(-1) and sampling quantity being 0.40 per spot(-1). This highly specific, accurate, selective and sensitive RTP sensor has been applied to the determination of GAS in biological samples and the diagnosis of diseases, and the results agreed well with those obtained by radioimmunometric assay (RIA). Meanwhile, the mechanism of SSRTPIA for the determination of GAS using CdTe-QDs-Ab(GAS) as the RTP sensor was discussed.

Affinity maturation of antibodies assisted by in silico modeling.

Rational engineering methods can be applied with reasonable success to optimize physicochemical characteristics of proteins, in particular, antibodies. Here, we describe a combined CDR3 walking randomization and rational design-based approach to enhance the affinity of the human anti-gastrin TA4 scFv. The application of this methodology to TA4 scFv, displaying only a weak overall affinity for gastrin17 (K(D) = 6 microM), resulted in a set of nine affinity-matured scFv variants with near-nanomolar affinity (K(D) = 13.2 nM for scFv TA4.112). First, CDR-H3 and CDR-L3 randomization resulted in three scFvs with an overall affinity improvement of 15- to 35-fold over the parental. Then, the modeling of two scFv constructs selected from the previous step (TA4.11 and TA4.13) was followed by a combination of manual and molecular dynamics-based docking of gastrin17 into the respective binding sites, analysis of apparent packing defects, and selection of residues for mutagenesis through phage display. Nine scFv mutants were obtained from the second maturation step. A final 454-fold improvement in affinity compared with TA4 was obtained. These scFvs showed an enhanced potency to inhibit gastrin-induced proliferation in Colo 320 WT and BxPc3 tumoral cells. In conclusion, we propose a structure-based rational method to accelerate the development of affinity-matured antibody constructs with enhanced potential for therapeutic use.

[Clinical value of serum gastrin determination (a review of literature)].

The generalization of the results of a considerable body of work on the use of serum gastrin permits one to conclude that only its properties to stimulate gastric mucosal hydrochloric acid and pepsin secretion in health and disease have been rather well studied. Single studies are devoted to changes in serum gastrin levels in diseases, including infection pathology, of the body's other organs and systems other than the gastrointestinal tract. The data available in the literature show that serum gastrin levels are directly related to humoral immunity parameters. It is concluded that determination of serum gastrin levels is an informative test of not only the gastrointestinal tract of a human being, but also his/her whole health, which makes it possible to predict, in case of illness, its exacerbation, outcomes and to timely correct therapy.

Development of a time-resolved fluorescence immunoassay based on immunomagnetic beads for gastrin-17.

OBJECTIVE: In this study, a novel, simple, and rapid immunoassay for the determination of gastrin-17 (G-17) in human serum was established by combining immunomagnetic beads with time-resolved fluorescence immunoassay (TRFIA). METHODS: Immunomagnetic beads were coated with anti-G-17 M01 antibody, anti-G-17 M02 antibody was labeled with Eu3+ chelates. The concentration of G-17 in the serum was detected with the double-antibody sandwich method. RESULTS: The limit of background(LOB), limit of detection (LOD), and limit of quantification (LOQ) were 0.09, 0.104, and 0.39 pmol/L, respectively. The detection range of G-17-TRFIA was 0.39-100 pmol/L. The average intra- and inter-assay coefficients of variation (CV) were 5.95%-9.07% and 6.09%-8.14%, respectively. The recoveries for the serum samples ranged from 94.70% to 100.95%. The specificity of our G-17-TRFIA was acceptable. The correlation coefficient between G-17-TRFIA and commercial G-17-ELISA methods was R2 = 0.9092. CONCLUSIONS: A novel G-17-TRFIA detection method was successfully established to provide a reference for the early diagnosis of patients with atrophic gastritis in clinical research.

Binding of CDR-derived peptides is mechanistically different from that of high-affinity parental antibodies.

We present data that reveal crucial differences between the binding mode of anti-gastrin17 (G17, pyroEGPWLEEEEEAYGWMDF-NH(2)) monoclonal antibodies (mAbs) and their CDR-derived synthetic binders (SBs) with G17. The mAbs recognize the N-terminal sequence of G17 (pyroEGPWL) with nanomolar affinity and high sequence selectivity. Molecular simulations suggest that G17 recognition is based primarily on a multitude of weak antibody-ligand interactions (H-bonding, van der Waals, etc.) inside a structurally well-defined cleft-like binding pocket. Relatively small structural changes (e.g. G-2 to A for G17) have a drastic impact on affinity, which is characteristic for antibody-like binding. In contrast, SBs recognize various sequences, including G17-unrelated targets with affinities of 1:1 complexes estimated in the 0.1-1.0 mM range. In most cases however, the G17/SB complex stoichiometries are not well-defined, giving rise to multimer aggregate formation with high apparent complex stabilities. Mutational studies on both G17 and SBs reveal the importance of positively charged (K/R) and aromatic residues (W/Y/F) for G17/SB complex formation. We propose that the synthetic binders use combinations of electrostatic, hydrophobic, and/or cation-π interactions in a variety of ways due to their intrinsic flexibility. This may also be the reason for their relatively low target specificity. We speculate that our findings are of general relevance, in showing that high-affinity mAbs do not necessarily provide the optimal basis for functional mimics design.

[Clinical and laboratory evaluation of efficiency of Helicobacter pylori eradication].

AIM: To assess the efficiency of eradication therapy in long-term period using the main signs of functional activity of gastric mucosa (gastrin-17, pepsinogen I, pepsinogen II) and serum antibodies to H. pylori. MATERIALS AND METHODS: 113 patients with chronic gastritis were examihed using clinical, endoscopic and laboratory-based methods of investigation, including GastroPanel Biohit, Finland. RESULTS: It was observed that after 12 month of successful eradication therapy the titer of IgG to H. pylori did not exceed 60 IU/l, with pepsinogen I and pepsinogen II cut-off values set under 150 microg/l and 15 microg/l respectively. CONCLUSION: The decrease of the titer of IgG to H. pylori and concentrations of pepsinogen I and II can be used as criteria of successful eradication therapy in long-term period.

Molluscan gastrin: concentration and molecular forms.

Blood and gastrointestinal tissues of the sea hare Aplysia californica and the land snail Otala lactea contain immunoreactive gastrin in heterogeneous forms similar to those of mammals. The observation that blood concentrations in terms of porcine gastrin standard are comparable to those of pig, man, and dog suggests significant homology between the structures of molluscan and mammalian gastrins.

The use of clinical, histologic, and serologic parameters to predict the intragastric extent of intestinal metaplasia: a recommendation for routine practice.

BACKGROUND: Surveillance of intestinal metaplasia (IM) of the gastric mucosa should be limited to patients at high risk of gastric cancer. Patients with extensive IM are at increased cancer risk; however, the intragastric extent of IM is usually unknown at the time of the initial diagnosis. OBJECTIVE: To assess the predictive value of clinical, histologic, and serologic parameters for the intragastric extent of IM. DESIGN AND SETTING: Prospective, multicenter study. PATIENTS: Eighty-eight patients with a previous diagnosis of IM of the gastric mucosa. INTERVENTION: Surveillance gastroscopy with extensive random biopsy sampling. MAIN OUTCOME MEASUREMENTS: Biopsy specimens were evaluated according to the Sydney classification system. In addition, serologic testing of Helicobacter pylori and cagA status, pepsinogens I and II, gastrin, and intrinsic factor antibodies was performed. The association between the available parameters and extensive IM was evaluated with logistic regression analysis. RESULTS: In 51 patients (58%), IM was present in the biopsy specimens from at least 2 intragastric locations. The most important predictors of extensive IM were a family history of gastric cancer, alcohol use > or = 1 unit/d (1 glass, approximately 10 mL or 8 g ethanol), moderate or marked IM of the index biopsy specimen, and a pepsinogen I to II ratio < 3.0. A simple risk score based on these factors could identify extensive IM in 24 of 25 patients (sensitivity 96%). LIMITATION: A prospective cohort study should confirm the proposed risk stratification. CONCLUSIONS: A risk score of clinical, histologic, and serologic parameters can predict extensive intragastric IM and may serve as a practical tool to select patients for surveillance endoscopy in routine clinical practice.

Antiulcer activity of preparations containing ultralow doses of antibodies in modeled chronic ulcer in rats.

Screening of three potential antiulcer preparations containing ultralow doses of antibodies to endogenous regulators of ulcer formation (gastrin, histamine, and H2 histamine receptors) on the model of acetic acid-induced gastric ulcer in rats revealed pronounced antiulcer effect of ultralow doses of antibodies to histamine. The dynamics of regeneration of the ulcer focus by morphological and histological characteristics was similar during treatment with ultralow doses of antibodies to histamine and with famotidine.

Validation of antibody-based assays for regulatory peptides: Do it once, get it right, and exploit the under-appreciated benefit of long-term antibody stability.

Many submitted manuscripts utilizing antibody-based assays for biologically active peptides frequently neither include nor cite adequate validation data with the risk that the report is adding to the reproducibility crisis in biological research. On the basis of recent experience in re-characterizing in a radioimmunoassay format a polycolonal antibody to gastrin that was first raised nearly five decades ago, it is argued that some antibodies can be stable for very many decades. Researchers concerned about the reproducibility of data using antibodies in assays for regulatory peptides should therefore note that by rigorous validation at an early stage they may not only contribute to the resolution of the reproducibility crisis but also establish a resource that could be useful for very many years.

Designing antibodies for the inhibition of gastrin activity in tumoral cell lines.

Gastrin and its derivatives are becoming important targets for immunotherapy of pancreatic, gastric and colorectal tumors. This study was conducted to design antibodies able to block gastrin binding to the gastrin/cholecystokinin-2 (CCK-2) receptor in order to delay tumor growth. The authors have used different gastrin molecules, combined with the diphtheria toxoid, to generate and select human single chain variable fragments (scFvs) as well as mouse monoclonal antibodies and scFvs against different regions of gastrin. There was a remarkable conservation in the antibody repertoire against gastrin, independently of the approach and the species. The germlines most frequently used in gastrin antibody formation were identified. Three different epitopes were identified in the gastrin molecule. The resulting mouse monoclonal antibodies and scFvs were analyzed for gastrin neutralization using Colo 320 WT cells, which overexpress the CCK-2 receptor. The gastrin neutralizing activity assay showed that N-terminal specific mouse monoclonal antibodies were more efficient to inhibit proliferation of Colo 320 WT cells than the anti-C terminal antibodies. Moreover, the human antigastrin scFvs obtained in this study inhibited significantly the proliferation of Colo 320 tumoral cells. These findings should contribute to a more rational design of antibody-based antigastrin therapies in cancer, including passive administration of human antibodies with blocking activity.

Functional nuclear medicine imaging of medullary thyroid cancer.

Medullary thyroid cancer (MTC) originates from parafollicular C cells of the thyroid and accounts for 3-12% of all thyroid cancers. As opposed to other types of dedifferentiated thyroid tumours, MTC cells are highly functional, producing and secreting high amounts of calcitonin and carcinoembryonic antigen. As parafollicular C cells are of neural crest origin, MTC acts as a neuroendocrine tumour also and expresses somatostatin receptors. Although conventional radiological methods such as ultrasonography, computed tomography and magnetic resonance imaging are widely used in the primary diagnosis and staging, they often fail to localize the residual or recurrent disease because the majority of MTC recurrence presents as occult disease. Thus, owing to functional characteristics of MTC, functional imaging modalities of nuclear medicine play a major role in the diagnostic and therapeutic strategies for MTC. Among nuclear medicine modalities, Tc(V) -dimercaptosuccinic acid, In-octreotide and I/I-meta-iodobenzylguanidine are commonly used in the diagnostic and even more in postoperative work-up of MTC. Alternatively, F-fluorodeoxyglucose and other positron emission tomography radiopharmaceuticals such as F-fluorodopa or F-fluorodopamine as well as radiolabelled antibodies such as Tc/I/I anticarcinoembryonic antigen, antigastrin, and anticholecystokinin-B have promising results. Functional imaging has a great advantage for nuclear medicine techniques in the routine work-up of MTC patients and also has a wide use in experimental studies.

Phage display selection of fully human antibody fragments to inhibit growth-promoting effects of glycine-extended gastrin 17 on human colorectal cancer cells.

Glycine-extended gastrin 17 (G17-Gly), a dominant processing intermediate of gastrin gene, has been implicated in the development or maintenance of colorectal cancers (CRCs). Hence, neutralizing G17-Gly activity by antibody entities can provide a potential therapeutic strategy in the patients with CRCs. To this end, we isolated fully human antibody fragments from a phage antibody library through biopanning against different epitopes of G17-Gly in order to obtain the highest possible antibody diversity. ELISA screening and sequence analysis identified 2 scFvs and 4 VL antibody fragments. Kinetic analysis of the antibody fragments by SPR revealed KD values to be in the nanomolar range (87.9-334 nM). The selected anti-G17-Gly antibody fragments were analyzed for growth inhibition and apoptotic assays in a CRC cell line, HCT-116, which is well-characterized for expressing gastrin intermediate species but not amidated gastrin. The antibody fragments exhibited significant inhibition of HCT-116 cells proliferation ranging from 36.5 to 73% of controls. Further, Annexin V/PI staining indicated that apoptosis rates of scFv H8 and VL G8 treated cells were 45.8 and 63%, respectively. Based on these results, we for the first time, demonstrated the isolation of anti-G17-Gly human scFv and VL antibodies with potential therapeutic applications in G17-Gly-responsive tumors.

Immunogenicity and safety of a novel tetanus toxoid-conjugated anti-gastrin vaccine in BALB/c mice.

The objective of this study is to determine the immunogenicity and safety of our novel anti-gastrin vaccine that is composed of the common amino-terminal portions of human carboxy-amidated gastrin-17 (G17) and glycine-extended gastrin-17 (gly-G17) as well as the common carboxy-terminal portion of the gastrin precursor progastrin (in a 50:50 mixture) all covalently linked to tetanus toxoid (TT) via peptide spacers. The vaccine, or immunogen, was injected intramuscularly into the legs of BALB/c mice, which produced high serum titres of specific IgG antibodies and IFN-γ in their spleen cells, identifiable by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT), respectively. TT as the protein carrier effectively enhanced the antigenic epitopes' humoural and cellular immune responses, unlike the antigenic epitopes alone or the immunogen's adjuvant emulsion system (AES), all of which failed to provoke any obvious immune response. Notably, the animals' body weights increased significantly after immunization (P < .01), while their haematology and serum biochemistry were all generally normal, and the gross anatomy of their main organs (e.g., heart, liver, spleen, lung, kidney) showed no obvious histopathological changes.