Immunogenic compsns to CCK-b/gastrin-receptor and methods for treatment of tumors
An immunogenic, gastrin technology, applied in the direction of anti-animal/human immunoglobulin, hormone receptor, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. problems such as promoting tumor growth
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Embodiment 1
[0054] Preparation of GRP1-DT and GRP4-DT conjugates
[0055] CCK-B / gastrin receptor peptides were prepared using standard solid-phase peptide synthesis methods. In order for the immunogen to induce a specific immune response, peptide 1 and peptide 4 were synthesized with the spacer sequence SSPPPPC (SEQ ID NO: 3 in the sequence listing) at the carboxy terminus. Subsequently, these are linked via the amino acid residue cysteine at the end of the spacer sequence using a heterobifunctional linker with a succinimidyl ester at one end and a maleimide at the other, using method A or method B below. The peptide is coupled to the amino group on the carrier (diphtheria toxoid (DT)).
[0056] Method A: As previously described in US Patent No. 5,023,077, attachment of peptide 1 or 4 to the carrier was achieved as follows. Dried peptides were dissolved in 0.1 M sodium phosphate buffer (pH 8.0) and a 30-fold molar excess of dithiothreitol (DTT). The solution was stirred for 4 hours u...
Embodiment 2
[0075] The following experiments were performed to establish the specificity of the antibody against the Ser spacer-containing GRP1-DT peptide prepared by method B in Example 1 in rabbits. A series of tests were performed to evaluate the specificity of rabbit antibodies induced by GRP1-DT immunization and affinity purified by GRP1-Ser Sepharose column immunoadsorption.
[0076] The GRP1-Ser peptide specificity of the affinity purified antibodies was evaluated by inhibition ELISA. The experiment was carried out as follows: 50 μl of 2 μg / ml conjugate solution (glycine buffer solution, 0.1M, pH=9.5) was incubated overnight at 4°C to coat the GRP1-Ser-BSA conjugate in 96 wells plate (Immulon U bottom). Affinity purified anti-GRP1 antibodies (10 ng / ml final concentration) were combined with different inhibitors (serial 1:10 dilutions) and incubated for 1 hour at room temperature. Inhibitors included GRP1-Ser, GRP1 EPT, Ser, human gastrin 17(1-9)-Ser spacer (hG17(9)-Ser), GRP1 EPT+S...
Embodiment 3
[0079] AR42J tumor cells (European Collection of Animal Cells, Porton Down, UK) were obtained from rat pancreatic carcinoma, which are clearly known to have CCK-B / gastrin receptors. Therefore, AR42J was tested to confirm the expression of the CCK-B / gastrin receptor and the specificity of the radioligand-inhibited hG17 receptor. AR42J cells in 7% CO in complete RPMI1640 (Sigma) 2 Incubated at 37°C in RPMI supplemented with 10% FCS (Gemini Bioproducts), 2 mM glutamine (JRH Biosciences), 1 mM sodium pyruvate (JRH B.) and 50 μg / ml gentamycin (Gemini Bioproducts). with 0.25% EDTA in PBS from 175 cm 2 Cells were harvested in T-flasks, washed twice with PBS (without EDTA), and centrifuged (400Xg for 10 minutes). Cells were maintained at 0-4°C for all manipulations. Prepare a single cell suspension with buffer and adjust the cell concentration to 10 6 cells / ml. Aliquot 1 ml of the cell suspension into 12 x 75 mm culture tubes, then centrifuge the cells and discard the supernatant...
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