Sandbox Reserved 1474: Difference between revisions
No edit summary |
No edit summary |
||
| Line 33: | Line 33: | ||
Because drugs of abuse are too small to generate an immune response on their own, a critical step toward making an effective drug-specific vaccine is to synthesize a hapten that maintains the chemical and structural properties of the original drug and has an added, carefully placed chemical linker with a distal moiety of a functional group that can be easily conjugated to a larger carrier antigenic protein.<ref name="Designing immunotherapies to thwart drug abuse"/>. The complete synthesis (+)-METH P6 was reported 2001<ref name="Generation of anti-(+)methamphetamine antibodies is not impeded by (+)methamphetamine administration during active immunization of rats"/> | Because drugs of abuse are too small to generate an immune response on their own, a critical step toward making an effective drug-specific vaccine is to synthesize a hapten that maintains the chemical and structural properties of the original drug and has an added, carefully placed chemical linker with a distal moiety of a functional group that can be easily conjugated to a larger carrier antigenic protein.<ref name="Designing immunotherapies to thwart drug abuse"/>. The complete synthesis (+)-METH P6 was reported 2001<ref name="Generation of anti-(+)methamphetamine antibodies is not impeded by (+)methamphetamine administration during active immunization of rats"/> | ||
. The chemical structures of other stereospecific (+) haptens were reported 2007<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. | . The chemical structures of other stereospecific (+) haptens were reported 2007<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. | ||
It was reported that 1)linkers located distal to the chiral center of the small drug molecule favors generation of stereospecific antibodies 2)longer flexible linker broadens recognition of meth like molecules 3) spacers equal to or greater than six atoms produce higher affinity mAb<>. | |||
| Line 43: | Line 46: | ||
===Immunization, Screening, and Hybridoma Generation=== | ===Immunization, Screening, and Hybridoma Generation=== | ||
Initial subcutaneous immunization of 100 micrograms of the (+)-METH P6 antigen emulsified with adjuvant was followed with monthly boosts of 50 microgram dosage. For all other antigen immunizations, dosage and boost intervals have also been reported<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. Serum samples were taken via tail bleed periodically to measure IgG titers by enzyme-linked immunosorbent essay (ELISA)<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. Wells were coated with the original hapten that conjugated to a different protein to avoid selecting carrier protein-reactive antibodies<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. The mouse with the highest anti-METH serum titer was chosen for monoclonal antibody production<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>.(cell line <ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>). After fusion of mouse B-cells with a myeloma cell line, hybridomas which has both the antibody-producing ability of the mouse B-cell and exaggerated longevity and reproductivity of the fusion partner myeloma were identified by ELISA and sub cloned to monoclonality<ref name ="Anti-phencyclidine monoclonal Fab fragments markedly alter phencyclidine pharmacokinetics in rats"/>. To generate large amounts of monoclonal antibody, mice were injected with hybridoma cells and ascites fluid that contained high concentration of IgG was collected after next several weeks<ref name ="Anti-phencyclidine monoclonal Fab fragments markedly alter phencyclidine pharmacokinetics in rats"/>. | Initial subcutaneous immunization of 100 micrograms of the (+)-METH P6 antigen emulsified with TiterMax adjuvant was followed with monthly boosts of 50 microgram dosage. For all other antigen immunizations, Freund's adjuvants were used<> and dosage and boost intervals have also been reported<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. Serum samples were taken via tail bleed periodically to measure IgG titers by enzyme-linked immunosorbent essay (ELISA)<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. Wells were coated with the original hapten that conjugated to a different protein to avoid selecting carrier protein-reactive antibodies<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. The mouse with the highest anti-METH serum titer was chosen for monoclonal antibody production<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>.(cell line <ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>). After fusion of mouse B-cells with a myeloma cell line, hybridomas which has both the antibody-producing ability of the mouse B-cell and exaggerated longevity and reproductivity of the fusion partner myeloma were identified by ELISA and sub cloned to monoclonality<ref name ="Anti-phencyclidine monoclonal Fab fragments markedly alter phencyclidine pharmacokinetics in rats"/>. To generate large amounts of monoclonal antibody, mice were injected with hybridoma cells and ascites fluid that contained high concentration of IgG was collected after next several weeks<ref name ="Anti-phencyclidine monoclonal Fab fragments markedly alter phencyclidine pharmacokinetics in rats"/>. | ||
| Line 61: | Line 64: | ||
====Radioimmunoassay determination of mAb6H4 cross-reactivity and dissociation constants==== | ====Radioimmunoassay determination of mAb6H4 cross-reactivity and dissociation constants==== | ||
| Line 82: | Line 83: | ||
==Clinical development== | ==Clinical development== | ||
===Pharmacological effects of two anti-methamphetamine monoclonal antibodies mAb4G9 and mAb7F9=== | ===Pharmacological effects of two anti-methamphetamine monoclonal antibodies mAb4G9 and mAb7F9=== | ||
After screening for more than 25000 potential hybridoma cell lines for mAb production, mAb with the most favorable immunochemical characteristics were extensively studied. Also the sequence features in each mAb variable regions were analyzed. A high degree of diversity in both compostition and length of CDRs are revealed. Although comparisons of CDR sequences are important, differences can be attributed to differences in germ-line sequences of particular V-region genes and to somatic mutation within the CDRs of these V-region genes. After analyzing sequence genes, each antibody was found unique and not clonal. That is, rather than coming from one germline gene arrangement early in B cell development, they resulted from unique V(D)J recombination events. Thus, no clear pattern of response was found. | |||
A common feature is a conserved proline at position 95 or 95a of all CDR L3 regions because of their ability to form 'hinges", except one case replaced with serine. And this residue was immediately followed by a hydrophobic amino acid or an aromatic residue which could be interacting with phenyl ring of (+)METH. | |||
mAb4G9, attributing to the design of hapten (+)METH MO10, was the only mAb to significantly cross-react with (+)AMP, in order to better understand its affinity for AMP, RIA analysis using [(+)-3H]AMP was conducted in addition to RIA analysis using [(+)-3H]METH. It was reported, the affinity for mAb4G9 for AMP is 51nM, demonstrating this molecule actually has the same KD value for (+)AMP and (+)METH. | |||
===Human-mouse chimeric monoclonal antibody(mAb) Ch-mAb7F9=== | ===Human-mouse chimeric monoclonal antibody(mAb) Ch-mAb7F9=== | ||
| Line 106: | Line 115: | ||
=Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine= | =Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine= | ||
==Materials and Methods== | ==Materials and Methods== | ||
=== | |||
===cDNA Cloning and sequencing of mAb=== | |||
The heavy and light chains cDNA of the mAb were cloned by reverse transcription-polymerase chain reaction, and then they were amplified and ligated into the cloning vector. The resulting plasmids of all mAb cloning was transformed into E. coli strain. All sequence submitted to GEN BANK. | |||
===Sub-cloning and Large-Scale Expression=== | ===Sub-cloning and Large-Scale Expression=== | ||
===Production and purification=== | ===Production and purification=== | ||