Sandbox Reserved 1474: Difference between revisions
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=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= | ||
when a short duration of action and greater extravascular penetration are needed, a significantly smaller fragment like Fab or scFv is used.() | when a short duration of action and greater extravascular penetration are needed, a significantly smaller fragment like Fab or scFv is used.() The genetic re-engineering of mAb6H4 IgG into scFv6H4 changed the protein from an ~150kDa protein with two anti-METH binding sites to an ~27.4kDa protein with one METH binding site achieved by a 15 amino acid linker to join the two separate gene product: VL and VH. A His6 affinity tag was encoded at the carboxyl terminus to aid in protein purification. | ||
==Materials and Methods== | ==Materials and Methods== | ||
===cDNA Cloning and sequencing of mAb=== | ===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 | 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 and then sequenced. All sequence submitted to GENBANK has been checked to assure integrity<>. | ||
===Sub-cloning and Large-Scale Expression=== | ===Sub-cloning and Large-Scale Expression=== | ||
the coding sequence of scFv6H4 was | After the sequence confirmation, the plasmid was linearized with SacI and the coding sequence of scFv6H4 was transformed to P.pastoris strain(yeast) by electroporation. It is expressed in yeast colony which exhibited high zeocin resistance. | ||
===Production and purification=== | ===Production and purification=== | ||
Large scale fermentation was done in batches in a 10 liter working volume biostatB bioreactor to express scFv6H4 in large scale for in vivo pharmacokinetic studies. ScFv was purified from the supernantant by metal affinity chromatography and metal affinity column in a naturally occurring mixture of monomer (~75%) amd dimer (~25%) analyzed with SEC. | |||
===Determination of Kd Values using bead-based radioimmunoassay=== | ===Determination of Kd Values using bead-based radioimmunoassay=== | ||
Changes have been made to RIA using TALON beads which binds to the His6 tag at the carboxyl terminus of the VL region of scFv6H4, which will orient the binding site of METH distal to the beads, allowing unhindered access to METH. Standard curve was constructed with [H3]METH competing with unlabeled METH. The KD was shown to be 10nM nearly identical to parent IgG mAb6H4 11nM. | |||
No cross reactivity with related compounds have been tested and reported. (pseudo-ephedrine, norepinephrine, dopamine, and serotonin), even at 100 microMolar. | |||
===Pharmacokinetic Studies of METH and scFv6H4 in Rats=== | ===Pharmacokinetic Studies of METH and scFv6H4 in Rats=== | ||
The very small molecular size(27k Da) of scFv monomers leads to rapid clearance (40) mins.scFv6H4 was engineered from mAb6H4. Monomer was reported to be quickly cleared or converted to multivalent forms with an apparent t1/2lamdaz of 5.8 min while multivalent forms showed a much longer t1/2lamdaz (228min). Multimers instead of monomers were considered the cause for the prolonged redistribution of METH into the serum. | After METH infusion 3.2mg/kg/day by osmotic minipumps which resulted average steady state serum concentration of 25ng/ml after 24h, mice were administered scFv6H4 which has led to drastic increase of serum concentration of METH. The compare of first 480 min METH concentration with control group was reported<>. scFv is unstable in vitro in Urine, stable in serum. The concentration of scFv in serum in vivo was determined by SEC, the method has been previously described<>. Monomer of scFv was reportd to have been completely eliminated in the serum within the first 30 minutes, yet the multivalent larger proteins persisted for >240 mins corresponding to the reported t1/2lamdaz to be 228 +/- 38min. It is interesting that it was reported the divalent form did not decrease for the first 10 minutes, as if while it is been eliminated, it is also been formed from the mono scFvs. Pharmacokinetic parameters were reported for mono and multi scFvs respectively<>. | ||
The very small molecular size(27k Da) of scFv monomers leads to rapid clearance (40) mins. scFv6H4 was engineered from mAb6H4. Monomer was reported to be quickly cleared or converted to multivalent forms with an apparent t1/2lamdaz of 5.8 min while multivalent forms showed a much longer t1/2lamdaz (228min). Multimers instead of monomers were considered the cause for the prolonged redistribution of METH into the serum. | |||
==Structural highlights from crystal structure of scFv6H4== | ==Structural highlights from crystal structure of scFv6H4== | ||
