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===Human-mouse chimeric monoclonal antibody(mAb) Ch-mAb7F9=== | ===Human-mouse chimeric monoclonal antibody(mAb) Ch-mAb7F9=== | ||
IgG mAbs are typically chimeric, humanized, or fully human proteins. The longest t1/2lamdaz values are usually achieved when the antibody does not bind to tissue sites and is not prematurely cleared due to antigenicity. | IgG mAbs are typically chimeric, humanized, or fully human proteins. The longest t1/2lamdaz values are usually achieved when the antibody does not bind to tissue sites and is not prematurely cleared due to antigenicity<ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use"/> | ||
Ch-mAb7F9, a chimeric mAb is produced as a treatment medication for METH abuse. In vitro, it is shown only binds to (+)METH (KD=6.9nM), (+)AMP( | . | ||
Ch-mAb7F9, a chimeric mAb is produced as a treatment medication for METH abuse based on the murine anti-METH mAb7F9<ref name="Pharmacological effects of two anti-methamphetamine monoclonal antibodies"/>. It is created by preserving mAb7f9 variable region with human IgG2 constant domains<ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use"/> to minimize the risk of effector function. In vitro, it is shown only binds to (+)METH (KD=6.9nM)<ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use"/> | |||
, (+)AMP(KI = 350 nM)<ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use"/> | |||
, (+)MDMA(kI=6.7nM)<ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use"/> | |||
. | |||
===Preclinical characterization of Ch-mAb7F9 for human use=== | ===Preclinical characterization of Ch-mAb7F9 for human use=== | ||
====Cross reactions in vitro ligand binding studies==== | |||
It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, result of the test that was conducted to determine the potential for complement activation, which is an undesired effector function, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. <ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use" /> | |||
Just as mAb7F9, ch-mAb counterpart does not bind any of these compounds except (+)AMP, (+)METH and (+)MDMA well enough to raise clinical effect and in vitro with only one interesting exception: (-)MDMA, which was capable of inhibiting [3H]METH binding at greater than 50% and it was the only ligand with a KI (ocncentration of inhibitor that prevents 50% of the [H3] from binding) less than 1 microMolar among other tested compounds. However, due to Ecstasy is a racemic mixture contains both (+) and (-) MDMA and the ability that mAb7F9 to bind both forms of MDMA may actually improve its utility as a potential treatment for MDMA abuse. | |||
<table><tr><td colspan='2'>Table 2 <ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use" />Ligands tested in Ch-mAb7F9 cross-reactivity study.<br></td></tr> | |||
<tr id='Related stimulates'><td class="sblockLbl"><b>Related stimulates</b></td><td class="sblockDat"> | |||
(+)-Methamphetamine, | |||
(+)-Amphetamine, | |||
(+)-MDMA, | |||
(-)-MDMA, | |||
(+)-MDA | |||
</td></tr> | |||
<tr id='Neurotransmitters'> | |||
==== | <td class="sblockLbl"><b>neurotransmitters </b></td><td class="sblockDat"> | ||
Dopamine, | |||
== | (-)-Norepinephrine, | ||
(-)-Epinephrine, | |||
Serotonin, | |||
γ-aminobutyric acid, | |||
L-Glutamate | |||
</td></tr> | |||
<tr id='Medications'><td class="sblockLbl"><b>Medications </b></td><td class="sblockDat"> | |||
(+)-Pseudoephedrine, | |||
(+)-Norpseudoephedrine, | |||
(-)-Phenylephrine, | |||
(±)-Ephedrine, | |||
2-Phenylethylamine, | |||
Tyramine, | |||
</td></tr> | |||
<tr id='Drugs of abuse'><td class="sblockLbl"><b>Drugs of abuse </b></td><td class="sblockDat"> | |||
Cocaine, | |||
Morphine, | |||
Phencyclidine | |||
</td></tr> | |||
</table> | </table> | ||
====Isothermal titration calorimetry potency studies==== | |||
Isothermal titration calorimetry analysis of ch-mAb7F9 binding to METH provided thermodynamic and stoichiometry measurements for its potency.<ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use" /> | |||
<table><tr><td colspan='2'> Antibody thermodynamic values and stoichiometry for target binding<ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use" />.<br></td></tr> | |||
<tr id='Antibody'><td class="sblockLbl"><b>Antibody </b></td><td class="sblockDat"> Ch-mAb7F9</td></tr> | |||
<tr id='Delta G'><td class="sblockLbl"><b>ΔG (kJ/mol) </b></td><td class="sblockDat"> -43 </td></tr> | |||
<tr id='Delta H'><td class="sblockLbl"><b>ΔH (kJ/mol) </b></td><td class="sblockDat"> -45 </td></tr> | |||
<tr id='-TdeltaS'><td class="sblockLbl"><b>-TΔS (kJ/mol) </b></td><td class="sblockDat">1 </td></tr> | |||
<tr id='Stoichiometry(N)'><td class="sblockLbl"><b>Stoichiometry </b></td><td class="sblockDat">1.89 </td></tr> | |||
</table> | |||
The gibbs free energy change is very similar to other antibodies reported in study. Like others, binding is mostly driven by a favorable enthalpy change that compensates for an almost nonexistent entropy penalty. The stoichiometry is expected to be 2 binding sites per antibody molecule and it is almost completely active. | |||
====Pharmacokinetics studies in rats==== | ====Pharmacokinetics studies in rats==== | ||
METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition. <ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use" /> | METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition. Both in vitro and in vivo demonstrated ch-mAb7F9 is pharmacologically similar to its murine counter part<ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use" />. Ch-mAb decreased the METH Vd by 5 and 25 fold at the 15 and 150mg/kg doses. Although METH t1/2lamdaz is increased 2 to 5 folds due to the decreased ClT to a greater degree, METH elimination was still rapid compared to ch-mAb elimination. (2-7 hrs v.s 10-13 d). | ||
In the current studies, a t1/2lamdaZ of 10-13 days for ch-mAb7F9 in rats was observed. The half life is predicted to be 3 weeks in human roughly 3 folds of that of rat due to the volume of distribution at steady state (Vdss) of IgG in both species is similar yet the clearance time (Clt) in humans is one third of that in rats. | |||
== Human Study == | == Human Study == | ||
===Phase 1 Study: First Human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers=== | ===Phase 1 Study: First Human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers=== | ||
The first study is conducted in healthy 42 volunteers, 10s of which received saline placebo as control group.<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>. Single, escalating doses of ch-mAb7F9 over the range of 0.2 to 20mg/kg (5 dose groups) were administered and followed for 147 d for pharmacokinetic and immugenicity studies<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>. No serious adverse reactions or discontinuations form the study due to adverse events<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>. No trends emerged of adverse events<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>. Half life of 17-19 d in the 3 highest does groups an volume of distribution of 5-6L suggesting antibody is confined primarily to the vascular compartment<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>. | |||
Serum ch-mAb7F9 concentration is plotted and reported for 147 d<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>. | |||
Most common AE include: increased blood creatine phosphokinase, upper respiratory tract infection, decreased hemoglobin, headache, increased aspartate aminotransferase and alanine aminotransferase, proteinuria, decreased white blood cell count, and nasal congestion<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>. AEs considered by the investigator to be related to study medication were limited to single events in the 2mg/kg group <ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>and included infusion reaction, bronchospasm, and proteinuria<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>. The 3 Grade 4 AEs(life-threatening) were all elevations in blood creatine phosphokinases levels and were considered unrelated to the ch-mAb and resolved without treatment. 6 events as Grade 3 and 47 grade 2 and 160 events as grade 1 (mild)<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>. | |||
Four(12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study (147 d)<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers"/>;however, this response did not appear to be dose related.<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers " /> | |||
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After METH infusion 3.2mg/kg/day<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> by osmotic minipumps which resulted average steady state serum concentration of 25ng/ml after 24h<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, mice were administered scFv6H4 which has led to drastic increase of serum concentration of METH<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, determined by liquid chromatography-tandem mass spectrometry as described previously<ref name ="Development of a liquid chromatography-tandem mass spectrometric method for the determination of methamphetamine and amphetamine using small volumes of rat serum"/>. The compare of the first 480 min METH concentration with control group was reported<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. scFv6H4 was reported stable in serum in vitro yet unstable in Urine in vitro<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. The concentration of scFv in serum in vivo was determined by SEC<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, the method has been previously described <ref name ="Pharmacokinetic mechanisms for obtaining high renal coelimination of phencyclidine and a monoclonal antiphencyclidine antigen-binding fragment of immunoglobulin G in the rat"/>. Monomer of scFv was reportd to have been completely eliminated in the serum within the first 30 minutes<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, yet the multivalent larger proteins persisted for >240 mins corresponding to the reported t1/2lamdaz to be 228 +/- 38 min<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. 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<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. | After METH infusion 3.2mg/kg/day<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> by osmotic minipumps which resulted average steady state serum concentration of 25ng/ml after 24h<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, mice were administered scFv6H4 which has led to drastic increase of serum concentration of METH<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, determined by liquid chromatography-tandem mass spectrometry as described previously<ref name ="Development of a liquid chromatography-tandem mass spectrometric method for the determination of methamphetamine and amphetamine using small volumes of rat serum"/>. The compare of the first 480 min METH concentration with control group was reported<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. scFv6H4 was reported stable in serum in vitro yet unstable in Urine in vitro<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. The concentration of scFv in serum in vivo was determined by SEC<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, the method has been previously described <ref name ="Pharmacokinetic mechanisms for obtaining high renal coelimination of phencyclidine and a monoclonal antiphencyclidine antigen-binding fragment of immunoglobulin G in the rat"/>. Monomer of scFv was reportd to have been completely eliminated in the serum within the first 30 minutes<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, yet the multivalent larger proteins persisted for >240 mins corresponding to the reported t1/2lamdaz to be 228 +/- 38 min<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. 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<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. | ||
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|RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4lar FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4lar OCA], [http://pdbe.org/4lar PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=4lar RCSB], [http://www.ebi.ac.uk/pdbsum/4lar PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=4lar ProSAT]</span> | |RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4lar FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4lar OCA], [http://pdbe.org/4lar PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=4lar RCSB], [http://www.ebi.ac.uk/pdbsum/4lar PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=4lar ProSAT]</span> | ||
}} | }} | ||
{{ STRUCTURE | {{ STRUCTURE | ||
|PDB=3gkz | |PDB=3gkz | ||
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|RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4lar FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4lar OCA], [http://pdbe.org/4lar PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=4lar RCSB], [http://www.ebi.ac.uk/pdbsum/4lar PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=4lar ProSAT]</span> | |RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4lar FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4lar OCA], [http://pdbe.org/4lar PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=4lar RCSB], [http://www.ebi.ac.uk/pdbsum/4lar PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=4lar ProSAT]</span> | ||
}} | }} | ||
===Aromatic-Aromatic Interaction: A Mechanism of Protein Structure Stabilization=== | ===Aromatic-Aromatic Interaction: A Mechanism of Protein Structure Stabilization=== | ||
The entrance of the binding pocket is lined with seven amino residues, one residue from each of them H1, H2, and H3 loops, 3 from the L3 loop, one from the beta-strand-3c of the heavy chain. These aromatic residues form a hydrophobic barrel around the aromatic portion of METH. | The entrance of the binding pocket is lined with seven amino residues, one residue from each of them H1, H2, and H3 loops, 3 from the L3 loop, one from the beta-strand-3c of the heavy chain. These aromatic residues form a hydrophobic barrel around the aromatic portion of METH<ref name="Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-methamphetamine and 3,4-methylenedioxymethamphetamine"/>. | ||
===Hydrophilic interactions of METH=== | ===Hydrophilic interactions of METH=== | ||
The protonated secondary amine of METH anchors the ligand deep in the pocket. There is a salt bridge between the cationic nitrogen of METH and the carboxyl oxygen of Glutamate. In addition, the cationic nitrogen forms a hydrogen bond to Hisdine of the light chain<ref name="Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-methamphetamine and 3,4-methylenedioxymethamphetamine"/>. | |||
===Water molecules in the binding cavity=== | ===Water molecules in the binding cavity=== | ||
two water molecules are in the pocket stablized by hydrogen bonding bewteen and with the side chain residues<ref name="Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-methamphetamine and 3,4-methylenedioxymethamphetamine"/>. | |||
{{ STRUCTURE | {{ STRUCTURE | ||
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}} | }} | ||