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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<>.




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===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<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/> 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"/>.




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Screening for anti(+)METH IgG response was conducted by radioimmunoassay (RIA)<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/> in which radio labelled [(+)-3H] METH is competing with unlabelled (+)-METH and (+)-AMP for the binding site of Abs in a manner similar to what has been previously described<ref name ="Antibodies against arylcyclohexylamines and their similarities in binding specificity with the phencyclidine receptor"/>. An IC50 value was determined for each compound after fitting a sigmoidal curve to the data points<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/><ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>.
Anti-(+)METH mAb6H4 was reported with Kd = 11 NM <ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>, having <.01% cross-reactivity with almost all compounds tested  except for (+)-methylenedioxymethamphetamine (MDMA) which has just slightly higher relative affinity than METH (9 nM to 11nM)<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>. Besides (+)METH and (+)MDMA, the tested chemicals also include: (-)-METH, (+/-)-AMP,(-)-MDMA,4-OH METH, Pseudoephedrine, Ephedrine, Dopamine, Norepinephrine, Serotonin, Epinephrine.<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>It was also reported stereospecific<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>, having an approximately 100 times higher relative affinity for the plus form than the minus forms of these substances<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>. No significant cross activity has been observed in the test for other compounds<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/> including methylenedioxyamphetamine, (+)-norpseudoephedrine, L-phenylephrine, (+)-phenylpropanolamine, beta-phenylethylamine, and tyramine<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>.
====Effect of mAb6H4 on METH and AMP pharmacokinetics====
the t1/2lamdaZ of mAb6H4 is reported to be around 8 days<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>, and the METH induced behavior effects on locomotor effects is within 400 min after METH administration for the maximum dosage (3.0mg/kg) reported<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>. Thus to compare the disposition of drugs with or without mAb6H4, AUC 4.5h 38min <<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>>was used because it was not possible to conduct complete pharmacokinetic profile for 8 days.
For METH doses of 0.3mg/kg <ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>and 1 mg/kg <ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>in rats, administration of mAb6H4 has led to significantly higher serum concentrations corresponding to significantly lower brain concentration of METH<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/> during the time frame with the drug dose was less than the mAb6H4 binding capacity<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>. It was obvious that mAb6H4 administration initially caused a rapid efflux of METH from the brain due to its high affinity<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>, however, compared to METH concentration in the brain without mAb6H4, after 4.5 hours, there seems to be a very slight rebound<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/> of the concentration in the brain. The reason is unknown, and could be explained as slower redistribution of the drug from other tissues<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>. Yet, however, at this point, even with the rebound, both the METH concentration in control and in the animals administered with mAb6H4 are well below the threshold associated with increased locomotor activity<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>.


mAb6H4 also appeared to have more mild effect to AMP<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/> because it had little cross activity with AMP in vitro. It was explained possibility because increased the amounts of METH in the serum available for metabolism<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>.


Screening for anti(+)METH IgG response was conducted by radioimmunoassay (RIA)<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/> in which radio labelled [(+)-3H] METH is competing with unlabelled (+)-METH and (+)-AMP for the binding site of Abs in a manner similar to what has been previously described<ref name ="Antibodies against arylcyclohexylamines and their similarities in binding specificity with the phencyclidine receptor"/>. An IC50 value was determined for each compound after fitting a sigmoidal curve to the data points<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/><ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>.
However, for METH dose of 3.0mg/kg<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>, which was greater than the mAb6H4 binding capacity, METH induced locomotor effects appeared to be increased<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>. Although there are possible explanations, the reason remained to be understood<>. The idea that mAb may slow the input of METH while prolonging exposure is proposed<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>.  


Anti-(+)METH mAb6H4 was reported with Kd = 11 nM<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>, having <.01% cross-reactivity with almost all compounds tested (including (-)-METH, (+/-)-AMP,(+/-)-MDMA,4-OH METH, Pseudoephedrine, Ephedrine, Dopamine, Norepinephrine, Serotonin, Epinephrine)<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/> except for (+)-methylenedioxymethamphetamine (MDMA) which has just slightly higher relative affinity than METH (9 nM to 11nM)<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>. It was also reported stereospecific<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>, having an approximately 100 times higher relative affinity for the plus form than the minus forms of these substances<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>. No significant cross activity has been observed in the test for other compounds<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/> including methylenedioxyamphetamine, (+)-norpseudoephedrine, L-phenylephrine, (+)-phenylpropanolamine, beta-phenylethylamine, and tyramine<ref name="Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats"/>.
==Clinical development==
===Pharmacological effects of anti-methamphetamine monoclonal antibodies mAb4G9===


After screening for more than 25000<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/> 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<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. 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<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. After analyzing sequence genes, each antibody was found unique and not clonal<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. 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<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>.


====Effect of mAb6H4 on METH and AMP pharmacokinetics====
A common feature is a conserved proline<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/> at position 95 or 95a of all CDR L3 regions<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/> because of their ability to form 'hinges", except one case replaced with serine<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. And this residue was immediately followed by a hydrophobic amino acid or an aromatic residue which could be interacting with phenyl ring of (+)METH<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>.


mAb4G9, attributing to the design of hapten (+)METH MO10<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>, was the only<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/> 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<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>. It was reported, the affinity for mAb4G9 for AMP is 51nM<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>, demonstrating this molecule actually has the same KD value for (+)AMP and (+)METH<ref name="Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse"/>.






==Clinical development==
===Pharmacological effects of two anti-methamphetamine monoclonal antibodies mAb4G9 and mAb7F9===
===Human-mouse chimeric monoclonal antibody(mAb) Ch-mAb7F9===
===Human-mouse chimeric monoclonal antibody(mAb) Ch-mAb7F9===


IgG2, Kappa;METH KD= 7nM
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 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====
====Cross reactions in vitro ligand binding studies====
It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. <ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use" />
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>
 
 
====Isothermal titration calorimetry potency studies====
====Isothermal titration calorimetry potency studies====
Binding is efficient. <ref name="Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use" />
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===
====Serum ch-mAb7F9 concentration====
====Immunogenicity analyses====
====IgG pharmacokinetic parameters====
half life 17-19 d, volume of distribution of 5-6 L in the 3 highest dose groups <ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers " />
====Human anti-chimeric antibody response====


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);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 " />
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 " />
 


=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.() 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==
===ScFv6H4 Cloning===
 
===cDNA Cloning and sequencing of mAb===
 
The heavy and light chains cDNA of the mAb were cloned by reverse transcription-polymerase chain reaction<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, and then they were amplified and ligated into the cloning vector<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. The resulting plasmids of all mAb cloning was transformed into E. coli strain and then sequenced and submitted to Genbank<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>.
 
===Sub-cloning and Large-Scale Expression===
===Sub-cloning and Large-Scale Expression===
After the sequence confirmation, the plasmid was linearized with SacI <ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> and the coding sequence of scFv6H4 was transformed to P.pastoris strain(yeast) by electroporation<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. It is expressed in yeast colony which exhibited high zeocin resistance<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>.
===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<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. ScFv was purified from the supernantant by metal affinity chromatography and metal affinity column<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> in a naturally occurring mixture of monomer (~75%) amd dimer (~25%)<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> 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<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, which will orient the binding site of METH distal to the beads, allowing unhindered access to METH<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. Standard curve was constructed with [H3]METH competing with unlabeled METH. The KD was reported to be 10nM nearly identical to parent IgG mAb6H4 11nM<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. It was reported just like parent mAb6H4, there is no cross reactivity with related compounds<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> including pseudo-ephedrine, norepinephrine, dopamine, and serotonin, even at 100 microMolar<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>.
===Pharmacokinetic Studies of METH and scFv6H4 in Rats===
===Pharmacokinetic Studies of METH and scFv6H4 in Rats===


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"/>.
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==
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|NAME=4laq
|NAME=4laq
|SCENE=80/800653/4laq_initial_scene/1
|SCENE=80/800653/4laq_initial_scene/1
|CAPTION=scFv6H4 free form  <scene name='80/800653/4laq_initial_scene/1'>initial scene(reset)</scene> <scene name='80/800653/Scfv6h4_heavy_chain/3' target='4laq'>Heavy chain</scene>  <scene name='80/800653/Scfv6h4_light_chain/8' target='4laq'>Light chain</scene>
|CAPTION=scFv6H4 free form  <scene name='80/800653/4laq_initial_scene/1' target='4laq' >initial scene(reset)</scene> <scene name='80/800653/Scfv6h4_heavy_chain/3' target='4laq'>Heavy chain</scene>  <scene name='80/800653/Scfv6h4_light_chain/8' target='4laq'>Light chain</scene>
|LIGAND= <scene name='pdbligand=MLT:D-MALATE' target='4laq'>MLT</scene>, <scene name='pdbligand=NI:NICKEL+(II)+ION' target='4laq'>NI</scene>, <scene name='pdbligand=SO4:SULFATE+ION' target='4laq'>SO4</scene>
|LIGAND= <scene name='pdbligand=MLT:D-MALATE' target='4laq'>MLT</scene>, <scene name='pdbligand=NI:NICKEL+(II)+ION' target='4laq'>NI</scene>, <scene name='pdbligand=SO4:SULFATE+ION' target='4laq'>SO4</scene>
|ACTIVITY= <span class='plainlinks'> </span>
|ACTIVITY= <span class='plainlinks'> </span>
Line 120: Line 214:
|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
Line 134: Line 225:
|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
Line 163: Line 247:
  }}
  }}


}}


==Structure optimization by point mutations==
===Methods===
===Saturation equilibrium dialysis to determine Kd===
===Inhibition equilibrium dialysis to determine IC50 values for AMP===
===Crystallization and structure highlights of scFv-S93T:METH complex===
=Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse=




Line 185: Line 262:
<ref name ="Pharmacodynamics of a monoclonal antiphencyclidine Fab with broad selectivity for phencyclidine-like drugs">PMID:9618414</ref>
<ref name ="Pharmacodynamics of a monoclonal antiphencyclidine Fab with broad selectivity for phencyclidine-like drugs">PMID:9618414</ref>
<ref name ="Pharmacokinetic antagonism of (+)-methamphetamine discrimination by a low-affinity monoclonal anti-methamphetamine antibody">PMID:12394422</ref>
<ref name ="Pharmacokinetic antagonism of (+)-methamphetamine discrimination by a low-affinity monoclonal anti-methamphetamine antibody">PMID:12394422</ref>
<ref name ="Pharmacokinetic mechanisms for obtaining high renal coelimination of phencyclidine and a monoclonal antiphencyclidine antigen-binding fragment of immunoglobulin G in the rat">PMID: 9808688</ref>
<ref name ="Development of a liquid chromatography-tandem mass spectrometric method for the determination of methamphetamine and amphetamine using small volumes of rat serum">PMID:15171915</ref>


<ref name="Designing immunotherapies to thwart drug abuse">PMID: 19592672</ref>
<ref name="Designing immunotherapies to thwart drug abuse">PMID: 19592672</ref>

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA, Christina R. Bourne, Zhaihui G. Modlin
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