We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 minutes after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [3H] cocaine binding assay. All three antibodies had long elimination half-lives, 2–5 nM Kds for cocaine, and prevented cocaine’s entry into the brain by sequestering it in the plasma. Pharmacokinetic and radio-ligand binding assays supported designation of the highest producing clone (85) as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy.
Background
Immunotherapy has shown potential as a treatment for cocaine abuse. The humanized recombinant anti-cocaine monoclonal antibody (mAb) with the preclinical designation h2E2 has been shown to decrease cocaine concentrations in the brain in rats, but its effects on cocaine self-administration behavior have never been tested.
Methods
The amount of cocaine needed to reinstate self-administration behavior (priming threshold) was calculated and the inter-injection intervals at unit doses of 0.3 μmol/kg and 3 μmol/kg during maintained self-administration were measured over a five-week baseline period. Rats trained to self-administer cocaine were infused with two doses of h2E2 (120 mg/kg i.v.) 35 days apart. Priming threshold and inter-injection intervals were measured for 35 days after both injections.
Results
After both injections of h2E2, priming thresholds were significantly increased (3-fold) compared to expected baseline and then gradually declined over 35 days. A significant decrease (15–33%) in inter-injection intervals during maintained self-administration was also observed following both h2E2 at the lower dose, and after the first injection at the higher dose. No significant decreases in body weight were observed after either injection, indicating a lack of toxicity following a second injection.
Conclusions
These data predict that the safety and efficacy of h2E2 will be maintained after multiple treatments of this potential immunotherapy for cocaine abuse.
Aims
A recombinant humanized anti-cocaine monoclonal antibody (mAb), h2E2, is at an advanced stage of pre-clinical development as an immunotherapy for cocaine abuse. It is hypothesized that h2E2 binds to and sequesters cocaine in the blood.
Main Methods
A three-compartment model of the effects of h2E2 on cocaine's distribution was constructed. The model assumes that h2E2 binds to cocaine and that the h2E2-cocaine complex does not enter the brain but distributes between the central and peripheral compartments. Free cocaine is eliminated from both the central and peripheral compartments, and h2E2 and the h2E2-cocaine complex are eliminated from the central compartment only. This model was tested against a new dataset measuring cocaine concentrations in the brain and plasma over one hour in the presence and absence of h2E2.
Key findings
The mAb significantly increased plasma cocaine concentrations with a concomitant significant decrease in brain concentration. Plasma concentrations declined over the 1-hour sampling period in both groups. With a set of parameters within reasonable physiological ranges, the three-compartment model was able to qualitatively and quantitatively simulate the increased plasma concentration in the presence of the antibody and the decreased peak brain concentration in the presence of antibody. Importantly, the model explained the decline in plasma concentrations over time as distribution of the cocaine-h2E2 complex into a peripheral compartment.
Significance
This model will facilitate the targeting of ideal mAb PK/PD properties thus accelerating the identification of lead candidate anti-drug mAbs.
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