Combining Active Immunization with Monoclonal Antibody Therapy To Facilitate Early Initiation of a Long-Acting Anti-Methamphetamine Antibody Response

Abstract

We hypothesized that an anti-METH mAb could be used in combination with a METH-conjugate vaccine (MCV) to safely improve the overall quality and magnitude of the anti-METH immune response. The benefits would include immediate onset of action (from the mAb), timely increases in the immune responses (from the combined therapy) and duration of antibody response that could last for months (from the MCV). A novel METH-like hapten (METH-SSOO9) was synthesized and then conjugated to immunocyanin monomers of keyhole limpet hemocyanin (IC(KLH)) to create the MCV ICKLH-SOO9. The vaccine, in combination with previously discovered anti-METH mAb7F9, was then tested in rats for safety and potential efficacy. The combination antibody therapy allowed safe achievement of an early high anti-METH antibody response, which persisted throughout the study. Indeed, even after 4 months the METH vaccine antibodies still had the capacity to significantly reduce METH brain concentrations resulting from a 0.56 mg/kg METH dose.

Figure 1

(A) The MCV used for active immunization (IC_KLH-SOO9; i.), the other MCV tested in vitro (IC_KLH-SOO9; ii.), the MCV used to generate mAb7F9 (BSA-MO9; iii.), and the MCV used to generate mAb4G9 (OVA-MO9; iv.). (B) In vitro percent inhibition of mAb7F9 or mAb4G9 [³H]-METH binding by IC_KLH-SOO9 (i.) or IC_KLH-SMO9 (ii.) MCVs. These data aided the decision to use IC_KLH-SOO9 and mAb7F9 for these studies, since this combination showed the least cross reactivity.

Figure 2

Total anti-METH antibody (mAb7F9 and/or polyclonal) binding shown as the percentage [³H]-METH bound by a 1:50 diluted rat serum sample over time. Dosing times and immunological treatments are denoted below the x-axis (combination treated rats were administered all MCV and mAb treatments). Statistical differences between METH binding in IC_KLH-SOO9 + mAb7F9, mAb7F9-only, and IC_KLH-SOO9-only treated animals were determined with a two-way repeated measures ANOVA with a post-hoc Bonferroni’s test. For clarity, only statistical analysis comparing IC_KLH-SOO9 + mAb7F9 combination group with the other two treatment groups are shown. The * symbol denotes statistical significance compared to mAb7F9 treated animals, and the # symbol denotes statistical significance compared to IC_KLH-SOO9 treated animals (p<0.05).

Figure 3

Data from the ELISA determination of immune binding of mAb7F9 in rat serum (week 8) to microtiter plates coated with OVA-SOO9, followed by detection with an anti-mouse secondary antibody.

Figure 4

Analysis of serum samples by ELISA for the presence of anti-METH pAb and mAb7F9 antibodies. Figures in the left panel show presence of anti-METH rat pAb over time (OVA-SOO9 ELISA plate coating, detection with anti-rat secondary Ab). Figures in the right panel show presence of mouse anti-METH mAb7F9 over time (OVA-SMO9 ELISA plate coating, detection with anti-mouse secondary Ab). See statistical comparisons in the results section.

Figure 5

METH serum and brain concentrations 2 hrs after a 0.56 mg/kg sc METH dose. The * denotes statistical significance compared to mAb7F9 treated animals (p<0.05).

Figure 6

(A) Average animal weight per group (n=8 rats) over time (upper panel). (B) Change in average body temperature per group over time compared to baseline values (lower panel). (C) Average organ weights per group determined at the end of the studies.

Scheme 1^a

^aReagents: (a) (S)-2-methylbenzlamine (2), Na(OAc)₃BH₃, (CH₂Cl)₂; (b) etheral HCl; (c) recrystallization from toluene; (d) HCO₂H, (CH₃CO)₂O, toluene; (e) BH₃, THF; (f) HCO₂NH₄, 5% Pd/C, CH₃OH; (g) BBr₃, CH₂Cl₂; (h) (BOC)₂O, Et₃N, CH₃OH; (i) NaH, Br(CH₂)₅CO₂Me, DMF; (j) LiOH, H₂O; (k) cystamine•2HCl, BOP, THF; (l) 2N, HCl

Scheme 2^a

^aReagents (a) cystamine•2HCl, HOAt, EDC•HCl, Et₃N, DMF; (b) HCl, Et₂O

Scheme 3^a

^aReagents: (a) H₂O; (b)TCEP, SSOO9 (12); (c) TCEP, SSMO9 (15)