Biopharmaceutics 4.0, Advanced Pre-Clinical Development of mRNA-Encoded Monoclonal Antibodies to Immunosuppressed Murine Models

Abstract

Administration of mRNA against SARS-CoV-2 has demonstrated sufficient efficacy, tolerability and clinical potential to disrupt the vaccination field. A multiple-arm, cohort randomized, mixed blind, placebo-controlled study was designed to investigate the in vivo expression of mRNA antibodies to immunosuppressed murine models to conduct efficacy, safety and bioavailability evaluation. Enabling 4.0 tools we reduced animal sacrifice, while interventions were designed compliant to HARRP and SPIRIT engagement: (a) Randomization, blinding; (b) pharmaceutical grade formulation, monitoring; (c) biochemical and histological analysis; and (d) theoretic, statistical analysis. Risk assessment molded the study orientations, according to the ARRIVE guidelines. The primary target of this protocol is the validation of the research hypothesis that autologous translation of Trastuzumab by in vitro transcribed mRNA-encoded antibodies to immunosuppressed animal models, is non-inferior to classical treatments. The secondary target is the comparative pharmacokinetic assessment of the novel scheme, between immunodeficient and healthy subjects. Herein, the debut clinical protocol, investigating the pharmacokinetic/pharmacodynamic impact of mRNA vaccination to immunodeficient organisms. Our design, contributes novel methodology to guide the preclinical development of RNA antibody modalities by resolving efficacy, tolerability and dose regime adjustment for special populations that are incapable of humoral defense.

Keywords: COVID-19; IVT mRNA; NOD/SCID/J; SARS–CoV-2; Trastuzumab; biopharmaceutics 4.0; immunodeficient; mRNA encoded antibodies; mRNA-vaccines; pharmacokinetics.

Figure 1

Schematic overview of the preparation and clinical validation of TZM antibodies encoded by mRNA.

Figure 2

Overview of the pre-clinical trial design points.

Figure 3

Concentration curves of TZM over time after intra venous infusion: (a) Predicted critical time window of interest based on projections of bibliographic data; (b) IWRES versus IPRED for predicted data of mAb infusion as full-length protein.

Figure 4

Box diagram showing the experimental design [48] for various factors [49]: (a) y-axis type of animal model categorical NOD/SCID/J (top) and C57BL/6/J (below), x-axis W.B.D. dosing in mg/kg, z-axis time in days, (b) y-axis TZM blood concentration in mg/mL, x-axis time in days, z-axis type of animal model C57BL/6/J (left) and NOD/SCID/J (right).

Figure 5

(a) Plasmid preparation backbone; (b) mRNA optimization chart showing the additions and mutations utilized for our clinical experiment.

Figure 6

Proof of clinical concept: (a) Confocal microcopy microphotograph of MCF7 cells by the live/dead fluorescent staining with Calcein AM/EthD-1; (b) confocal microcopy microphotograph by the live/dead fluorescent staining with Calcein AM/EthD-1 showing low levels of cell viability of MCF-7 cells, incubated for 72 h with the supernatant of transfected CHO-K1 with mRNA Trastuzumab (photographs were taken at 10× magnification; scale bars represent 50 μm).

Figure 7

Fish bone diagram of risk assessment demonstrating the factors that critically alternate the validity of the obtained results and effect reproducibility and extrapolation, spanning the sectors of animal type, experimental unit preference, mRNA, analysis methods, carrier and statistical methods employed.

Figure 8

Model structure of the PBPK TZM mRNA IVT for the plasma baseline concentrations. $\mathrm{Whereas} {\mathrm{C}}_{\mathrm{RC},\mathrm{P}}$ is the mAb mRNA TZM concentration found in plasma volume ${\mathrm{V}}_{\mathrm{P}}$, ${\mathrm{C}}_{\mathrm{d}}$ and${ \mathrm{C}}_{\mathrm{lq} }$are the mAb concentrations in ISF in the tissues dense and leaky of volumes ${\mathrm{V}}_{\mathrm{d}}$ and ${\mathrm{V}}_{\mathrm{lq}}$ and of continuous, and fenestrated capillaries, respectively, ${\mathrm{V}}_{\mathrm{l}}$ is the lymph volume and assumed almost equal to blood volume. The ${\mathrm{F}}_{\mathrm{L} }$ is the total lymph flow which is considered equal to the sum of ${\mathrm{F}}_{\mathrm{L},1 }$and ${\mathrm{F}}_{\mathrm{L},2 }$ being the lymph flux for ${\mathrm{V}}_{\mathrm{d}}$ and ${\mathrm{V}}_{\mathrm{lq}}$ respectively. The ${\mathsf{\sigma }}_{\mathrm{d}}$ and ${\mathsf{\sigma }}_{\mathrm{l}}$ and the ${\mathsf{\sigma }}_{\mathrm{d}*}$ and ${\mathsf{\sigma }}_{\mathrm{l}*}$ being the coefficients of vascular reflection ${\mathrm{V}}_{\mathrm{d}}$ and ${\mathrm{V}}_{\mathrm{lq}}$ for the mRNA formulation and the correspondent TZM IgG, respectively. The ${\mathrm{CL}}_{\mathrm{P}},{ \mathrm{CL}}_{\mathrm{in},1}$, ${\mathrm{CL}}_{\mathrm{in},1*}$ and ${\mathrm{CL}}_{\mathrm{in},2}$, ${\mathrm{CL}}_{\mathrm{in},2*}$are the clearances for the plasma and ISF dense, leaky tissues for the mRNA formulation and the correspondent TZM IgG, respectively. Simulations of the predicted active tissue concentrations will be compared to the experimental measurements in order to evaluate the model performance. The areas under the concentration versus time curves (AUC0-t) will be calculated using the NCA bundle of Phoenix 8.1 (2021, Pharsight Corporation, Mountain View, CA) and predictions will be extrapolated using the same software for the relevant human populations.