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Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.
Monoclonal antibodies (mAbs) are an innovative group of drugs with increasing clinical importance in oncology, combining high specificity with generally low toxicity. There are, however, numerous challenges associated with the development of mAbs as therapeutics. Mechanistic understanding of factors that govern the pharmacokinetics (PK) of mAbs is critical for drug development and the optimisation of effective therapies; in particular, adequate dosing strategies can improve patient quality life and lower drug cost. Physiologically-based PK (PBPK) models offer a physiological and mechanistic framework, which is of advantage in the context of animal to human extrapolation. Unlike for small molecule drugs, however, there is no consensus on how to model mAb disposition in a PBPK context. Current PBPK models for mAb PK hugely vary in their representation of physiology and parameterisation. Their complexity poses a challenge for their applications, e.g., translating knowledge from animal species to humans.
In this thesis, we developed and validated a consensus PBPK model for mAb disposition taking into account recent insights into mAb distribution (antibody biodistribution coefficients and interstitial immunoglobulin G (IgG) pharmacokinetics) to predict tissue PK across several pre-clinical species and humans based on plasma data only. The model allows to a priori predict target-independent (unspecific) mAb disposition processes as well as mAb disposition in concentration ranges, for which the unspecific clearance (CL) dominates target-mediated CL processes. This is often the case for mAb therapies at steady state dosing.
The consensus PBPK model was then used and refined to address two important problems:
1) Immunodeficient mice are crucial models to evaluate mAb efficacy in cancer therapy. Protection from elimination by binding to the neonatal Fc receptor is known to be a major pathway influencing the unspecific CL of both, endogenous and therapeutic IgG. The concentration of endogenous IgG, however, is reduced in immunodeficient mouse models, and this effect on unspecific mAb CL is unknown, yet of great importance for the extrapolation to human in the context of mAb cancer therapy.
2) The distribution of mAbs into solid tumours is of great interest. To comprehensively investigate mAb distribution within tumour tissue and its implications for therapeutic efficacy, we extended the consensus PBPK model by a detailed tumour distribution model incorporating a cell-level model for mAb-target interaction. We studied the impact of variations in tumour microenvironment on therapeutic efficacy and explored the plausibility of different mechanisms of action in mAb cancer therapy.
The mathematical findings and observed phenomena shed new light on therapeutic utility and dosing regimens in mAb cancer treatment.