AbstractThe thesis is focused on the development of antibody based therapeutic molecules for the treatment of HER2 positive breast cancer. The generated molecules consist in antibody-drug conjugates (ADCs) including whole antibody and antibody fragment conjugates, and an immunocytokine, all of these molecules being based on the model monoclonal antibody Trastuzumab. This work comprises the whole production and characterization process of the molecules, going from the cloning of the genetic sequences of the antibody molecules to the in vitro analysis of their antiproliferation activity, and, for one of the developed candidates, an in vivo assessment of its therapeutic potential was also performed.
In a first step, the reference antibody Trastuzumab was produced using the mammalian cell line HEK293, its production being characterized in shake flasks as well as in 5L and 50L single use culture strategies. An operation sequence was defined for recovering and purifying the product, obtaining purity levels in the order of 99% and physicochemical characterization tools were implemented in order to assess the quality of the product. The production, purification and characterization strategies developed for Trastuzumab were applied to the other antibody-derived molecules later produced.
In a first attempt to develop a Trastuzumab antibody-derived molecule with an enhanced therapeutic activity, Trastuzumab was fused to the cytokine interferon-2, forming an immunocytokine. This construct, however, did not result in an improved antiproliferative activity with respect to Trastuzumab, as assessed in an in vitro 2D proliferation assay. It did not result neither in an activation of the immune system, as analyzed in an in vitro lymphocyte proliferation assay.
An increased potency of the antiproliferative activity of Trastuzumab was achieved by heterogeneously conjugating it to the cytotoxic drugs DM1 and vcMMAE, with drug-antibody ratios (DARs) of 3.1 and 4, respectively, applying two of the main reference conjugation strategies found in commercially approved ADCs: conjugation to endogenous lysine residues of the antibody (DM1), and conjugation to endogenous interchain cysteine residues after a partial reduction step (vcMMAE). After successfully implementing the conjugation strategies and DAR characterization tools, site-directed conjugation strategies (yielding homogeneous products that have therapeutic advantages in the form of a wider therapeutic window) were attempted.
Three different strategies were implemented for the homogeneous conjugation of Trastuzumab. In a first simple and straightforward strategy, Trastuzumab was conjugated to vcMMAE with an aimed DAR of 8, by applying a complete reduction of the antibody. High DARs, however, can result in instability and impaired in vivo therapeutic efficacy of the ADC. Therefore, the homogeneous conjugation for a DAR of 2 was attempted by inserting a cysteine in the sequence of Trastuzumab and conjugating the vcMMAE drug to it, obtaining a DAR of 1.79. An innovative alternative strategy to obtain an homogeneous ADC with a DAR of 2 was also developed, consisting in the cysteine conjugation of an independently produced light chain, which is then assembled with independently produced heavy chains, forming an homogeneous ADC with a DAR of 2.
The heterogeneous and homogeneous conjugation strategies implemented for whole Trastuzumab antibody were also applied to Trastuzumab-based scFv antibody fragments, resulting in conjugation processes with a low recovery yield due to precipitation issues.
The different generated ADCs had their antiproliferation activity tested in 2D in vitro models using the breast cancer model SKBR3 cell line, confirming their antitumor potential. 3D culture models were also generated, showing a higher resistance to the developed drugs than the 2D display. Finally, the therapeutic effect of the homogeneous ADC formed by the chains assembly was tested on a developed mouse model in vivo, displaying a strong antitumor response, and therefore validating the whole developed ADC production process.
|Date of Award||21 Oct 2019|
|Supervisor||Antoni Casablancas Mira (Director), Jordi Joan Cairo Badillo (Director), Jordi Joan Cairo Badillo (Tutor) & Marti Lecina Veciana (Director)|