Linkers for bioconjugation

Abstract

Cancer is a heterogeneous disease that represents one of the principal causes of mortality in developed countries. Due to the social and economic implications of this pathology, tremendous efforts have been making over the past decades to improve available therapeutic options to tackle this illness. Although a large number of potent chemotherapeutic agents have been identified and successfully used in clinical practice, the development of new anticancer chemotherapeutic drugs with higher antitumor efficacy and less toxicity remains as a research challenge.

Current advances in the mechanistic understanding of the molecular drivers of malignancy have led to many anticancer drugs, which are targeted directly at the cancerous cells and not the neighbouring healthy cells. Thus, in order to enhance the efficacy of existing anticancer agents, several drug delivery approaches have been developed. Appropriately, ADCs strategy consists on bind determinate cell-killing drug to a monoclonal antibody (mAb) through a specific linker. Due to their high-binding specificity for tumor-specific antigens, mAb can be used as vehicles to target cell- killing payloads to tumor cells. Accordingly, two kinds of linkers are suitable to attach the payload to the antibody. On one hand, non-cleavable linkers afford and stable binding between the drug and the antibody, and alternatively, the drug could be released in the proximity or even inside of affected cells using cleavable linkers.

Based on the potential of ADCs as prodrugs, our main goal is develop linkers, which could promote the drug release close to the affected tumor cells. Taking into account that connection between the antibody and the cytotoxic agent has significant effects on the selectivity, pharmacokinetics and therapeutic index of the ADC an efficient binding is needed. Therefore, the properties of a successful linker can be split up into different modules that have been combined to form an effective bridge between the cytotoxic payload and the carrier antibody.

In the present thesis, we focus on the development of new linkers for bioconjugation and more specifically for Antibody–Drug conjugates (ADCs). With this purpose in mind, a conscious design of the linkers for ADCs has to be done. In the present thesis, different cleavable (Chapter 1) and non-cleavable (Chapter 2) linkers are studied and it will be explained in detail along the thesis.

As a consequence, the design has to be focused on linkers stables in systemic circulation. Additionally, cleavable linkers should be unstable in tumor cells environment, such as high concentration of glutathione, highest acidic concentration,

and enzymatic cleavage, among others as above-mentioned. These conditions should allow, chemically or enzymatically, the release of the drug from the carrier (antibody- linker).

On one hand, the systems studied in chapter I involve N-alkylated dialkylglycines and the tetrahydropyran moiety as cleavable Linkers. On the other hand in chapter II, the mesitylene ring and perfluoroarylated compounds were studied as non-cleavable linkers for bioconjugation. The present thesis include and extensive study regarding both cleavable and non-cleavable linker. Particularly, the preparation and chemical modification of the handles in order to test and optimize the bioconjugation step. Also an interesting study was carried out in order to determine how the bioconjugation step affect to the macromolecular entities. Specifically we focus on the conjugation to antibodies and how the covalent binding of the studied linkers affect to the antigen- antibody bind affinity.

El cáncer está considerado como una de las principales causas de mortalidad a nivel mundial. Y ello conlleva elevados esfuerzos socio-económicos para tratar y curar esta enfermedad a pesar de disponer un amplio número de agentes anticancerígenos descritos, la obtención de nuevos agentes terapéuticos con mejor eficacia y menores efectos secundarios continua siendo a día de hoy, un reto científico a lograr.

Con el fin de mejorar estos tratamientos, se han desarrollado diversos sistemas de liberación controlada de fármacos. Un ejemplo interesante son, los sistemas conocidos como ADCs (Antibody-Drug Conjugates) que consisten en la unión de un fármaco a un anticuerpo para permitir direccionar el fármaco a célula diana. Dicha unión fármaco-anticuerpo se produce mediante un conector o linker que pueden ser no hidrolizables (Non-cleavable linkers) o hidrolizables (Cleavable Linkers).

La presente tesis centra su objeto de estudio en el diseño de conectores tanto hidrolizables como no hidrolizables. En el primer capítulo de la presente Tesis se abordó la utilización de α, α-dialquilglicinas N-alquiladas como posibles conectores hidrolizables para bioconjugación. Este tipo de sistemas tipo 1,4-dicarbonílicos, presentan acidólisis de la amida del extremo C- terminal. Otro sistema estudiado como conector están basados en los enlaces tipo acetal, tioacetal y aminal sobre el anillo de tetrahidropirano. Fruto del estudio de el anillo de tetrahidropirano como conector, se estudio la aplicabilidad de este tipo de anillo como grupo protector del tiol de la cisteína para la síntesis de péptidos de fase sólida.

En el segundo capitulo se abordó el estudio de conectores no hidrolizables con potencial aplicabilidad den bioconjugación. En primer lugar se estudiaron los compuestos aromáticos tipo mesitileno utilizando los precursores dibromados para la formación de tioeteres estables a partir de dos cisteínas en péptidos, proteínas y anticuerpos. Además en este mismo capítulo se emprendió la unión de compuestos perfluorados en bioconjugación.