Browsing by Author "Dilchert, Janine"

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Open Access
Engineering of bispecific T cell receptors using mammalian display technologies
(2022) Dilchert, Janine; Kontermann, Roland (Prof. Dr.)
The main goal of immunotherapy in oncology is to use the power of the patient’s own immune system to fight cancer. Therefor many bispecific antibodies were developed mainly for redirecting T cells to cancer cells. A novel and promising class of biotherapeutics are increasingly recognized, namely bispecific T cell receptor (TCR)- based molecules capable of redirecting and activating T cells towards tumor-specific peptides presented by human leucocyte antigens (HLA). The usage of TCR-based molecules allows for targeting of novel tumor antigens including intracellular antigens and thus significantly widens the accessible target space in cancer immunotherapy. In contrast to antibodies, TCRs naturally exhibit a low binding affinity and stability and thus a complex maturation process is required for successful generation of TCR-based biotherapeutics. We developed a Chinese hamster ovary (CHO) cell display system for the maturation of TCR-based biomolecules, such as T cell engaging receptor (TCER®). Unlike previously used phage or yeast display systems, the mammalian system is capable of engineering TCRs in the final TCER® format making the step of reformatting of matured TCRs dispensable. The display approach is based on a recombinase-mediated cassette exchange for efficient and stable single copy integration of bispecific agents into a predefined genetic locus of the CHO cell. This work describes the setup of the CHO display, the membrane-bound expression of different TCR-based formats as well as its successful application for engineering of TCER® molecules using TCR variable domains from a model TCR recognizing preferentially expressed antigen in melanoma (PRAME). Affinity-improved TCER® molecules were isolated from a library encoding different complementarity determining region (CDR) variants in the final format. The selected TCER® candidates were evaluated in the CHO display system regarding their binding to the PRAME pHLA target as well as 11 peptides with high degree of sequence similarity to the PRAME peptide as part of specificity testing. TCER® variants expressed as soluble proteins showed strong reactivity against PRAME-positive tumor cells linked with a pronounced cytokine release from activated T cells. This study supports feasibility of the CHO-based maturation system for TCR affinity maturation in the final TCER® format and demonstrate data consistency between membrane-bound and soluble TCER® format.
Open Access
Mammalian display platform for the maturation of bispecific TCR-based molecules
(2022) Dilchert, Janine; Hofmann, Martin; Unverdorben, Felix; Kontermann, Roland; Bunk, Sebastian
Bispecific T cell receptor (TCR)-based molecules capable of redirecting and activating T cells towards tumor cells represent a novel and promising class of biotherapeutics for the treatment of cancer. Usage of TCRs allows for targeting of intracellularly expressed and highly selective cancer antigens, but also requires a complex maturation process to increase the naturally low affinity and stability of TCRs. Even though TCR domains can be matured via phage and yeast display, these techniques share the disadvantages of non-human glycosylation patterns and the need for a later reformatting into the final bispecific format. Here, we describe the development and application of a Chinese Hamster Ovary (CHO) display for affinity engineering of TCRs in the context of the final bispecific TCR format. The recombinase-mediated cassette exchange (RCME)-based system allows for stable, single-copy integration of bispecific TCR molecules with high efficiency into a defined genetic locus of CHO cells. We used the system to isolate affinity-increased variants of bispecific T cell engaging receptor (TCER) molecules from a library encoding different CDR variants of a model TCR targeting preferentially expressed antigen in melanoma (PRAME). When expressed as a soluble protein, the selected TCER molecules exhibited strong reactivity against PRAME-positive tumor cells associated with a pronounced cytokine release from activated T cells. The obtained data support the usage of the CHO display-based maturation system for TCR affinity maturation in the context of the final bispecific TCER format.