Ida Laurén: Next generation CD40 targeting immunotherapy: Development of antibody-based personalized cancer treatment

Abstract

Antibody-based therapeutics have revolutionised the field of immune-oncology and other diseases with their diverse targeted effects. Immune-stimulatory antibodies, such as agonistic CD40 antibodies, have shown promising preclinical results in inducing specific anti-tumour responses. This thesis presents the development of a novel bispecific antibody platform based on agonistic CD40 antibodies designed to enable flexible peptide cargo delivery.

In paper I, the design of a tetravalent bispecific antibody platform based on two well-established agonistic anti-CD40 antibodies was evaluated. The bispecific antibodies retained their agonistic activity and demonstrated high-affinity binding to a defined peptide tag, enabling efficient intracellular peptide antigen delivery. This affinity binding was crucial for inducing antigen-specific T cell proliferation in vivo. The lead candidate identified in paper I was further assessed for large-scale production in paper II, using the PiggyBack transfection system to generate a stable cell pool. Optimisation of nutrients and feeding schedules allowed for large-scale bioreactor culturing, followed by purification and quality analysis. 

In paper III, a novel set of anti-CD40 antibodies was generated through phage display, and the lead antibody clone, which exhibited agonistic activity and low off-target binding, was converted to the bispecific format described in paper I, but with a humanised version of the single chain fragment variable (scFv). This candidate drug, in combination with peptide cargo, generated potent anti-tumour effects in several tumour models and demonstrated low toxicity and immunogenicity. 

In paper IV, the peptide tag binding to the bispecific antibody was modified by amino acid substitution to assess whether affinity binding and/or biological effects could be modified. The substitution with a non-conserved amino acid led to a slightly decreased affinity binding. This substitution led to modified in vitro antigen processing and/or presentation of the CD4+ and CD8+ T cell antigens, while in vivo activity appeared to depend more on conjugate stability.

In conclusion, this bispecific antibody platform facilitates flexible peptide cargo loading by affinity binding and provides a promising opportunity for personalised therapy by using patient-specific neoantigen peptides as cargo to generate effective anti-tumour T cell responses.