Programmable half-life and anti-tumour effects of bispecific T-cell engager-albumin fusions with tuned FcRn affinity

Abstract

Fc-less bispecific T-cell engagers have reached the immuno-oncology market but necessitate continual infusion due to rapid clearance from the circulation. This work introduces a programmable serum half-life extension platform based on fusion of human albumin sequences engineered with either null (NB), wild type (WT) or high binding (HB) FcRn affinity combined with a bispecific T-cell engager. We demonstrate in a humanised FcRn/albumin double transgenic mouse model (AlbuMus) the ability to tune half-life based on the albumin sequence fused with a BiTE-like bispecific (anti-EGFR nanobody x anti-CD3 scFv) light T-cell engager (LiTE) construct [(t½ 0.6 h (Fc-less LiTE), t½ 19 hours (Albu-LiTE-NB), t½ 26 hours (Albu-LiTE-WT), t½ 37 hours (Albu-LiTE-HB)]. We show in vitro cognate target engagement, T-cell activation and discrimination in cellular cytotoxicity dependent on EGFR expression levels. Furthermore, greater growth inhibition of EGFR-positive BRAF mutated tumours was measured following a single dose of Albu-LiTE-HB construct compared to the Fc-less LiTE format and a full-length anti-EGFR monoclonal antibody in a new AlbuMus RAG1 knockout model introduced in this work. Programmable half-life extension facilitated by this albumin platform potentially offers long-lasting effects, better patient compliance and a method to tailor pharmacokinetics to maximise therapeutic efficacy and safety of immuno-oncology targeted biologics.

Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity

“…5-fold more of the Albumin HB was released back into the medium for the non-fused Albumin HB compared with the Albumin WT ( Figure 2 E). This effect was observed for the hLiTCo-Albu construct, with a higher concentration detected in the medium, possibly owing to anti-EGFR V HH bound to hEGFR on the cell surface of the HMEC-1 cells ( Mandrup et al., 2021 ) ( Figure 2 E).…”

“…In this study, we generated a small (≈47 kDa) human bispecific costimulatory antibody termed human LiTCo (hLiTCo, li ght T cell co stimulatory ) by fusing the anti-EGFR (human/mouse) EGa1 V HH ( Compte et al., 2020 ) to the N-terminal end of the anti-human 4-1BB SAP3.28 scFv (V H -V L orientation) ( Compte et al., 2021 ) with a flexible GGGGS linker ( Figures 1 , S1 A, and S1B). The hLiTCo-Albu was generated by genetic fusion of the hLiTCo to the N terminus of an albumin sequence with high binding to human FcRn (Albumin HB ) ( Figures 1 , S1 C, and S1D) used previously by ourselves in an Albu-LiTE construct ( Mandrup et al., 2021 ). Both hLiTCo and hLiTCo-Albu were efficiently secreted by human HEK293 cells, and in Western blot analysis under reducing conditions exhibited a migration pattern consistent with the molecular weights calculated from the amino acid sequences ( Figure S2 A).…”

“…Inspection of the binding interface of albumin with FcRn has allowed the design of recombinant variants with different FcRn affinities by single-point amino acid mutations ( Andersen et al., 2012 ) that have been subsequently used to tune the pharmacokinetic (PK) profile ( Andersen et al., 2014 ). Recently, we generated bispecific li ght T cell e ngagers (LiTEs) genetically fused to engineered HSA variants with different FcRn affinities to achieve programmable PK properties ( Mandrup et al., 2021 ).…”

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment

“…Multi-specific nanobodies retain at least part of the advantages of nanobodies, providing a mass that is still significantly smaller than IgGs and the capacity of targeting hidden epitopes by means of their protruding paratopes. Furthermore, their in vivo half-life can be fine-tuned according to the application needs ( 199 ). It must be also considered that nanobody biophysical features allow their administration via delivery routes that are not accessible to conventional IgGs.…”

Antibody variable region engineering for improving cancer immunotherapy

“…BiTEs are formed by scFv or nanobodies; therefore, they potentially offer higher tumor penetration due to their smaller size than full size IgG. For example, a bispecific nanobody against CD3 and EGFR bound specifically with EGFR‐overexpressing tumor cells, thereby mediating lysis via both T cell activation and EGFR signaling blockade [ 80 ]. In 2009, catumaxomab, an anti‐CD3 and EpCAM‐bispecific antibody targeting EpCAM‐positive tumors for T cell‐mediated lysis, became the first US FDA‐approved BiTE for intraperitoneal treatment of patients with malignant ascites [ 48 , 81 ].…”