Generation of a safe and efficacious llama single-domain antibody fragment (vHH) targeting the membrane-proximal region of 4-1BB for engineering therapeutic bispecific antibodies for cancer.

BACKGROUND: The discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only offered an average of 20%-30% response rates across the tumor spectrum and the combination of agonists towards the tumor-necrosis superfamily members, such as 4-1BB and CD40, has shown potent efficacy in preclinical studies; however, these agonists have exhibited high degrees of toxicity with limited efficacy in human trials. In this study, we have generated a single-domain antibody towards a unique epitope of 4-1BB that limits its potential on-target toxicity while maintaining sufficient potency. This 4-1BB binder is ideal for use in the engineering of multispecific antibodies to localize 4-1BB activation within the tumor microenvironment, as shown here by a anti-PD-L1/4-1BB bispecific candidate (PM1003). METHODS: To determine the functional activity of the 4-1BB- and PD-L1-binding elements of PM1003, in vitro luciferase reporter and primary cell assays were used to test the potency of programmed cell death 1 ligand 1 (PD-L1) blockade and PD-L1-mediated 4-1BB activation via cross-bridging. X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms, and accurate binding kinetics were determined using standard affinity measurement techniques. Human 4-1BB and/or PD-L1 knock-in mice were used in cancer models for testing the in vivo antitumor efficacy of PM1003, and safety was evaluated further. RESULTS: PM1003 shows potent activation of 4-1BB and blockade of PD-L1 in cell-based assays. 4-1BB activation was exerted through the bridging of PD-L1 on target cells and 4-1BB on effector cells. No PD-L1-independent activation of 4-1BB was observed. Through X-ray crystallography, a unique binding epitope in the cysteine-rich domain 4 (CRD4) region was resolved that provides high potency and potentially low on-target toxicity as determined by primary immune cell assays and toxicity evaluation in vivo. CONCLUSIONS: A unique single-domain antibody was discovered that binds to the CRD4 domain of 4-1BB. When incorporated into a 4-1BB/PD-L1 bispecific (PM1003), we have shown the potent inhibition of PD-L1 activity with 4-1BB agonism upon cross-bridging with PD-L1 in vitro. Antitumor activity with minimal toxicity was found in vivo. Thus, PM1003 is a uniquely differentiating and next generation therapeutic agent for cancer therapy.

Optimization and modification of anti-rhTNF-α single chain variable fragment antibody: effective in vitro affinity maturation and functional expression of chimeric Fab.

AIMS: Single chain variable fragment (scFv) is one of the most popular recombinant antibody (rAb) formats. However, sometimes scFv with the most favorable specificity profile lack sufficient affinity or acceptable pharmacokinetics for clinical applications. To address these problems, we described a method to modify recombinant anti-rhTNF-α scFv-F6D2E7. RESULTS: Random mutations were inserted into CDR-H3 by performing PCR with tailored degenerate primers. After construction of a mutated antibody gene library, affinity selection was performed. Meanwhile the scFv (scFv-G10) selected from the library exhibited the most improved affinity to rhTNF-α (2.9-fold higher than the parental scFv-F6D2E7). The scFv-G10 sequence and human constant (CH1 & CL) regions were used to construct a novel vector for developing an expression system that allows the production of a completely functional antigen-binding fragment (Fab) in Escherichia coli. The bioactivity of the Fab was determined by L929 cell cytotoxicity assay. Fab-G10 could neutralize rhTNF-α-induced cytotoxicity to L929 cells, and the calculated 50% inhibition rate (IC50) was 5.0×10⁻7 M. CONCLUSION: We generated an artificial antibody fragment (scFv-G10) that had improved affinity and desirable specificity. Further, the Fab-G10 was constructed and expressed in E. coli, where the bioactivity was further detected.

Phage-derived fully human antibody scFv fragment directed against human vascular endothelial growth factor receptor 2 blocked its interaction with VEGF.

Vascular endothelial growth factor receptor 2 (VEGFR-2) plays a critical role in tumor angiogenesis. None therapeutic antibodies targeting VEGFR-2 are available in clinical use. Herein, we describe the screening of a new single-chain antibody fragment (scFv) targeting extracellular domain 3 of human VEGFR-2 (kinase insert domain-containing receptor [KDR]3) from Griffin phage display scFv library. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions. The scFv exerted particular binding sites to KDR3 on molecular docking, and the binding affinity was further convinced by binding analysis both in quantitative ELISA and real-time kinetic determination by biosensors (K(D) = 40 nM). Finally, the scFv was revealed to inhibit VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVECs; IC(50) = 5 nM) and to inhibit HUVEC migration significantly at 17 nM. Taken together, our results indicate that we have successfully isolated a scFv which differentially recognizes KDR3 and has potential clinical applications in the treatment of angiogenesis related diseases.