Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.
We previously showed that a human anti-TACE antibody, D1(A12), is a potent inhibitor of TNF-α converting enzyme (TACE) ectodomain proteolysis and has pharmacokinetic properties suitable for studies of the inhibition of TACE-dependent growth factor shedding in relation to possible therapeutic applications. However, the lack of murine TACE immunoreactivity limits pre-clinical in vivo studies to human xenograft models which are poor analogies to in situ pathology and are not considered clinically predictive. Here, to overcome these limitations, we set out to develop a 'mouse and human cross-reactive' specific anti-TACE antibody. We first re-investigated the originally selected anti-TACE ectodomain phage-display clones, and isolated a lead 'mouse-human cross-reactive' anti-TACE scFv, clone A9. We reformatted scFv-A9 into an IgG2 framework for comprehensive biochemical and cellular characterization and further demonstrated that A9 is an exosite TACE inhibitor. However, surface plasmon resonance analysis and quenched-fluorescent (QF) peptide assay indicated that IgG reformatting of A9 caused low binding affinity and an 80-fold reduction in TACE ectodomain inhibition, severely limiting its efficacy. To address this, we constructed second generation phage-display randomization libraries focused on the complementarity-determining region 3, and carried out affinity selections shuffling between human and mouse TACE ectodomain as antigen in addition to an off-rate selection to increase the chance of affinity improvement. The bespoke 'three-step' selections enabled a 100-fold affinity enhancement of A9 IgG, and also improved its IC50 in a QF peptide assay to 0.2 nM. In human and mouse cancer cell assays, matured A9 IgG showed significant cell-surface TACE inhibition as a monotherapy or combination therapy with chemotherapeutic agent. Collectively, these data suggest that we successfully developed an exosite inhibitor of TACE with sub-nanomolar affinity, which possesses both murine and human immunoreactive properties that can be used for in vivo application in murine pre-clinical cancer models.
Proteases and Tumour Microenvironment Group, Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK Faculty of Health Sciences, University of Macau, Taipa, Macau SAR, China henry.kwok@cruk.cam.ac.uk gm290@cam.ac.uk.
Hang Fai
HF
Kwok
Proteases and Tumour Microenvironment Group, Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK Department of Molecular Biology and Genetics, University of Aarhus, Denmark.
Kenneth A
KA
Botkjaer
Proteases and Tumour Microenvironment Group, Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK Present address: Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
Christopher J
CJ
Tape
Proteases and Tumour Microenvironment Group, Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK.
Yanchao
Y
Huang
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.
John
J
McCafferty
Proteases and Tumour Microenvironment Group, Department of Oncology, Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK henry.kwok@cruk.cam.ac.uk gm290@cam.ac.uk.
Gillian
G
Murphy
D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D000072198:ADAM17 Protein; D000494:Allosteric Regulation; D000818:Animals; D000918:Antibody Specificity; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D057809:HEK293 Cells; D006801:Humans; D007167:Immunotherapy; D020128:Inhibitory Concentration 50; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000494:pharmacology*; D000093542:Gemcitabine
D000970:Antineoplastic Agents; D057127:Single-Chain Antibodies; D003841:Deoxycytidine; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606463:Adam17 protein, mouse; D000093542:Gemcitabine
anti-TACE antibody; exosite; immunoreactivity; phage-display randomization libraries; sub-nanomolar affinity