High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement

被引：21

作者：

De Tavernier, Evelyn ^{[1
]}

Detalle, Laurent ^{[1
]}

Morizzo, Erika ^{[1
]}

Roobrouck, Annelies ^{[1
]}

De Taeye, Severine ^{[1
]}

Rieger, Melanie ^{[1
,2
]}

Verhaeghe, Tom ^{[1
]}

Correia, Andreia ^{[1
]}

Van Hegelsom, Rob ^{[1
]}

Figueirido, Rita ^{[1
,3
]}

Noens, Jeroen ^{[1
]}

Steffensen, Soren ^{[1
]}

Stohr, Thomas ^{[1
,4
]}

Van de Velde, Willem ^{[1
]}

Depla, Erik ^{[1
,5
]}

Dombrecht, Bruno ^{[1
]}

机构：

[1] Ablynx NV, Technol Pk 21, B-9052 Ghent, Belgium

[2] Univ Hosp Munster, Inst Expt Musculoskeletal Med, Domagkstrasse 3, D-48149 Munster, Germany

[3] Immunocore Ltd, 90 Pk Dr,Milton Pk, Abingdon OX14 RY, Oxon, England

[4] A2M Pharma GmbH, Alfred Nobel Str 10, D-40789 Monheim, Germany

[5] VIB Discovery Sci, Gaston Geenslaan 1, B-3001 Leuven, Belgium

来源：

JOURNAL OF BIOLOGICAL CHEMISTRY | 2016年 / 291卷 / 29期

关键词：

GRAM-NEGATIVE BACTERIA; PSEUDOMONAS-AERUGINOSA; III SECRETION; V-ANTIGEN; MURINE MODEL; INFECTION; PROTEIN; TRANSLOCATION; IMMUNIZATION; MACROPHAGES;

D O I：

10.1074/jbc.M115.684241

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Improving potencies through concomitant blockage of multiple epitopes and avid binding by fusing multiple (different) monovalent Nanobody building blocks via linker sequences into one multivalent polypeptide chain is an elegant alternative to affinity maturation. We explored a large and random formatting library of bivalent (combinations of two identical) and biparatopic (combinations of two different) Nanobodies for functional blockade of Pseudonionas aeruginosa PcrV. PcrV is an essential part of the P. aeruginosa type III secretion system (T3SS), and its oligomeric nature allows for multiple complex binding and blocking options. The library screening yielded a large number of promising biparatopic lead candidates, revealing significant (and non-trivial) preferences in terms of Nanobody building block and epitope bin combinations and orientations. Excellent potencies were confirmed upon further characterization in two different P. aeruginosa T3SS- mediated cytotoxicity assays. Three biparatopic Nanobodies were evaluated in a lethal mouse P. aeruginosa challenge pneumonia model, conferring 100% survival upon prophylactic administration and reducing lung P. aeruginosa burden by up to 2 logs. At very low doses, they protected the mice from P. aeruginosa infection -related changes in lung histology, myeloperoxidase production, and lung weight. Importantly, the most potent Nanobody still conferred protection after therapeutic administration up to 24 h post -infection. The concept of screening such formatting libraries for potency improvement is applicable to other targets and biological therapeutic platforms.

引用

页码：15243 / 15255

页数：13

共 39 条

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