Construction and evaluation of an antibody phage display library targeting heparan sulfate

Willeke F Daamen,Elly M.M Versteeg,Thierry Van Wessel,Toin H Van Kuppevelt,Els M.A Van De Westerlo,Lars A.A Damen

doi:10.1007/s10719-020-09925-z

Abstract

Heparan sulfate (HS) is a linear polysaccharide with high structural diversity. Different HS epitopes have been detected and localized using single chain variable fragment (scFv) antibodies from a ‘single pot’ phage display library containing a randomized complementarity determining region of the heavy chain (CDR3). In this study, we created a new library containing anti-HS scFvs that all harbor a dp-38 heavy chain segment where the CDR3 region was engineered to contain the XBBXBX heparin binding consensus site (X = any amino acid, B = R, K or H). The library contained ~1.73 × 106 unique antibodies and was biopanned against HS from several sources. The selected antibodies were sequenced and chemically/immunohistologically characterized. A number of 67 anti-HS scFv antibodies were selected, of which 31 contained a XBBXBX CDR3 sequence. There was a clear preference for glycine at the first and proline at the fourth position of the CDR3. The sequence GZZP(R/K)X (Z = R, K or H, but may also contain N, S, or Q) was unusually overrepresented. Selected antibodies reacted with HS/heparin, but not with other glycosaminoglycans. Antibodies reacted differentially with respect to N-, 2-O, or 6-O-desulfated heparin preparations, and showed distinct topologies of HS epitopes in rat kidney sections. The library may be instrumental in the selection of a large pool of HS epitope-specific antibodies, and - since all antibodies differ only in their 6 amino acid CDR region - may be a tool for a rational design of antibodies recognizing specific HS sulfation patterns.

Highlights

Heparan sulfate (HS) is a long heterogeneously sulfated glycosaminoglycan, predominantly present in the extracellular matrix and at the cell surface
Screening of complementarity determining region 3 (CDR3) sequences demonstrated that 23.3% (44/194) of the randomly selected clones contained a XBBXBX amino acid sequence, similar to what was theoretically estimated (24.4%) (Table 2)
The location of the epitope defined by the anti-HS scFv anti- In this study, we successfully developed a new antibody phage bodies was analyzed in rat kidney cryosections and compared display library designed to target HS by engineering the CDR3 in Position in the CDR3 VH

Summary

Introduction

Heparan sulfate (HS) is a long heterogeneously sulfated glycosaminoglycan, predominantly present in the extracellular matrix and at the cell surface. Glycoconj J (2020) 37:445–455 antibody phage-display library [8], many single chain variable fragment (scFv) antibodies have been selected against different sources of HS [9,10,11,12,13,14,15]. The recognition of specific HS structures by antibodies from this library is primarily generated by the VH chain, especially the complementarity determining region 3 (CDR3) [8, 22]. Heparin binding consensus sites include XBBXBX, XBBBXXBX and XBBBXXBBBXXBBX amino acid sequences where ‘B’ is a basic and ‘X’ is a random amino acid residue [13, 25, 26]. These sequences are found in several, but not all, HS-binding proteins [25,26,27,28]

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