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

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. Electronic supplementary material The online version of this article (10.1007/s10719-020-09925-z) contains supplementary material, which is available to authorized users.


Introduction
Heparan sulfate (HS) is a long heterogeneously sulfated glycosaminoglycan, predominantly present in the extracellular matrix and at the cell surface. During its biosynthesis, HS is modified by sulfation and epimerization reactions, resulting in a high structural and functional diversity [1,2]. Specific sulfation motifs within HS are involved in several physiological processes like cell signaling [2,3], blood clotting [4], and pathological processes such as tumorigenesis [5] and Alzheimer's disease [6,7]. However, it remains a challenge to identify and localize HS with specific structural motifs in tissues. With the development of the 'single pot' Nissim 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]. These scFv antibodies consist of the variable part of the heavy (V H ) and light chain (V L ) of an immunoglobulin, joined together by a linker. In the Nissim library, the V H chain is variable, whereas the V L is fixed [8,16]. About 45% of the scFv antibodies selected against HS harbor a V H dp-38 germline gene segment [13][14][15][17][18][19][20][21]. The recognition of specific HS structures by antibodies from this library is primarily generated by the V H chain, especially the complementarity determining region 3 (CDR3) [8,22]. The CDR3 is the region with most conformational variability and flexibility and is thought to have a major influence in epitope binding [11-14, 23, 24]. About 17% of the anti-HS antibodies published contain a heparin binding consensus site in the CDR3 region of the V H chain [13][14][15][17][18][19][20][21]. 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].
To expand the number of antibodies against HS, we here engineered and evaluated an antibody phage display library targeted for HS with a single V H germline segment (dp-38) and a CDR3 consisting of an XBBXBX heparin binding consensus site. As a template we used a previously obtained scFv antibody (HS4C3) which harbors both a dp-38 germline gene segment and a CDR3 sequence containing an XBBXBX heparin binding consensus site (GRRLKD) [11,13].

Materials & methods
Construction of an antibody phage display library with a XBBXBX CDR3 sequence The phage display library was constructed to contain only scFv antibodies harboring the V H 3 dp-38 germline with an XBBXBX CDR3 amino acid sequence. Using the wellcharacterized HS4C3 scFv antibody from the Nissim library as template, the library was constructed in a series of 5 PCR procedures (PCR 1-PCR 5) (Fig. 1) [8,13]. In all procedures, a general PCR mixture was used containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM MgCl 2 , 0.5 μM primers (Table 1), 2.5 units Taq polymerase (Promega), and 0.75-1 mM dNTP in a total volume of 50 μL. The cycling temperatures were 5 min 95°C followed by 25-40 cycles of 1 min at 95°C, 1 min at 46-50°C for annealing and 74°C for 1.5-2 min for extension. Detailed PCR procedures are listed in Supplementary Table 1. In PCR 1, the V H (without the CDR3 and framework 4) of the HS4C3 gene was amplified. In PCR 2, the V L (including the c-Myc tag), the linker sequence and the framework 4 of the V H of the HS4C3 gene were amplified. The PCR 3 procedure was performed to introduce a new CDR3 into the V H gene using an 87 nucleotide degenerate reverse primer. This primer contained the CDR3 encoded as 5'-SNN NYK SNN NYK NYK SNN -3′, where S encodes for G or C; Y for C or T; K for G or T; and N for A, C, G, or T, to obtain the XBBXBX sequence (Table 1). Next, we combined the new V H genes and the V L gene in PCR4 using overlapping sequences (FR4 of the V H ). The product of PCR 4 was evaluated on 1% agarose gel and isolated using the QiaEX II agarose gel extraction kit (Qiagen GmbH, Hilden, Germany). The purified product was amplified in PCR 5. The final product was purified after 1% agarose gel electrophoresis using phenol/chloroform extraction. DNA was precipitated using ethanol, the pellet was washed with 70% ethanol and dissolved in 50 μL water. The final product was digested using NcoI and NotI restriction enzymes (Life Technologies BV, Breda, the Netherlands) and ligated overnight at 16°C into a NcoI and NotI digested pHEN1 vector (a kind gift of prof. G. Winter, Cambridge) using the Sureclone Ligation Kit (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The ligation product was purified using phenol/ chloroform extraction. The ligation product was transformed via electroporation into E. coli TG1 (K12, supE, hsdΔ5, thi, Δ(lac-proAB, F′(traD36, proAB + , lacI q , lac-ZΔM15) electroporation competent cells, efficiency ≥1.0*10 10 cfu/μg (Stratagene Cloning Systems, La Jolla, USA). To calculate the titer of transformants, dilutions of the original electroporated bacteria were plated onto 2xTY agar plates containing 0.1% (w/v) ampicillin and 1% (w/v) glucose and incubated overnight at 37°C.

Random selection of clones
The library was plated on large 2xTY agar plates containing 0.1% (w/v) ampicillin and 1% (w/v) glucose and incubated overnight at 37°C. Subsequently, 240 clones were randomly picked from the plate and cultured overnight in LB medium containing 1% (w/v) glucose and 0.1% (w/v) ampicillin. Plasmid DNA was collected using the QIAprep spin Miniprep Kit (Qiagen, Leusden, the Netherlands) and the V H and CDR3 was sequenced using the PelB, forlinkseq and VL3 primer ( Table 1).

Selection of new scFv antibodies
Phage display biopanning procedures was performed as described previously [10]. Tubes were coated by incubation with 10 μg/mL HS from bovine kidney (Seikagaku Kogyo Co, Tokyo, Japan), HS from intestinal mucosa (Sigma), or HS isolated from human lung [17], all in 90% (w/v) (NH 4 ) 2 SO 4 [29]. Phages were produced from the library by addition of helper phage VCS-M13 (Stratagene, La Jolla, CA). Phages were precipitated and isolated using 20% (w/v) PEG/2.5 M NaCl solution and added to the coated tubes. Phages were allowed to bind for 2 h under rotation. Non-bound phages were washed away, and bound phages were eluted with 1 mL 100 mM triethylamine for 10 min. In some cases, the 10 min step was followed by an additional 20 min elution step with 1 mL 100 mM triethylamine, to retrieve phages that remained bound in the first elution step. The triethylamine was neutralized by addition of 0.5 mL 1 M Tris-HCl (pH 7.3). TG1 cells were infected with the collected phages and plated onto 2xTY agar plates containing 0.1% (w/v) ampicillin.

Initial screening for unique antibodies against HS
Screening of scFv was performed as described previously [10,13]. A 96-wells flat-bottom polystyrene plate was incubated overnight with 100 μL of 90% (w/v) (NH 4 ) 2 SO 4 containing 10 μg/mL HS from bovine kidney (Seikagaku), HS from porcine intestinal mucosa, heparin from porcine intestinal mucosa, dermatan sulfate from pig skin, chondroitin sulfate A (CSA) from bovine trachea, chondroitin sulfate C (CSC) from shark cartilage, thymus DNA, and hyaluronic acid from rooster comb (all from Sigma, St. Louis, MO). After washing, Fig. 1 Schematic overview of PCR reactions used to construct the XBBXBX CDR3 antibody library. In PCR 1, the heavy variable chain (V H ) up to framework 3 (FR3) of the HS4C3 coding sequence was amplified. In PCR 2, framework 4 (FR4) of V H and the light variable chain (V L ) of the HS4C3 coding sequence was amplified. The XBBXBX complementarity determining region 3 (CDR3) was engineered using the degenerate oligonucleotide CDR3 primer in PCR 3. The new V H genes were combined with the V L gene in PCR 4 and subsequently amplified in PCR 5. Note that the CDR3 primer contains, next to the CDR3 region, the FR4 region and part of the FR3 region. For primers, see Table 1. scFv: single chain variable fragment antibody.

Large-scale antibody production
For further characterization of the antibodies, scFv were obtained from the periplasmic fraction as described [13]. Bacteria were grown overnight at 37°C in 15 mL LB-medium (100 μg/mL ampicillin and 1% (w/v) glucose). Subsequently, 10 mL of the overnight culture was added to 1 L induction medium (2xTY medium containing 0.1% (w/v) glucose and 0.1% (w/v) ampicillin). At an OD 600 of 0.6, the antibody expression was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG, UBPBio, Oxfordshire, UK) in a final concentration of 1 mM, and incubated for three h at 30°C. The cultures were centrifugated for 15 min at 3561 g to separate bacteria from culture medium. Bacterial pellet was resuspended and incubated for 15 min on ice in 10 mL 0.2 mM sodium borate buffer (pH 8.0) containing 0.16 M NaCl to accommodate osmotic shock. This buffer was enriched with protease inhibitors (10 mM N-ethylmaleimide, 100 mM 6-amino hexane acid, 10 mM benzamidine-HCl, 5 mM iodoacetamide, 0.15 μM pepstatin, 30 nM apoprotein, and 100 μM phenylmethylsulfonyl fluoride), supplemented with 1% (w/v) NaN 3 . After centrifugation for 15 min at 10,000 g at 4°C, supernatant was passed over a PBS wetted 0.45 μm filter and subsequently dialyzed against PBS, using a Visking 12-14 kDa MWCO dialysis hose (VWR, Amsterdam, NL).

Characterization of scFv antibodies
ELISA. Epitope specificity of the scFv antibodies was further analyzed using an ELISA setting. The wells of a 96-wells flat bottom polystyrene microplate (Greiner Bio-One, Alphen aan de Rijn, the Netherlands) were coated by incubating with 100 μL of 90% (w/v) (NH 4 ) 2 SO 4 containing 10 μg/mL heparin, N-desulfated/N-acetylated heparin, 2-O desulfated heparin or 6-O desulfated heparin (kind gifts of dr. A. Naggi and prof. Casu of the G. Ronzoni Institute for Chemical and Biochemical Research, Milan, Italy [30,31]). Composition of the modified heparins are listed in Supplementary Table 2 [31]. Plates were washed with PBST and blocked with 3% (w/v) BSA with 1% (v/v) Tween 20 in PBS. Periplasmic fractions containing the scFv antibodies were diluted 1:1 in 1% (w/v) BSA/PBST and incubated for 1.5 h. Bound scFv antibodies were detected as described above, using mouse anti-c-Myc antibody 9E10 and alkaline phosphatase conjugated goat anti-mouse antibodies, and visualized using alkaline phosphatase substrate disodium p-4-nitrophenylphosphate.

Library
The library was constructed using a degenerated primer, aimed to introduce a new CDR3 consisting of an XBBXBX heparin binding consensus sequence. The primer design was such that there is a 62.5% chance of a basic amino acid (R, K or H) at the 'B' site, the other possibilities being Q, S or N. The latter amino acids are also frequently involved in the binding of heparin/HS [25,32].
The theoretical diversity of the library is 1.34 × 10 8 unique clones, encoding for 1.73 × 10 6 unique full-length proteins. To obtain an impression of the actual diversity of the library, 222 randomly picked clones were sequenced, yielding 194 full sequences of which 189 (97.4%) were unique (  Table 2). Selection against HS (see below) yielded 67 unique scFv antibodies ( Table 3). The XBBXBX CDR3 was more abundant in the scFv selected against HS (46%, 33/67) than in randomly picked clones (23.3%) (p < 0.01) ( Table 2). In addition, 35 of the 67 (52%) scFv antibodies selected against HS had glycine (G) as the first amino acid residue, and 30 of the 67 (44.5%) contained proline (P) as the fourth amino acid residue (Table 4). A combination of G at the first position and P at the fourth, together with a R or K at the fifth position, was observed 23 times (34.4%) ( Table 2). This combination was completely absent in randomly selected clones (theoretically 0.2% of all clones harbor this sequence ( Table 2)).

Biopanning of library against various HS and characterization of antibodies
After biopanning against immobilized HS, 25 unique antibodies were selected against bovine kidney (10 min elution), and 30 after an additional 20 min elution (Table 3). Further, 5 antibodies were selected against HS from human lung, and 7 against porcine intestinal mucosa after 10 min elution ( Table 3). The reactivity of all antibodies with various types of glycosaminoglycans and modified heparin preparations was determined (Supplementary Table 4 and 5), as well as the location of antibody-defined HS epitopes in the kidney (Supplementary Table 6). To illustrate the characteristics of some anti-HS antibodies, we here highlight two antibodies selected against bovine kidney after 10 min elution (MP3C1 and MP4A11'10.b.k.), two against bovine kidney after an additional 20 min elution (MP4F11 and MP3B2'20.b.k.), two against HS from porcine mucosa (MP4G5, and MP3G7), and one against human lung HS (MP3A5) ( Table 3). One randomly picked scFv antibody (MPB49) that was not reactive with HS was also included. Antibody specificity was screened using ELISA. Anti-HS antibodies were moderate to strong binders to heparin ( Table 5). All antibodies were moderate to strong binders to HS from bovine kidney, whereas antibodies MP4G5 and MP4F11 showed strong binding towards HS from intestinal mucosa. Antibodies were not reactive with other glycosaminoglycans (chondroitin sulfate A and C, dermatan sulfate, hyaluronic acid) and DNA, with the exception of MP4F11 which showed moderate binding to dermatan sulfate ( Table 5).

Analysis of sulfate groups in HS-epitope of scFv antibodies
Reactivity of the anti-HS antibodies was further studied using modified heparin preparations (N-desulfated/N-acetylated, 2-O desulfated, and 6-O desulfated heparin; Table 6). Reactivity of MP4F11 and MP3G7 was observed towards all desulfated forms of heparin, indicating that both antibodies recognize

Localization of HS epitopes in rat kidney sections
The location of the epitope defined by the anti-HS scFv antibodies was analyzed in rat kidney cryosections and compared to the original HS4C3 antibody. Although all scFvs have a unique CDR3 sequence, three main patterns of staining were observed (Fig. 2, Table 7). MP3C1 ( Fig. 2A), MP3B2'20.b.k. and MP3A5 mainly stained the glomerulus and the peritubular capillaries, while MP4A11'10.b.k. (Fig. 2B) and MP3G7 primarily recognized Bowman's capsule. Both Bowman's capsule and the glomerulus were recognized by MP4F11 (Fig.  2C), MP4G5 and MP4F11). Antibody MPB49 (randomly selected) was not reactive with kidney sections (Fig. 2E).

Library development
In this study, we successfully developed a new antibody phage display library designed to target HS by engineering the CDR3 in  CDR3  MP4F5  TRRLKS  MP3E5  WRQRQA  MP3D9  LRRAQP  MP3H3  GRRHSA  MP4F10  SRRHRS  MP4E8  VRRSRT  MP3A11  GSRLRR  MP4A9  GQRMKH  MP3C1  SRRHKN  MP4H12  TRRPKT  MP4H8  GRKPRT  MP3F5  GQRHSA  MP3H1  RRNPKL  MP4B11  TRKPRR  MP4G5  GRKPRL  MP3D6  GNRLRS  MP3C8  RRHPQR  MP3C2  TKHQKR  MP3A4  GKSPRK  MP3A5  GNQQRH  MP4B9  NRRSKT  MP4B9  SRNTKA  MP3G7  GKQPRK  MP3D5  MRKTHT  MP4G9  RRQPRT  MP3B9  GKKPRR  MP3H10  IRSLKP  MP3B2  NRRHRT  MP3C2  GSKQRV  MP4B12  NRRARQ  MP3G6 GSKPRR ARHIQT Antibodies highlighted in yellow contain a XBBXBX CDR3 sequence; antibodies in red contain a GZZP(R/K)X CDR3 sequence, where 'B' encodes the basic residues H, R, or K; 'X' encodes any amino acid and 'Z' encodes H, R, K, N, Q, or S. a the 20 min. elution was performed after an initial 10 min elution. HS: heparan sulfate the V H of the antibodies. The CDR3 is thought to be the largest contributor to antigen recognition and in this study we designed it to contain a heparin binding consensus site [33]. In proteins, XBBXBX, XBBBXXBX, and XBBBXXBBBXXBBX sequences have been described as heparin binding consensus sites [26]. We selected the XBBXBX sequence, since a number of the previously obtained anti-HS scFvs contain short (<10 amino acids) CDR3 sequences, which may be favorable for binding [13,14,34]. To engineer the XBBXBX CDR3, we used a degenerate primer, which also allowed S, N, and Q in the 'B' positions. As a result, 23.5% of our library contained the actual XBBXBX amino acid sequence in the CDR3. The XBBXBX consensus site is not essential for protein-HS interaction, although many proteins use it [28,35]. Of the selected anti-HS antibodies 46% contained a XBBXBX CDR3 sequence. The presence of S, N, or Q at the 'B' positions of the CDR3 was found in a number of anti-HS scFv antibodies indicating that the XBBXBX is not an absolute requirement for binding. Interestingly, these residues have been described in heparinbinding peptides [25], and a number of previously obtained anti-HS scFv antibodies contain these residues within their CDR3 [12][13][14][15].
When evaluating the 'X' residues within the XBBXBX CDR3 of anti-HS scFvs, an abundance of G and P at the first and fourth position respectively, was noticed. These residues have been described to provide structural stability to a loop structure in the CDR3 [36]. Moreover, these residues have been frequently found in heparin-binding peptides alongside R, K, H, S, N, and Q [25]. Anti-HS scFv antibodies from the library seem to have a preference for the GZZP(R/K)X sequence, where 'Z' encodes R, K, H, S, N, or Q. Since the amino acids G and P are frequently found in turns, bends and folds [37][38][39][40] the GZZP(R/K)X sequence may form a charged pocked structure that binds to a specific monosaccharide or a specific spatial orientation of sulfate groups, which are generally present on the outside of a HS molecule. The G and P would then contribute to the formation of a pocket, and the positively charged amino acids would confer binding to the negatively charged saccharide.

Anti-HS scFvs on design
In this study we aimed to obtain new anti-HS scFv directed towards different HS-epitopes by engineering the CDR3. Since this approach was successful, applying specific modifications to the anti-HS scFvs may prove to be effective to obtain antibodies against specific sulfation patterns. These modifications may not be limited towards the CDR3, since regions located outside the linear heparin binding site have been demonstrated to influence HS binding [45][46][47]. Knowledge on the specific amino acids involved in HS binding may allow for a rational design of antibodies targeting specific sulfation patterns. Strategies such as "protect and label" can identify essential amino acids involved in HS binding [48]. Once identified, site-directed mutagenesis of the CDR3 and other regions within V H and/or V L may result in the generation of anti-HS scFvs recognizing specific sulfation patterns. These tools may be instrumental in elucidating of the function of HS sulfation patterns in (patho)physiological processes.
In conclusion, we successfully constructed a new anti-HS scFv library, engineering the CDR3 to contain the heparinbinding consensus site XBBXBX. The library may be a rich source of new antibodies directed against different HS-epitope, and may allow the design of novel antibodies recognizing specific sulfation patterns.

Compliance with ethical standards
Conflicts of interest The authors declare that they have no conflicts of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.
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