Identification of heparin-binding amino acid residues in antibody HS4C3 with the potential to design antibodies against heparan sulfate domains.

Heparan sulfate (HS) is a linear polysaccharide with high structural and functional diversity. Detection and localization of HS in tissues can be performed using single chain variable fragment (scFv) antibodies. Although several anti-HS antibodies recognizing different sulfation motifs have been identified, little is known about their interaction with HS. In this study the interaction between the scFv antibody HS4C3 and heparin was investigated. Heparin-binding lysine and arginine residues were identified using a protect and label methodology. Site-directed mutagenesis was applied to further identify critical heparin-binding lysine/arginine residues using immunohistochemical and biochemical assays. In addition, computational docking of a heparin tetrasaccharide towards a 3-D homology model of HS4C3 was applied to identify potential heparin-binding sites. Of the 12 lysine and 15 arginine residues within the HS4C3 antibody, 6 and 9, respectively, were identified as heparin-binding. Most of these residues are located within one of the complementarity determining regions (CDR) or in their proximity. All basic amino acid residues in the CDR3 region of the heavy chain were involved in binding. Computational docking showed a heparin tetrasaccharide close to these regions. Mutagenesis of heparin-binding residues reduced or altered reactivity towards HS and heparin. Identification of heparin-binding arginine and lysine residues in HS4C3 allows for better understanding of the interaction with HS and creates a framework to rationally design antibodies targeting specific HS motifs.

Development of prediction models for complications after primary total hip and knee arthroplasty: a single-centre retrospective cohort study in the Netherlands.

OBJECTIVE: The aim of this study was to develop prediction models for patients with total hip arthroplasty (THA) and total knee arthroplasty (TKA) to predict the risk for surgical complications based on personal factors, comorbidities and medication use. DESIGN: Retrospective cohort study. SETTING: Tertiary care in outpatient clinic of university medical centre. PARTICIPANTS: 3776 patients with a primary THA or TKA between 2004 and 2018. PRIMARY AND SECONDARY OUTCOME MEASURES: Multivariable logistic regression models were developed for primary outcome surgical site infection (SSI), and secondary outcomes venous thromboembolism (VTE), postoperative bleeding (POB), luxation, delirium and nerve damage (NER). RESULTS: For SSI, age, smoking status, body mass index, presence of immunological disorder, diabetes mellitus, liver disease and use of non-steroidal anti-inflammatory drugs were included. An area under the receiver operating characteristic curve (AUC) of 71.9% (95% CI=69.4% to 74.4%) was found. For this model, liver disease showed to be the strongest predictor with an OR of 10.7 (95% CI=2.4 to 46.6). The models for POB and NER showed AUCs of 73.0% (95% CI=70.7% to 75.4%) and 76.6% (95% CI=73.2% to 80.0%), respectively. For delirium an AUC of 85.9% (95% CI=83.8% to 87.9%) was found, and for the predictive algorithms for luxation and VTE we found least favourable results (AUC=58.4% (95% CI=55.0% to 61.8%) and AUC=66.3% (95% CI=62.7% to 69.9%)). CONCLUSIONS: Discriminative ability was reasonable for SSI and predicted probabilities ranged from 0.01% to 51.0%. We expect this to enhance shared decision-making in considering THA or TKA since current counselling is predicated on population-based probability of risk, rather than using personalised prediction. We consider our models for SSI, delirium and NER appropriate for clinical use when taking underestimation and overestimation of predicted risk into account. For VTE and POB, caution concerning overestimation exceeding a predicted probability of 0.08 for VTE and 0.05 for POB should be taken into account. Furthermore, future studies should evaluate clinical impact and whether the models are feasible in an external population.

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.