Antibody CDR amino acids underlying the functionality of antibody repertoires in recognizing diverse protein antigens.

Antibodies recognize protein antigens with exquisite specificity in a complex aqueous environment, where interfacial waters are an integral part of the antibody-protein complex interfaces. In this work, we elucidate, with computational analyses, the principles governing the antibodies' specificity and affinity towards their cognate protein antigens in the presence of explicit interfacial waters. Experimentally, in four model antibody-protein complexes, we compared the contributions of the interaction types in antibody-protein antigen complex interfaces with the antibody variants selected from phage-displayed synthetic antibody libraries. Evidently, the specific interactions involving a subset of aromatic CDR (complementarity determining region) residues largely form the predominant determinant underlying the specificity of the antibody-protein complexes in nature. The interfacial direct/water-mediated hydrogen bonds accompanying the CDR aromatic interactions are optimized locally but contribute little in determining the epitope location. The results provide insights into the phenomenon that natural antibodies with limited sequence and structural variations in an antibody repertoire can recognize seemingly unlimited protein antigens. Our work suggests guidelines in designing functional artificial antibody repertoires with practical applications in developing novel antibody-based therapeutics and diagnostics for treating and preventing human diseases.

Sensitivity of Ca2+-sensing receptor-transient receptor potential-mediated Ca2+ influx to extracellular acidity in bEND.3 endothelial cells.

Ca2+-sensing receptors (CaSRs) are G protein-coupled receptors activated by elevated concentrations of extracellular Ca2+. In our previous works, we showed protein and functional expression of CaSR in mouse cerebral endothelial cell (EC) (bEND.3); the CaSR response (high Ca2+-elicited cytosolic [Ca2+] elevation) was unaffected by suppression of phospholipase C but in part involved Ca2+ influx through transient receptor potential V1 (TRPV1) channels. In this work, we investigated if extracellular acidity affected CaSR-mediated Ca2+ influx triggered by high (3 mM) Ca2+ (CaSR agonist), 3 mM spermine (CaSR agonist), and 10 mM cinacalcet (positive allosteric modulator of CaSR). Extracellular acidosis (pH 6.8 and pH 6.0) strongly suppressed cytosolic [Ca2+] elevation triggered by high Ca2+, spermine, and cinacalcet; acidosis also inhibited Mn2+ influx stimulated by high Ca2+ and cinacalcet. Purinoceptor-triggered Ca2+ response, however, was not suppressed by acidosis. Extracellular acidity also did not affect membrane potential, suggesting suppressed CaSR-mediated Ca2+ influx in acidity did not result from the reduced electrical driving force for Ca2+. Our results suggest Ca2+ influx through a putative CaSR-TRP complex in bEND.3 EC was sensitive to extracellular pH.

Functionalized Terpolymer-Brush-Based Biointerface with Improved Antifouling Properties for Ultra-Sensitive Direct Detection of Virus in Crude Clinical Samples.

New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.

Eradicating mesothelin-positive human gastric and pancreatic tumors in xenograft models with optimized anti-mesothelin antibody-drug conjugates from synthetic antibody libraries.

Mesothelin (MSLN) is an attractive candidate of targeted therapy for several cancers, and hence there are increasing needs to develop MSLN-targeting strategies for cancer therapeutics. Antibody-drug conjugates (ADCs) targeting MSLN have been demonstrated to be a viable strategy in treating MSLN-positive cancers. However, developing antibodies as targeting modules in ADCs for toxic payload delivery to the tumor site but not to normal tissues is not a straightforward task with many potential hurdles. In this work, we established a high throughput engineering platform to develop and optimize anti-MSLN ADCs by characterizing more than 300 scFv CDR-variants and more than 50 IgG CDR-variants of a parent anti-MSLN antibody as candidates for ADCs. The results indicate that only a small portion of the complementarity determining region (CDR) residues are indispensable in the MSLN-specific targeting. Also, the enhancement of the hydrophilicity of the rest of the CDR residues could drastically increase the overall solubility of the optimized anti-MSLN antibodies, and thus substantially improve the efficacies of the ADCs in treating human gastric and pancreatic tumor xenograft models in mice. We demonstrated that the in vivo treatments with the optimized ADCs resulted in almost complete eradication of the xenograft tumors at the treatment endpoints, without detectable off-target toxicity because of the ADCs' high specificity targeting the cell surface tumor-associated MSLN. The technological platform can be applied to optimize the antibody sequences for more effective targeting modules of ADCs, even when the candidate antibodies are not necessarily feasible for the ADC development due to the antibodies' inferior solubility or affinity/specificity to the target antigen.

SIGLEC-3 (CD33) serves as an immune checkpoint receptor for HBV infection.

Chronic hepatitis B (CHB) infection is rarely eradicated by current antiviral nucleos(t)ide analogues. We found that α2,6-biantennary sialoglycans of HBV surface antigen (HBsAg) bound human SIGLEC-3 (CD33) by IP and ELISA, and the binding affinity between SIGLEC-3 and α2,6-biantennary sialoglycans was determined by biolayer interferometry (equilibrium dissociation constant [KD]: 1.95 × 10-10 ± 0.21 × 10-10 M). Moreover, HBV activated SIGLEC-3 on myeloid cells and induced immunosuppression by stimulating immunoreceptor tyrosine-based inhibitory motif phosphorylation and SHP-1/-2 recruitment via α2,6-biantennary sialoglycans on HBsAg. An antagonistic anti-SIGLEC-3 mAb reversed this effect and enhanced cytokine production in response to TLR-7 agonist GS-9620 in PBMCs from CHB patients. Moreover, anti-SIGLEC-3 mAb alone was able to upregulate the expression of molecules involved in antigen presentation, such as CD80, CD86, CD40, MHC-I, MHC-II, and PD-L1 in CD14+ cells. Furthermore, SIGLEC-3 SNP rs12459419 C, which expressed a higher amount of SIGLEC-3, was associated with increased risk of hepatocellular carcinoma (HCC) in CHB patients (HR: 1.256, 95% CI: 1.027-1.535, P = 0.0266). Thus, blockade of SIGLEC-3 is a promising strategy to reactivate host immunity to HBV and lower the incidence of HCC in the CHB patient population.

Endosomal TLR3 co-receptor CLEC18A enhances host immune response to viral infection.

Human C-type lectin member 18A (CLEC18A) is ubiquitously expressed in human, and highest expression levels are found in human myeloid cells and liver. In contrast, mouse CLEC18A (mCLEC18A) is only expressed in brain, kidney and heart. However, the biological functions of CLEC18A are still unclear. We have shown that a single amino acid change (S339 →R339) in CTLD domain has profound effect in their binding to polysaccharides and house dust mite allergens. In this study, we further demonstrate that CLEC18A and its mutant CLEC18A(S339R) associate with TLR3 in endosome and bind poly (I:C) specifically. Compared to TLR3 alone, binding affinity to poly (I:C) is further increased in TLR3-CLEC18A and TLR3-CLEC18A(S339R) complexes. Moreover, CLEC18A and CLEC18A(S339R) enhance the production of type I and type III interferons (IFNs), but not proinflammatory cytokines, in response to poly (I:C) or H5N1 influenza A virus (IAV) infection. Compared to wild type (WT) mice, ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice generate higher amounts of interferons and are more resistant to H5N1 IAV infection. Thus, CLEC18A is a TLR3 co-receptor, and may contribute to the differential immune responses to poly (I:C) and IAV infection between human and mouse.

A panel of anti-influenza virus nucleoprotein antibodies selected from phage-displayed synthetic antibody libraries with rapid diagnostic capability to distinguish diverse influenza virus subtypes.

Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10-15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90-94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.

Effective binding to protein antigens by antibodies from antibody libraries designed with enhanced protein recognition propensities.

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.

Antibody-drug conjugates with HER2-targeting antibodies from synthetic antibody libraries are highly potent against HER2-positive human gastric tumor in xenograft models.

HER2-ECD (human epidermal growth factor receptor 2 - extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies' propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.

Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy for peritoneal malignancy: preliminary results of a multi-disciplinary teamwork model in Asia.

BACKGROUND: Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) is an emerging surgical procedure for peritoneal carcinomatosis (PC). CRS/HIPEC is a complicated treatment that requires multi-disciplinary teamwork (MDT), which may be lacking when establishing a CRS/HIPEC programme. Herein, we report our preliminary treatment outcomes with the early implementation of an MDT model for CRS/HIPEC. METHODS: From April 2015 to December 2016, 45 patients with a diagnosis of PC who received CRS/HIPEC were reviewed retrospectively in a single institution in Taiwan. RESULTS: Among the 45 patients, CRS was mainly performed by laparotomy (n = 42), and only three patients with limited PC underwent laparoscopic CRS. The first 13 patients received treatment before the MDT had been established (group 1), and the other 32 patients were treated after the MDT had been established (group 2). The highest peri-HIPEC body temperature in group 2 was significantly lower than that in group 1 (36.8 °C vs. 37.5 °C, p < 0.001). Overall, eight patients experienced major complications. The trend of a lower major complication rate was observed after the MDT model had been implemented (30.7% in group 1 vs. 12.4% in group 2, p = 0.202). Pre-CRS/HIPEC abdominal pain significantly increased the risk of post-operative major complications (p = 0.017). CONCLUSIONS: Our experience suggests that the early implementation of an MDT model when establishing a CRS/HIPEC programme at a single institution may result in a higher complete cytoreduction rate and lower major complication rate, and also shorten the learning curve of this complicated procedure.

High throughput discovery of influenza virus neutralizing antibodies from phage-displayed synthetic antibody libraries.

Pandemic and epidemic outbreaks of influenza A virus (IAV) infection pose severe challenges to human society. Passive immunotherapy with recombinant neutralizing antibodies can potentially mitigate the threats of IAV infection. With a high throughput neutralizing antibody discovery platform, we produced artificial anti-hemagglutinin (HA) IAV-neutralizing IgGs from phage-displayed synthetic scFv libraries without necessitating prior memory of antibody-antigen interactions or relying on affinity maturation essential for in vivo immune systems to generate highly specific neutralizing antibodies. At least two thirds of the epitope groups of the artificial anti-HA antibodies resemble those of natural protective anti-HA antibodies, providing alternatives to neutralizing antibodies from natural antibody repertoires. With continuing advancement in designing and constructing synthetic scFv libraries, this technological platform is useful in mitigating not only the threats of IAV pandemics but also those from other newly emerging viral infections.

High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries.

Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates.

Discovering neutralizing antibodies targeting the stem epitope of H1N1 influenza hemagglutinin with synthetic phage-displayed antibody libraries.

Broadly neutralizing antibodies developed from the IGHV1-69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. Such understanding could inform designs of synthetic antibody libraries targeting the stem epitope on hemagglutinin, leading to artificially designed antibodies that are functionally advantageous over antibodies from natural antibody repertoires. In this work, the sequence space of the complementarity determining regions of a broadly neutralizing antibody (F10) targeting the stem epitope on the hemagglutinin of a strain of H1N1 influenza virus was systematically explored; the elucidated antibody-hemagglutinin recognition principles were used to design a phage-displayed antibody library, which was then used to discover neutralizing antibodies against another strain of H1N1 virus. More than 1000 functional antibody candidates were selected from the antibody library and were shown to neutralize the corresponding strain of influenza virus with up to 7 folds higher potency comparing with the parent F10 antibody. The antibody library could be used to discover functionally effective antibodies against other H1N1 influenza viruses, supporting the notion that target-specific antibody libraries can be designed and constructed with systematic sequence-function information.

Predominant structural configuration of natural antibody repertoires enables potent antibody responses against protein antigens.

Humoral immunity against diverse pathogens is rapidly elicited from natural antibody repertoires of limited complexity. But the organizing principles underlying the antibody repertoires that facilitate this immunity are not well-understood. We used HER2 as a model immunogen and reverse-engineered murine antibody response through constructing an artificial antibody library encoded with rudimentary sequence and structural characteristics learned from high throughput sequencing of antibody variable domains. Antibodies selected in vitro from the phage-displayed synthetic antibody library bound to the model immunogen with high affinity and specificities, which reproduced the specificities of natural antibody responses. We conclude that natural antibody structural repertoires are shaped to allow functional antibodies to be encoded efficiently, within the complexity limit of an individual antibody repertoire, to bind to diverse protein antigens with high specificity and affinity. Phage-displayed synthetic antibody libraries, in conjunction with high-throughput sequencing, can thus be designed to replicate natural antibody responses and to generate novel antibodies against diverse antigens.

Antibody variable domain interface and framework sequence requirements for stability and function by high-throughput experiments.

Protein structural stability and biological functionality are dictated by the formation of intradomain cores and interdomain interfaces, but the intricate sequence-structure-function interrelationships in the packing of protein cores and interfaces remain difficult to elucidate due to the intractability of enumerating all packing possibilities and assessing the consequences of all the variations. In this work, groups of ß strand residues of model antibody variable domains were randomized with saturated mutagenesis and the functional variants were selected for high-throughput sequencing and high-throughput thermal stability measurements. The results show that the sequence preferences of the intradomain hydrophobic core residues are strikingly flexible among hydrophobic residues, implying that these residues are coupled indirectly with antigen binding through energetic stabilization of the protein structures. By contrast, the interdomain interface residues are directly coupled with antigen binding. The interdomain interface should be treated as an integral part of the antigen-binding site.

Effect of hyperbaric oxygenation on intervertebral disc degeneration: an in vivo study with sprague-dawley rats.

STUDY DESIGN: An in vivo study was conducted to test the effect of hyperbaric oxygenation (HBO) on intervertebral disc degeneration in Sprague-Dawley rats. OBJECTIVE: To observe the changes in intervertebral disc height and levels of glycosaminoglycan, collagen, interleukin-1ß (IL-1ß), prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) in degenerated intervertebral discs after HBO therapy. SUMMARY OF BACKGROUND DATA: Although the involvement of IL-1ß, PGE-2, NO, and low O2 concentration has been demonstrated in intervertebral disc degeneration, the actual mechanism is not clear. It has been reported that HBO influences changes in IL-1ß, PGE-2, NO, and O2 concentration. Previously, a study demonstrated an in vitro positive effect of HBO on the human nucleus pulposus. Thus, an in vivo study in animals was necessary. METHODS: Twelve Sprague-Dawley rats were each injected with chondroitinase ABC in 2 proximal intervertebral discs of the tail. After treating with 100% oxygen at 2.5 atmospheres 2 hours per days for 10 days, the change in disc height was determined by radiography. The amounts of PGE-2, iNOS, glycosaminoglycan, and total collagen in the intervertebral disc were quantified by enzyme-linked immunosorbent assay. Tissue morphology and the distribution of glycosaminoglycan, IL-1ß, and iNOS in the intervertebral disc were assessed by histology and immunohistochemistry. The area of IL-1ß in the intervertebral discs was quantified using image analysis software. RESULTS: HBO therapy stopped the decrease in intervertebral disc height, caused an increase in the amount of glycosaminoglycan, and inhibited IL-1ß, PGE-2, and iNOS production. CONCLUSION: HBO provides a potential treatment modality for intervertebral disc degeneration.

Classification and analysis of pathology of the long head of the biceps tendon in complete rotator cuff tears.

BACKGROUND: Pathology of the long head of the biceps tendon (LHB) is commonly associated with rotator cuff tears (RCTs). Superior labral anterior-posterior (SLAP) lesions can also occur with RCTs. The purpose of this study was to include SLAP lesions as part of LHB pathology in surgical cases of RCT and define the role of SLAP lesions in RCTs. METHODS: We retrospectively evaluated clinical data from 176 cases of complete RCT undergoing surgery. During surgery, the LHB was arthroscopically examined. A modified 6-type classification was used to describe the LHB pathology in these cases: tendinitis, subluxation, dislocation, partial tear, complete rupture and SLAP lesions. The relationship of LHB pathology to different characteristics of RCTs was statistically analyzed. RESULTS: Of RCT cases, 33% had Type 1 (tendinitis), 11% had Type 2 (subluxation), 9% had Type 3 (dislocation), 16% had Type 4 (partial tear), 7% had Type 5 (complete rupture) and 6% had Type 6 (SLAP) lesions. The remaining 18% of cases had no obvious LHB pathology. LHB pathology were associated with RCTs of a long duration (> 3 months), large area (> 5 cm(2)), and multiple or subscapularis tendon involvement. Seventy four percent of patients with affected shoulders underwent simultaneous surgery for both LHB pathology and RCTs. CONCLUSION: Most patient with RCTs with chronic, massive, and multiple or subscapularis tendon involvement also had LHB injury. SLAP lesions, which we classified as a subgroup of LHB pathology, should be identified during rotator cuff surgery and treated appropriately.

Rationalization and design of the complementarity determining region sequences in an antibody-antigen recognition interface.

Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes.

Effects of signal sequence on phage-displayed disulfide-stabilized single chain antibody variable fragment (sc-dsFv) libraries.

Phage-displayed single chain variable fragment (scFv) libraries are powerful tools in antibody engineering. Disulfide-stabilized scFv (sc-dsFv) with an interface disulfide bond is structure-wise more stable than the corresponding scFv. A set of recently discovered signal sequences replacing the wild type (pelB) signal peptidase cleavage site in the c-region has been shown to be effective in rescuing the expression of sc-dsFv libraries on the phage surface. However, the effects of the other regions of the signal sequence on the expression of the sc-dsFv libraries and on the formation of the interface disulfide bond in the phage-displayed sc-dsFv have not been clear. In this work, selected novel signal sequence variants in the h-region were shown to be equally effective in promoting sc-dsFv library expression on the phage surface; the expression level and complexity of the sc-dsFv libraries were comparable to the corresponding scFv libraries produced with the wild-type (pelB) signal sequence. The interface disulfide bond in the phage-displayed sc-dsFv was proven to form to a large extent in the library variant ensemble generated with signal sequence variants in both the h-region and the c-region. The sc-dsFv engineering platform established in this work can be applied to many of the known scFv molecules which are in need of a more stable version for the applications under harsh conditions or for longer shelf-life.

Enhancement of rotator cuff tendon-bone healing with injectable periosteum progenitor cells-BMP-2 hydrogel in vivo.

PURPOSE: The fixation and incorporation of ruptured rotator cuff tendon to bone is a major concern in rotator cuff repair surgery. Rotator cuff repair usually fails at the tendon-bone interface, especially in case of large or massive tears. To enhance tendon-bone healing, an injectable hydrogel made with periosteal progenitor cells(PPCs) and poly (ethylene glycol) diacrylate (PEGDA) tethered with bone morphogenic protein-2(BMP-2) was developed to encourage extracellular matrix synthesis for tendon-to-bone healing in rotator cuff repair. METHODS: The infraspinatus tendon was cut from the greater tuberosity and repaired through a transosseous tunnel with the injectable progenitor cell-BMP-2 hydrogel applied between the tendon-bone interface. The injectable hydrogel was prepared from 10% poly (ethylene glycol) diacrylate (PEGDA) containing 0.05% of the photoinitiator. BMP-2 tethered with poly(ethylene glycol) (PEG) was blended to the hydrogel. Rabbit periosteal progenitor cells (PPCs) isolated from periosteum were mixed with hydrogel and injected on the tendon-bone interface. Ultraviolet radiation (365 nm) was applied for 60 s to photopolymerize the injection and solidify the hydrogel. The rabbits were killed at 4 and 8 weeks. The morphological characteristics of the healing tendon-to-bone interface were evaluated by histological and immunohistochemical methods. The biomechanical test was done to determine healing attachment strength. RESULTS: At both the 4- and 8-week killing, histological analysis of the tendon-bone interface showed an increasing fibrocartilage and bone layer formed in the tendon-bone interface in PEGDA group. At 4 weeks, fibrocartilage-like tissue was observed in a focal area. At 8 weeks, further matrix deposition occurred with fibrocartilage formation in the tendon-bone junction, and bone formation appeared near host bone. Immunohistochemistry revealed the presence of aggrecan and type II collagen. Biomechanical testing revealed a higher maximum pull-out load at all time points with a statistically significant difference at 4 and 8 weeks postoperatively. CONCLUSION: PEGDA hydrogel was approved as an adequate matrix for the encapsulation of cells and signal factor, and as an effective local delivery method to the tendon-bone interface through injection and photopolymerization. The PPCs-BMP2-hydrogel provides a powerful inductive ability between the tendon and the bone and enhances tendon-bone healing through the neoformation of fibrocartilage.