Effect of Clp protease from Corynebacterium glutamicum on heterologous protein expression.

The protease present in a host may reduce the yield and biological activity of heterologous proteins. In this study, we used protease overexpression and deletion strategies to examine the effect of the Clp protease system in Corynebacterium glutamicum on the recombinant protein and to produce a highly efficient heterologous protein expression host. In this study, we identified seven genes in the Clp protease family in Corynebacterium glutamicum ATCC 13032 through bioinformatics analysis, and studied their effects on the enhanced green fluorescent protein (EGFP) reporter protein. The fluorescence intensity of the knockout strain was significantly higher, and the effect of the clpS deletion strain was the most obvious. To verify the universal effect of the lack of clpS, the excellent industrial strain C. glutamicum 1.15647 was transformed to form recombinant 15647-ΔclpS. Based on the results, 15647-ΔclpS had a more significant effect on improving protein expression. Furthermore, recombinant human teriparatide (rhPTH) and variable domain of heavy chain of heavy-chain antibody (VHH) were selected to verify the universal applicability of the knockout strain for expressing heterologous proteins. Accordingly, we found that protease deficiency could increase the production of heterologous proteins. Finally, through a large-scale fermentation, the 15647-ΔclpS strain was used to produce VHH. Its yield was approximately 530 mg/L, which was 65% higher than that of WT-15647. In this study, a host that could effectively increase heterologous protein expression was successfully obtained.

Spy chemistry-enabled protein directional immobilization and protein purification.

Site-directed protein immobilization allows the homogeneous orientation of proteins with high retention of activity, which is advantageous for many applications. Here, we report a facile, specific, and efficient strategy based on the SpyTag-SpyCatcher chemistry. Two SpyTag-fused model proteins, that is, the monomeric red fluorescent protein (RFP) and the oligomeric glutaryl-7-aminocephalosporanic acid acylase, were easily immobilized onto a SpyCatcher-modified resin directly from cell lysates, with activity recoveries in the range of 85-91%. This strategy was further adapted to protein purification, which proceeded through the selective capture of the SpyCatcher-fused target proteins by a SpyTag-modified resin, with the aid of an intein to generate authentic N-termini. For two model proteins, that is, RFP and a variable domain of a heavy chain antibody, the yields were ∼3-7 mg/L culture with >90% purities. This approach could provide a versatile tool for producing high-performance immobilized protein devices and proteins for industrial and therapeutic uses.

Validation of a modified pre-lysis sample preparation technique for flow cytometric minimal residual disease assessment in multiple myeloma, chronic lymphocytic leukemia, and B-non Hodgkin lymphoma.

BACKGROUND: Minimal residual disease (MRD) assessment of hematopoietic neoplasia below 10-4 requires more leukocytes than is usually attainable by post-lysis preparation. However, not all laboratories are resourced for consensus Euroflow pre-lysis methodology. Our study aim was to validate a modified pre-lysis protocol against our standard post-lysis method for MRD detection of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and B-non Hodgkin lymphoma (B-NHL), to meet demand for deeper MRD assessment by flow cytometry. METHOD: Clinical samples for MRD assessment of MM, CLL, and B-NHL (50, 30, and 30 cases, respectively) were prepared in parallel by pre and post-lysis methods for the initial validation. Total leukocytes, MRD, and median fluorescence intensity of antigen expression were compared as measures of sensitivity and antigen stability. Lymphocyte and granulocyte composition were compared, assessing relative sample processing stability. Sensitivity of the pre-lysis assay was monitored post validation for a further 18 months. RESULTS: Pre-lysis achieved at least 10-4 sensitivity in 85% MM, 81% CLL, and 90% B-NHL samples versus 24%, 48%, and 26% by post-lysis, respectively, with stable antigen expression and leukocyte composition. Post validation over 18 months with technical expertise improving, pre-lysis permitted 10-5 MRD assessment in 69%, 86%, and 82% of the respective patient groups. CONCLUSION: This modified pre-lysis procedure provides a sensitive, robust, time efficient, and relatively cost-effective alternative for MRD testing by MFC at 10-5 , facilitating clinically meaningful deeper response assessment for MM, CLL, and B-NHL. This method adaptation may facilitate more widespread adoption of highly sensitive flow cytometry-based MRD assessment.

Identifying Conformation-Selective Heavy-Chain-Only Antibodies Against Membrane Proteins by a Thermal-Shift Scintillation Proximity Assay.

Over the last decades, the use of heavy-chain-only antibodies has received growing attention in academia and industry as research and diagnostic tools as well as therapeutics. Their generation has improved with the help of innovative new methods such as the sybody technology; however, identifying conformation-selective compounds against membrane proteins remains a major challenge. In this chapter, we apply a thermal shift scintillation proximity assay (SPA-TS) to identify sybodies from an in vitro display campaign with the ability to selectively stabilize the inhibitor-bound conformation of the human solute carrier (SLC) family transporter SC6A9 (GlyT1). Using detergent-purified GlyT1 protein and a tritium-labeled glycine uptake inhibitor small molecule, we find sybody candidates that increase the apparent melting temperature in SPA-TS by several degrees. The thermal shift stabilizes the GlyT1-inhibitor complex and qualifies the sybodies for structural studies and inhibitor-selective small molecule screening assays. The SPA-TS assay in its current form is adaptable to any antibody discovery campaign for membrane proteins and permits the generation of highly valuable tools in most stages of drug discovery and development.

A novel intracellularly expressed NS5B-specific nanobody suppresses bovine viral diarrhea virus replication.

Bovine viral diarrhea virus (BVDV) infection causes significant economic losses to the cattle industry worldwide and still represents a huge pressure on agricultural production. Thus, the development of novel anti-BVDV strategies are urgently needed. The nonstructural protein 5 (NS5B) of BVDV is essential for viral replication. Further, the camel single-domain antibody (nanobody) represents a promising antiviral approach with the advantages of small size, stable structure, high specificity and solubility, and the recognition of specific epitopes. However, no NS5B-specific nanobodies against BVDV have been reported. In this study, NS5B-specific nanobodies were isolated from a phage display library of variable domains of Camellidae heavy chain-only antibodies (VHHs). Further, an MDBK cell line stably expressing Nb1 was established to explore antiviral activity. Results showed that Nb1 could markedly suppress BVDV replication and interact with the BVDV NS5B protein. This suggests that nanobodies have potential for the development of novel antiviral drugs against BVDV infection.

VHH characterization.Recombinant VHHs: Production, characterization and affinity.

Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology.

Characterization of ADCs by Capillary Electrophoresis.

Capillary electrophoresis (CE) is a highly efficient separation technique that resolves ions based on their electrophoretic mobility in the presence of an applied voltage. It has been broadly applied for characterizing biotherapeutics including ADCs. In this chapter, step-by-step procedures for characterizing ADCs using CE will be described with focus placed on reduced and non-reduced capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for purity determination and imaged capillary isoelectric focusing (iCIEF) for charge heterogeneity analysis.

Isolation and characterization of camelid single-domain antibodies against HER2.

OBJECTIVE: To isolate and characterize novel high-affinity llama single-domain antibodies against human HER2. RESULTS: We immunized a llama with human HER2, constructed a phage-displayed VHH library from the lymphocytes of the animal, and isolated six unique HER2-specific VHHs by panning. All six VHHs were unique at the amino acid level and were clonally unrelated, as reflected by their distinct CDR3 lengths. All six VHHs recognized recombinant human HER2 ectodomain with monovalent affinities ranging from 1 to 51 nM, had comparable affinities for cynomolgus monkey HER2, and bound HER2+ SKOV3 cells by flow cytometry. Three of the VHHs recognized recombinant murine HER2 with no loss of affinity compared with human and cynomolgus monkey HER2. The VHHs recognized three major epitopes on HER2 (including one conserved across the human, simian and murine orthologues), all of which were distinct from that of trastuzumab. These VHHs may be useful in the design of modular cancer immunotherapeutics.

Isolation and characterization of a monoclonal antibody containing an extra heavy-light chain Fab arm.

Isolation and characterization of monoclonal antibody (mAb) variants to understand the impact of their structure on function is a typical activity during early-stage candidate selection that contributes to derisking clinical development. In particular, efforts are devoted to characterizing oligomeric variants, owing to their potential immunogenic nature. We report here a mAb variant consisting of a canonical mAb monomer associated in a non-covalent fashion with an antigen-binding fragment (Fab) arm amputated from its Fc domain. The truncated heavy chain is encoded in the cell line genome and is the likely product of a genomic recombination during cell line generation. The addition of the Fab arm results in severe loss of potency, indicating its interaction with the Fab domain of the monomer. The presence of such a variant can easily be mitigated by an adequate purification step.

Physicochemical improvement of rabbit derived single-domain antibodies by substitutions with amino acids conserved in camelid antibodies.

Recently, we showed that immunized rabbit heavy chain variable regions (rVHs) can have strong antigen binding activity comparable to that of the camelid variable domain of the heavy chain of heavy chain antibody (VHH). These rVHs lack the light chain variable regions (rVLs), which exist in the authentic Fab format; thus, molecular surfaces at the interface region of rVHs are exposed to solvent. This physical feature may change physicochemical properties, such as causing reduced stability. By overcoming potential physicochemical issues through engineering the interface region, rVHs could become more useful as single-domain antibodies. In this study, we substituted amino acid residues conserved at the interface region of rVHs with those of VHHs. These substitutions included V37F, involving substitution of a residue in the hydrophobic core with a bulkier hydrophobic amino acid, and G44E/L45R, involving double substitutions of highly exposed residues with more hydrophilic ones. As expected, biophysical and structural characterizations showed that the V37F substitution markedly enhanced the thermal stability through increased hydrophobic packing, while G44E/L45R substitutions greatly reduced hydrophobicity of the interface. The quadruple substitutions of V37F/G44E/L45R/F91Y resulted in not only enhancements of thermal stability and reduction in hydrophobicity, both in an additive manner, but also synergistic improvement of purification yield. This quadruple mutant exhibited greatly reduced non-specific binding with improved colloidal stability owing to the reduced hydrophobicity. The approach used in this study should further enhance the utility of rVHs and promote research and development of single-domain antibodies.

Selection, characterization, and thermal stabilization of llama single domain antibodies towards Ebola virus glycoprotein.

BACKGROUND: A key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity. RESULTS: An immune phage display library containing more than 107 unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15-17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies. CONCLUSIONS: SdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola.

Purification and quantification of heavy-chain antibodies from the milk of bactrian camels.

Camel milk has a unique composition with naturally occurring heavy-chain antibodies (HCAbs), which exert rehabilitating potencies in infection and immunity. To characterize HCAb in camel milk, immunoglobulin G (IgG) was isolated from the milk of Camelus bactrianus by a combination of affinity chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis to purify and size-fractionate protein A and protein G, which were further identified by Western blotting, and were quantified by bicinchoninic acid (BCA) and ELISA. The results indicated that IgG1 fraction contains molecules of 50 kDa heavy chains and 36 kDa light chains. The HCAbs (IgG2 and IgG3 fractions) devoid of light chains, contain heavy chains of 45 kDa and 43 kDa, respectively, the amounts of which were significantly higher than that of the IgG1 in the milk of bactrian camels. Above all, we revealed the considerable amounts of HCAbs in the milk of bactrian camels, and developed a novel method for their purification and quantification. These findings provide the basis for developing potential effects of camel milk and its interface with the dairy industry, as well as future investigations of HCAb and its roles in human health and diseases.

Assembly and Expression of Shark Ig Genes.

Sharks are modern descendants of the earliest vertebrates possessing Ig superfamily receptor-based adaptive immunity. They respond to immunogen with Abs that, upon boosting, appear more rapidly and show affinity maturation. Specific Abs and immunological memory imply that Ab diversification and clonal selection exist in cartilaginous fish. Shark Ag receptors are generated through V(D)J recombination, and because it is a mechanism known to generate autoreactive receptors, this implies that shark lymphocytes undergo selection. In the mouse, the ∼2.8-Mb IgH and IgL loci require long-range, differential activation of component parts for V(D)J recombination, allelic exclusion, and receptor editing. These processes, including class switching, evolved with and appear inseparable from the complex locus organization. In contrast, shark Igs are encoded by 100-200 autonomously rearranging miniloci. This review describes how the shark primary Ab repertoire is generated in the absence of structural features considered essential in mammalian Ig gene assembly and expression.

Selection of therapeutic H5N1 monoclonal antibodies following IgVH repertoire analysis in mice.

The rapid rate of influenza virus mutation drives the emergence of new strains that inflict serious seasonal epidemics and less frequent, but more deadly, pandemics. While vaccination provides the best protection against influenza, its utility is often diminished by the unpredictability of new pathogenic strains. Consequently, efforts are underway to identify new antiviral drugs and monoclonal antibodies that can be used to treat recently infected individuals and prevent disease in vulnerable populations. Next Generation Sequencing (NGS) and the analysis of antibody gene repertoires is a valuable tool for Ab discovery. Here, we describe a technology platform for isolating therapeutic monoclonal antibodies (MAbs) by analyzing the IgVH repertoires of mice immunized with recombinant H5N1 hemagglutinin (rH5). As an initial proof of concept, 35 IgVH genes were selected using a CDRH3 search algorithm and co-expressed in a murine IgG2a expression vector with a panel of germline murine kappa genes. Culture supernatants were then screened for antigen binding. Seventeen of the 35 IgVH MAbs (49%) bound rH5VN1203 in preliminary screens and 8 of 9 purified MAbs inhibited 3 heterosubtypic strains of H5N1 virus when assayed by HI. Two of these MAbs demonstrated prophylactic and therapeutic activity in virus-challenged mice. This is the first example in which an NGS discovery platform has been used to isolate anti-influenza MAbs with relevant therapeutic activity.

Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies.

Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA(-) TcdB(+) strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients.

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity.

An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.

Selection of human single domain antibodies recognizing the CMYC protein using enhanced intracellular antibody capture.

Protein functions that are mediated by interaction with other proteins (protein-protein interactions, PPI) are important for normal cell biology and also in disease. Molecules that can interfere with PPI are required as laboratory tools to dissect function, as lead drug surrogates for target validation and as templates for drug discovery. We describe enhanced developments to Intracellular Antibody Capture (IAC) technology that can select antibody fragments able to interact with targets in cells. This is illustrated by the isolation of single heavy chain variable region domains binding to the basic-helix-loop-helix and leucine zipper region of the CMYC oncogenic protein. The enhanced IAC (eIAC) methodology deploys screening in yeast cells of a single diverse library initially with randomization only of CDR3. Further sequential randomization of CDR2 and CDR1 of three independently selected anti-CMYC clones illustrates an in vivo affinity maturation process. This concise eIAC approach facilitates the rapid selection of antibody fragments to explore the proteome interaction spectrum of mammalian cells and disease targeting.

HC-HA/PTX3 Purified From Amniotic Membrane Promotes BMP Signaling in Limbal Niche Cells to Maintain Quiescence of Limbal Epithelial Progenitor/Stem Cells.

To explore how limbal niche cells (LNCs) may control quiescence, self-renewal, and corneal epithelial lineage commitment/differentiation of limbal epithelial progenitor/stem cells (LEPCs), we have established an in vitro sphere assay by reunion between the two cell types in three-dimensional Matrigel. The resultant sphere exhibits inhibition of corneal epithelial lineage commitment/differentiation and marked clonal growth of LEPCs, of which the latter is correlated with activation of canonical Wnt signaling. Herein, we have created a similar reunion assay in immobilized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3), which is purified from amniotic membrane (AM) and consists of a complex formed by hyaluronic covalently linked to heavy chain 1 of inter-α-inhibitor and noncovalently linked to pentraxin 3. The resultant spheres exhibited similar suppression of corneal epithelial lineage commitment/differentiation but upregulation of quiescence markers including nuclear translocation of Bmi-1, and negligible clonal growth of LEPCs. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (Planar cell polarity) Wnt signaling as well as BMP signaling in both LEPCs and LNCs. The activation of BMP signaling in LNCs was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1(DCKO) mice. Furthermore, ablation of BMP signaling in LEPCs led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPCs. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNCs which leads to BMP signaling in LEPCs to achieve quiescence, helping explain how AM transplantation is clinically useful to be used as a matrix for ex vivo expansion of LEPCs and to treat corneal blindness caused by limbal stem cells deficiency.

Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning.

Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG.

Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies.