ABSTRACT
We previously described an in vitro single-chain fragment (scFv) library platform originally designed to generate antibodies with excellent developability properties. The platform design was based on the use of clinical antibodies as scaffolds into which replicated natural complementarity-determining regions purged of sequence liabilities were inserted, and the use of phage and yeast display to carry out antibody selection. In addition to being developable, antibodies generated using our platform were extremely diverse, with most campaigns yielding sub-nanomolar binders. Here, we describe a platform advancement that incorporates Fab phage display followed by single-chain antibody-binding fragment Fab (scFab) yeast display. The scFab single-gene format provides balanced expression of light and heavy chains, with enhanced conversion to IgG, thereby combining the advantages of scFvs and Fabs. A meticulously engineered, quality-controlled Fab phage library was created using design principles similar to those used to create the scFv library. A diverse panel of binding scFabs, with high conversion efficiency to IgG, was isolated against two targets. This study highlights the compatibility of phage and yeast display with a Fab semi-synthetic library design, offering an efficient approach to generate drug-like antibodies directly, facilitating their conversion to potential therapeutic candidates.
Subject(s)
Antibody Affinity , Immunoglobulin Fab Fragments , Peptide Library , Single-Chain Antibodies , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/chemistry , Humans , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Single-Chain Antibodies/chemistry , Antibody Affinity/immunology , Cell Surface Display Techniques/methods , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin G/chemistryABSTRACT
Antibody discovery processes are continually advancing, with an ever-increasing number of potential binding sequences being identified out of in vivo, in vitro, and in silico sources. In this work we describe a rapid system for high yield recombinant antibody (IgG and Fab) expression using Gibson assembled linear DNA fragments (GLFs). The purified recombinant antibody yields from 1 ml expression for this process are approximately five to ten-fold higher than previous methods, largely due to novel usage of protecting flanking sequences on the 5' and 3' ends of the GLF. This method is adaptable for small scale (1 ml) expression and purification for rapid evaluation of binding and activity, in addition to larger scales (30 ml) for more sensitive assays requiring milligram quantities of antibody purified over two columns (Protein A and size exclusion chromatography). When compared to plasmid-based expression, these methods provide nearly equivalent yield of high-quality material across multiple applications, allowing for reduced costs and turnaround times to enhance the antibody discovery process.
Subject(s)
Immunoglobulin Fab Fragments , Immunoglobulin G , Recombinant Proteins , Immunoglobulin G/genetics , Immunoglobulin G/chemistry , Immunoglobulin G/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Fab Fragments/chemistry , Gene Expression , Humans , Antibodies/genetics , Antibodies/chemistryABSTRACT
The self-association of therapeutic antibodies can result in elevated viscosity and create problems in manufacturing and formulation, as well as limit delivery by subcutaneous injection. The high concentration viscosity of some antibodies has been reduced by variable domain mutations or by the addition of formulation excipients. In contrast, the impact of Fc mutations on antibody viscosity has been minimally explored. Here, we studied the effect of a panel of common and clinically validated Fc mutations on the viscosity of two closely related humanized IgG1, κ antibodies, omalizumab (anti-IgE) and trastuzumab (anti-HER2). Data presented here suggest that both Fab-Fab and Fab-Fc interactions contribute to the high viscosity of omalizumab, in a four-contact model of self-association. Most strikingly, the high viscosity of omalizumab (176 cP) was reduced 10.7- and 2.2-fold by Fc modifications for half-life extension (M252Y:S254T:T256E) and aglycosylation (N297G), respectively. Related single mutations (S254T and T256E) each reduced the viscosity of omalizumab by ~6-fold. An alternative half-life extension Fc mutant (M428L:N434S) had the opposite effect in increasing the viscosity of omalizumab by 1.5-fold. The low viscosity of trastuzumab (8.6 cP) was unchanged or increased by ≤2-fold by the different Fc variants. Molecular dynamics simulations provided mechanistic insight into the impact of Fc mutations in modulating electrostatic and hydrophobic surface properties as well as conformational stability of the Fc. This study demonstrates that high viscosity of some IgG1 antibodies can be mitigated by Fc mutations, and thereby offers an additional tool to help design future antibody therapeutics potentially suitable for subcutaneous delivery.
Subject(s)
Immunoglobulin Fc Fragments , Immunoglobulin G , Mutation , Omalizumab , Trastuzumab , Humans , Trastuzumab/chemistry , Viscosity , Omalizumab/chemistry , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/genetics , Immunoglobulin G/chemistry , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/geneticsABSTRACT
Antibodies are engineerable quantities in medicine. Learning antibody molecular recognition would enable the in silico design of high affinity binders against nearly any proteinaceous surface. Yet, publicly available experiment antibody sequence-binding datasets may not contain the mutagenic, antigenic, or antibody sequence diversity necessary for deep learning approaches to capture molecular recognition. In part, this is because limited experimental platforms exist for assessing quantitative and simultaneous sequence-function relationships for multiple antibodies. Here we present MAGMA-seq, an integrated technology that combines multiple antigens and multiple antibodies and determines quantitative biophysical parameters using deep sequencing. We demonstrate MAGMA-seq on two pooled libraries comprising mutants of nine different human antibodies spanning light chain gene usage, CDR H3 length, and antigenic targets. We demonstrate the comprehensive mapping of potential antibody development pathways, sequence-binding relationships for multiple antibodies simultaneously, and identification of paratope sequence determinants for binding recognition for broadly neutralizing antibodies (bnAbs). MAGMA-seq enables rapid and scalable antibody engineering of multiple lead candidates because it can measure binding for mutants of many given parental antibodies in a single experiment.
Subject(s)
High-Throughput Nucleotide Sequencing , Immunoglobulin Fab Fragments , Mutation , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , High-Throughput Nucleotide Sequencing/methods , Protein Engineering/methods , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Complementarity Determining Regions/genetics , Complementarity Determining Regions/chemistry , Antibody Affinity , Antigens/immunology , Antigens/geneticsABSTRACT
Directed evolution is a pivotal strategy for new antibody discovery, which allowed the generation of high-affinity Fabs against gliadin from two antibody libraries in our previous studies. One of the libraries was exclusively derived from celiac patients' mRNA (immune library) while the other was obtained through a protein engineering approach (semi-immune library). Recent advances in high-throughput DNA sequencing techniques are revolutionizing research across genomics, epigenomics, and transcriptomics. In the present work, an Oxford Nanopore in-lab sequencing device was used to comprehensively characterize the composition of the constructed libraries, both at the beginning and throughout the phage-mediated selection processes against gliadin. A customized analysis pipeline was used to select high-quality reads, annotate chain distribution, perform sequence analysis, and conduct statistical comparisons between the different selection rounds. Some immunological attributes of the most representative phage variants after the selection process were also determined. Sequencing results revealed the successful transfer of the celiac immune response features to the immune library and the antibodies derived from it, suggesting the crucial role of these features in guiding the selection of high-affinity recombinant Fabs against gliadin. In summary, high-throughput DNA sequencing has improved our understanding of the selection processes aimed at generating molecular binders against gliadin.
Subject(s)
Gliadin , Immunoglobulin Fab Fragments , Nanopore Sequencing , Peptide Library , Humans , Celiac Disease/immunology , Celiac Disease/genetics , Cell Surface Display Techniques/methods , Gliadin/immunology , Gliadin/genetics , High-Throughput Nucleotide Sequencing/methods , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunologyABSTRACT
The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.
Subject(s)
Antigen-Antibody Complex , Immunoglobulin Fab Fragments , Humans , Crystallization/methods , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/chemistry , Antigen-Antibody Complex/chemistry , Antigens/chemistry , Crystallography, X-Ray , Protein ConformationABSTRACT
The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10-45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.
Subject(s)
Escherichia coli , Immunoglobulin Fab Fragments , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/chemistry , Escherichia coli/genetics , Pichia , Adalimumab/therapeutic use , Carbon Isotopes , Antibodies, Monoclonal , TrastuzumabABSTRACT
We have previously published protocols for high-throughput IgG reformatting and expression, that enable rapid reformatting of phage-displayed antibody Fab fragments into a single dual expression vector for full IgG expression in Expi293F cells (Chen et al. Nucleic Acids Res 42:e26, 2014; Chen et al. Methods in Molecular Biology, vol 1701, 2018). However, when working with phage clones from a naïve library containing highly diverse N-terminal sequences, where the 5' PCR primers bind, the PCR step can become cumbersome. To overcome this limitation, we have investigated and found that the C-terminal 7 amino acid residues of the human antibody VH1 secretion signal can be replaced with those from ompA or pelB bacterial signals to form hybrid signal sequences that can drive strong IgG expression in Expi293F cells. The use of such hybrid signals allows any Fab fragment in the library to be amplified and cloned into the IgG expression vector using only a single 5' PCR primer targeting the bacterial secretion signal of the light or heavy chain, thus dramatically simplifying the IgG reformatting workflow.
Subject(s)
Bacteriophages , Humans , Bodily Secretions , Cell Surface Display Techniques , Immunoglobulin Fab Fragments/genetics , Technology , Immunoglobulin G/geneticsABSTRACT
Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.
Subject(s)
COVID-19 , SARS-CoV-2 , Female , Animals , Cattle , Antibodies , Immunoglobulin Fab Fragments/genetics , DisulfidesABSTRACT
Antibodies are used for many therapeutic and biotechnological purposes. Because the affinity of an antibody to the antigen is critical for clinical efficacy of pharmaceuticals, many affinity maturation strategies have been developed. Although we previously reported an affinity maturation strategy in which the association rate of the antibody toward its antigen is improved by introducing a cluster of arginine residues into the framework region of the antibody, the detailed molecular mechanism responsible for this improvement has been unknown. In this study, we introduced five arginine residues into an anti-hen egg white lysozyme antibody (HyHEL10) Fab fragment to create the R5-mutant and comprehensively characterized the interaction between antibody and antigen using thermodynamic analysis, X-ray crystallography, and molecular dynamics (MD) simulations. Our results indicate that introduction of charged residues strongly enhanced the association rate, as previously reported, and the antibody-antigen complex structure was almost the same for the R5-mutant and wild-type Fabs. The MD simulations indicate that the mutation increased conformational diversity in complementarity-determining region loops and thereby enhanced the association rate. These observations provide the molecular basis of affinity maturation by R5 mutation.
Subject(s)
Antigen-Antibody Complex , Antigens , Protein Conformation , Antigens/chemistry , Antigen-Antibody Complex/chemistry , Complementarity Determining Regions/genetics , Complementarity Determining Regions/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/chemistry , Crystallography, X-RayABSTRACT
In vitro antibody display libraries have emerged as powerful tools for a streamlined discovery of novel antibody binders. While in vivo antibody repertoires are matured and selected as a specific pair of variable heavy and light chains (VH and VL) with optimal specificity and affinity, during the recombinant generation of in vitro libraries, the native sequence pairing is not maintained. Here we describe a cloning method that combines the flexibility and versatility of in vitro antibody display with the advantages of natively paired VH-VL antibodies. In this regard, VH-VL amplicons are cloned via a two-step Golden Gate cloning procedure, allowing the display of Fab fragments on yeast cells.
Subject(s)
Antibodies , Immunoglobulin Fab Fragments , Immunoglobulin Fab Fragments/genetics , Cloning, Molecular , Peptide LibraryABSTRACT
Antigen-binding fragments (Fabs) of antibodies are both key biopharmaceuticals and valuable tools for basic life science. To streamline the production of diverse Fabs by capitalizing on standard and highly optimized protein production protocols, we here explore a method to prepare recombinant Fabs as secreted fusion proteins with an N-terminal human growth hormone domain and an octa-histidine tag. These tagged Fabs can be purified with standard immobilized metal chelate affinity chromatography. We first demonstrated Fab overproduction using the rat monoclonal antibody NZ-1. Optimization of linker residues enabled the complete removal of the tags by TEV protease, leaving only two additional residues at the N-terminus of the heavy chain. We purified NZ-1 Fab at â¼4 µg/mL of culture supernatant and further confirmed that the NZ-1 Fab from the fusion protein maintained its native fold and binding affinity for target cell-surface antigens. We also showed that several other Fabs of mouse IgG1s, the major subclass in mice, could be produced with the same procedure. Our preparation method can provide greater flexibility in functional and structural modifications of target Fabs because specialized purification techniques are not necessary.
Subject(s)
Human Growth Hormone , Animals , Humans , Mice , Antibodies, Monoclonal/chemistry , Cell Line , Human Growth Hormone/genetics , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/chemistry , Recombinant Proteins/chemistryABSTRACT
Whole-mount in situ hybridization (WISH) is widely used to visualize transcribed gene sequences (mRNA) in developing embryos, larvae, and other nucleotide probe permeable tissue samples. This methodology involves the hybridization of an antisense nucleotide probe to the target mRNA, followed by chromogen or fluorescence-based detection. Here we describe a protocol for the spatiotemporal analysis of mRNA transcripts in axolotl embryos/larvae using digoxigenin-labeled riboprobes, anti-digoxigenin alkaline phosphatase, Fab fragments antibody, and NBT/BCIP chromogen detection.
Subject(s)
Alkaline Phosphatase , Urodela , Animals , In Situ Hybridization , Larva/genetics , Urodela/genetics , RNA, Messenger/genetics , RNA, Messenger/analysis , Nucleotides , Immunoglobulin Fab Fragments/geneticsABSTRACT
Engineering increased stability into antibodies can improve their developability. While a range of properties need to be optimized, thermal stability and aggregation are two key factors that affect the antibody yield, purity, and specificity throughout the development and manufacturing pipeline. Therefore, an ideal goal would be to apply protein engineering methods early-on, such as in parallel to affinity maturation, to screen out potential drug molecules with the desired conformational and colloidal stability. This chapter introduces our methods to computationally characterize an antibody Fab fragment, propose stabilizing variants, and then experimentally verify these predictions.
Subject(s)
Antibodies , Immunoglobulin Fab Fragments , Immunoglobulin Fab Fragments/genetics , Protein Engineering , Molecular Conformation , Protein Stability , Antibody AffinityABSTRACT
Antigen binding fragments (Fab) are a promising class of therapeutics as they maintain high potency while having significantly smaller size relative to full-length antibodies. Because Fab molecules are aglycosylated, many expression platforms, including prokaryotic, yeast, and mammalian cells, have been developed for their expression, with Escherichia coli being the most commonly used Fab expression system. In this study, we have examined production of a difficult to express Fab molecule in a targeted integration (TI) Chinese Hamster Ovary (CHO) host. Without a need for extensive host or process optimization, as is usually required for E. coli, by simply using different vector configurations, clones with very high Fab expression titers were obtained. In this case, by increasing heavy chain (HC) gene copy numbers, clones with titers of up to 7.4 g/L in the standard fed-batch production culture were obtained. Our findings suggest that having a predetermined transgene integration site, as well as the option to optimize gene copy number/dosage, makes CHO TI hosts an effective system for expression of Fab molecules, allowing Fab expression using platform process and without significant process development efforts.
Subject(s)
Immunoglobulin Fab Fragments , Recombinant Proteins , Animals , Cricetinae , CHO Cells , Cricetulus , Gene Dosage , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Fab Fragments/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , TransgenesABSTRACT
Matrix metalloproteinase 9 (MMP9) contributes to several aspects of inflammation and cancer pathology, including invasion, metastasis, and angiogenesis. In this study, we expressed a recombinant fragment antigen-binding (Fab)-type anti-MMP9 antibody in Escherichia coli with high purity within five days and confirmed the nanomolar order of antigen-binding efficiency of the recombinant Fab. Moreover, we optimized the experimental time for performing enzyme-linked immunosorbent assay (ELISA), and decreased the reaction time from the conventional 20.5 h to 3.5 h. The rapid and sensitive MMP9 detection system developed in this study can be applied to a range of applications, including the diagnosis of diseases with MMP9 overexpression including inflammatory and cancer-related diseases.
Subject(s)
Escherichia coli , Immunoglobulin Fab Fragments , Immunoglobulin Fab Fragments/genetics , Recombinant Proteins , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , MetalloproteasesABSTRACT
Antibody-directed nanotherapeutics (ADNs) represent a promising delivery platform for selective delivery of an encapsulated drug payload to the site of disease that improves the therapeutic index. Although both single-chain Fv (scFv) and Fab antibody fragments have been used for targeting, no platform approach applicable to any target has emerged. scFv can suffer from intrinsic instability, and the Fabs are challenging to use due to native disulfide over-reduction and resulting impurities at the end of the conjugation process. This occurs because of the close proximity of the disulfide bond connecting the heavy and light chain to the free cysteine at the C-terminus, which is commonly used as the conjugation site. Here we show that by engineering an alternative heavy chain-light chain disulfide within the Fab, we can maintain efficient conjugation while eliminating the process impurities and retaining stability. We have demonstrated the utility of this technology for efficient ADN delivery and internalization for a series of targets, including EphA2, EGFR, and ErbB2. We expect that this technology will be broadly applicable for targeting of nanoparticle encapsulated payloads, including DNA, mRNA, and small molecules.
Subject(s)
Nanoparticles , Single-Chain Antibodies , Disulfides/chemistry , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Nanoparticles/chemistryABSTRACT
Yeast surface display (YSD) is a powerful methodology for discovery and engineering of antibodies, and the yeast mating has been used to overcome low transformation efficiency of yeast in antibody library generation. We developed an optimized method of yeast mating for generating a large, combinatorial antibody fragment library and heterodimeric protein library by cellular fusion between two haploid cells carrying different library each other. This method allows for increased diversity in screening of target-specific fragment antigen-binding (Fab) antibodies as well as in the development of heterodimeric Fc variants for bi-specific antibody generation and T-cell receptor (TCR). Here we describe the efficient isolation of human antibodies against the activated GTP-bound form of the oncogenic Ras mutant (KRasG12D-GTP) by sequential isolation of their heavy chains (HCs) followed by combination with light chains (LCs) via the yeast mating process. This strategy facilitates guided selection of the antigen-specific HC with either a fixed functional LC, which has cytosol penetrating ability, or an LC library to generate the Fab. It also allows for deeper exploration of a sequence space with fixed diversity, leading to a higher probability of successful isolation of human antibodies with high specificity and affinity.
Subject(s)
Peptide Library , Saccharomyces cerevisiae , Antibodies/metabolism , Guanosine Triphosphate/metabolism , Humans , Immunoglobulin Fab Fragments/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division. The complex contains 4 protein components: Aurora B kinase, survivin, borealin and INCENP. The CPC acts as a node to dynamically organize other partnering subcomplexes to build multiple functional structures during mitotic progression. Using phage display mutagenesis, a cohort of synthetic antibodies (sABs) were generated against different domains of survivin, borealin and INCENP. Immunofluorescence established that a set of these sABs can discriminate between the form of the CPC complex in the midbody versus the spindle. Others localize to targets, which appear to be less organized, in the nucleus or cytoplasm. This differentiation suggests that different CPC epitopes have dynamic accessibility depending upon the mitotic state of the cell. An Immunoprecipitation/Mass Spectrometry analysis was performed using sABs that bound specifically to the CPC in either the midbody or MT spindle macromolecular assemblies. Thus, sABs can be exploited as high performance reagents to profile the accessibility of different components of the CPC within macromolecular assemblies during different stages of mitosis suggesting this high throughput approach will be applicable to other complex macromolecular systems.
Subject(s)
Antibodies , Aurora Kinase B , Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Immunoglobulin Fab Fragments , Multiprotein Complexes , Survivin , Antibodies/chemistry , Antibodies/genetics , Aurora Kinase B/analysis , Aurora Kinase B/immunology , Cell Cycle Proteins/analysis , Cell Cycle Proteins/immunology , Chromosomal Proteins, Non-Histone/analysis , Chromosomal Proteins, Non-Histone/immunology , Cytoskeleton/metabolism , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Mitosis , Multiprotein Complexes/analysis , Multiprotein Complexes/immunology , Peptide Library , Phosphorylation , Spindle Apparatus/metabolism , Survivin/chemistry , Survivin/metabolismABSTRACT
The introduction of intermolecular disulfide bonds by amino acid mutations is an effective method for stabilizing dimeric proteins. X-ray crystal structure of Fab of a therapeutic antibody, adalimumab, revealed the first loop of the CH1 domain to be partially unsolved at position 135-141. To find new sites for the introduction of intermolecular disulfide bonds in adalimumab Fab, Fab mutants targeting the unsolved region were predicted using molecular simulation software. Four Fab mutants, H:K137C-L:I117C, H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C, were expressed in the methylotrophic yeast Pichia pastoris. SDS-PAGE analysis of these mutants indicated that H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C mutants mostly formed intermolecular disulfide bonds, whereas some H:K137C-L:I117C mutants formed intermolecular disulfide bonds and some did not. Differential scanning calorimetry measurements showed increased thermal stability in all Fab mutants with engineered disulfide bonds. The bio-layer interferometry measurements, for binding of the antigen tumor necrotic factor α, indicated that Fab mutants had less antigen-binding activity than wild-type Fab. In particular, the KD value of H:K137C-L:F209C was ~17 times higher than that of wild-type Fab. Thus, we successfully introduced intermolecular disulfide bonds between the first loop region of the CH1 and CL domains and observed that it increases the thermostability of Fab and affects the antigen-binding activity.