Full-length IgG Antibodies Research Articles

YEAST SURFACE DISPLAY OF FULL-LENGTH IGG FOR ENGINEERING MONOCLONAL AND BISPECIFIC ANTIBODIES

Abstract The single-chain variable fragment (scFv) and antigen-binding fragment (Fab) are widely used in yeast surface display for antibody engineering. However, these non-IgG antibody fragments are not always well-expressed or functional. We developed a novel approach to display full-length IgG on yeast surfaces for engineering monoclonal and bispecific antibodies. The yeast surface display of full-length IgG was generated by transforming glycoengineered yeast P. pastoris with plasmids encoding for anchor protein, heavy chains, and light chains. IgG-displaying yeast cells were incubated with the fluorophore antigen and secondary detection antibody for FACS sorting. The displayed antibody with bound antigen and detection antibody was detected by FACS sorting based on both antibody affinity and display level. FACS sorting could eliminate the deviation caused by expression levels. The yeast surface display of IgG-like bispecific antibodies was also constructed, which exhibited specific binding of two distinct fluorophore antigens in flow-cytometry analysis. Thus, our yeast surface display of full-length IgG can be used to screen light chain libraries to isolate common light chains for bispecific antibodies. To assess antigen-binding efficiency between different displaying formats, an antibody was respectively displayed as full-length IgG or Fab on the surface of yeast cells for flow-cytometry analysis. Cells displaying full-length IgG showed a higher level of fluorescence than those displaying Fab fragments, indicating that yeast surface display of full-length IgG is superior for high-throughput screening. To test the specificity of the displayed antibody, we performed a demo experiment in which TNF-binding adalimumab-displaying cells were mixed with trastuzumab-displaying cells at a 1 to 1,000,000 ratio, mimicking the immune library. After three consecutive rounds of sorting, the yeast cells with high fluorescence were plated on a selective medium and the individual antibody clones were sequenced. All ten sequenced clones were confirmed to be adalimumab, demonstrating the maintenance of genotype-phenotype linkage for library screening in yeast surface display of full-length IgG. A mutation library was also generated from the initial hit and displayed at the surface of yeast cells for the screening of higher affinity maturation antibodies by FACS. A pool of high-affinity binders was plated on a selective medium. Individual clones were analyzed by flow cytometry and sequenced to identify unique antibodies with higher affinity. In summary, full-length IgG antibodies can be displayed on the yeast cell surface, mimicking their native forms in molecular structure and biophysical properties. The technique combines the high throughput of yeast display with mammalian-cell quality control. This novel approach can be used to engineer monoclonal and bispecific antibodies for high affinity and improved developability profiles.

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.

Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells.

Yeast-surface display (YSD) is commonly applied to screen Fab immune or naïve libraries for binders of predefined target molecules. However, reformatting of isolated variants represents a time-intensive bottleneck. Herein, we present a novel approach to facilitate a lean transition from antibody screening using YSD Fab libraries to the production of full-length IgG antibodies in Expi293-F cells. In this study, utilizing Golden Gate Cloning (GGC) and a bidirectional promoter system, an exemplary Fab-displaying YSD library was generated based on immunised transgene rats. After subsequent screening for antigen-specific antibody candidates by fluorescence-activated cell sorting (FACS), the Fab-encoding genes were subcloned into a bidirectional mammalian expression vector, exhibiting CH2-CH3 encoding genes, in a GGC-mediated, PCR-free manner. This novel, straightforward and time-saving workflow allows the VH/VL pairing to be preserved. This study resulted in antibody variants exhibiting suitable biophysical properties and covered a broad VH diversity after two rounds of FACS screening, as revealed by NGS analysis. Ultimately, we demonstrate that the implication of such a gene transfer system streamlines antibody hit discovery efforts, allowing the faster characterisation of antibodies against a plethora of targets that may lead to new therapeutic agents.

A comparative analysis of recombinant Fab and full‐length antibody production in Chinese hamster ovary cells

Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen-binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full-length antibodies are mammalian cell-based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small- and large-scale Fab production. Using a panel of full-length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full-length antibody format resulted in the recovery of fewer mini-pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody-producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full-length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats.

A novel and effective approach to generate germline-like monoclonal antibodies by integration of phage and mammalian cell display platforms

Phage display technology allows for rapid selection of antibodies from the large repertoire of human antibody fragments displayed on phages. However, antibody fragments should be converted to IgG for biological characterizations and affinity of antibodies obtained from phage display library is frequently not sufficient for efficient use in clinical settings. Here, we describe a new approach that combines phage and mammalian cell display, enabling simultaneous affinity screening of full-length IgG antibodies. Using this strategy, we successfully obtained a novel germline-like anti-TIM-3 monoclonal antibody named m101, which was revealed to be a potent anti-TIM-3 therapeutic monoclonal antibody via in vitro and in vivo experiments, indicating its effectiveness and power. Thus, this platform can help develop new monoclonal antibody therapeutics with high affinity and low immunogenicity.

Construction of a full-length antibody phage display vector

Antibody phage display technology plays an important role in the development of monoclonal antibodies, humanization, and affinity evolution of antibodies. Thus far, antibody phage display mainly focuses on the display of antibody variable region or antigen-binding fragments. In this study, we constructed a new phage display system that can display full-length IgG antibodies on M13 phage. The phage display vector contains open reading frames (ORFs) encoding full-length the heavy and light chains of the antibody. NcoI/XhoI restriction enzyme sites were used to clone the variable region of the heavy chain into the heavy chain ORF, and SalI/NotI sites were used to clone the light chain variable region. SnaBI and SbfI restriction enzyme sites were designed between the cloning sites of heavy and light chains, respectively, to increase the cloning efficiency. The full-length antibodies of nivolumab against programmed death factor 1, trastuzumab against human epidermal growth factor 2, diL2K against the cluster of differentiation 3 epsilon, and adalimumab against tumor necrosis factor- alpha were displayed on phage with the vector. Phage-displayed antibodies showed their original antigen-binding activity. An amber codon shifted the vector to express IgG in non-suppressed Escherichia coli. The heavy and light chains of the E. coli-expressed antibodies could be detected through western blotting, and the antigen-binding activity was confirmed using an enzyme-linked immunosorbent assay. Biopanning was carried out with a model phage display antibody library, and the results showed that the novel phage system could be used for antibody library construction and highly efficient antibody screening. The reported system is the first full-length antibody phage display system.

Integration of the B-Cell Receptor Antigen Neurabin-I/SAMD14 Into an Antibody Format as New Therapeutic Approach for the Treatment of Primary CNS Lymphoma.

Recently, neurabin-I and SAMD14 have been described as the autoantigenic target of approximately 66% of B-cell receptors (BCRs) of primary central nervous system lymphomas (PCNSL). Neurabin-I and SAMD14 share a highly homologous SAM domain that becomes immunogenic after atypical hyper-N-glycosylation (SAMD14 at ASN339 and neurabin-I at ASN1277). This post-translational modification of neurabin-I and SAMD14 seems to lead to a chronic immune reaction with B-cell receptor activation contributing to lymphoma genesis of PCNSLs. The selective tropism of PCNSL to the CNS corresponds well to the neurabin-I and SAMD14 protein expression pattern. When conjugated to Pseudomonas Exotoxin A (ETA´), the PCNSL reactive epitope exerts cytotoxic effects on lymphoma cells expressing a SAMD14/neurabin-I reactive BCR. Thus, the reactive epitopes of SAMD14/neurabin-I might be useful to establish additional therapeutic strategies against PCNSL. To test this possibility, we integrated the PCNSL-reactive epitope of SAMD14/neurabin-I into a heavy-chain-only Fab antibody format in substitution of the variable region. Specific binding of the prokaryotically produced SAMD14/neurabin-I Fab-antibody to lymphoma cells and their internalization were determined by flow cytometry. Since no established EBV-negative PCNSL cell line exists, we used the ABC-DLBCL cell lines OCI-Ly3 and U2932, which were transfected to express a SAMD14/neurabin-I reactive BCR. The SAMD14/neurabin-I Fab antibody bound specifically to DLBCL cells expressing a BCR with reactivity to SAMD14/neurabin-I and not to unmanipulated DLBCL cell lines. Eukaryotically produced full-length IgG antibodies are well established as immunotherapy format. Therefore, the PCNSL-reactive epitope of SAMD14/neurabin-I was cloned into a full-length IgG1 format replacing the variable domains of the light and heavy chains. The IgG1-format SAMD14/neurabin-I construct was found to specifically bind to target lymphoma cells expressing a SAMD14/neurabin-I reactive B cell receptor. In addition, it induced dose-dependent relative cytotoxicity against these lymphoma cells when incubated with PBMCs. Control DLBCL cells are not affected at any tested concentration. When integrated into the Fab-format and IgG1-format, the PCNSL-reactive epitope of SAMD14/neurabin-I functions as B-cell receptor Antigen for Reverse targeting (BAR). In particular, the IgG1-format BAR-body approach represents a very attractive therapeutic format for the treatment of PCNSLs, considering its specificity against SAMD14/neurabin-I reactive BCRs and the well-known pharmacodynamic properties of IgG antibodies.

Effect of Modulating FcRn Binding on Direct and Pretargeted Tumor Uptake of Full-length Antibodies.

Full-length antibodies lack ideal pharmacokinetic properties for rapid targeted imaging, prompting the pursuit of smaller peptides and fragments. Nevertheless, studying the disposition properties of antibody-based imaging agents can provide critical insight into the pharmacology of their therapeutic counterparts, particularly for those coupled with potent payloads. Here, we evaluate modulation of binding to the neonatal Fc receptor (FcRn) as a protein engineering-based pharmacologic strategy to minimize the overall blood pool background with directly labeled antibodies and undesirable systemic click reaction of radiolabeled tetrazine with circulating pretargeted trans-cyclooctene (TCO)-modified antibodies. Noninvasive SPECT imaging of mice bearing HER2-expressing xenografts was performed both directly (111In-labeled antibody) and indirectly (pretargeted TCO-modified antibody followed by 111In-labeled tetrazine). Pharmacokinetic modulation of antibodies was achieved by two distinct methods: Fc engineering to reduce binding affinity to FcRn, and delayed administration of an antibody that competes with binding to FcRn. Tumor imaging with directly labeled antibodies was feasible in the absence of FcRn binding, rapidly attaining high tumor-to-blood ratios, but accompanied by moderate liver and spleen uptake. Pretargeted imaging of tumors with non-FcRn-binding antibody was also feasible, but systemic click reaction still occurred, albeit at lower levels than with parental antibody. Our findings demonstrate that FcRn binding impairment of full-length IgG antibodies moderately lowers tumor accumulation of radioactivity, and shifts background activity from blood pool to liver and spleen. Furthermore, reduction of FcRn binding did not eliminate systemic click reaction, but yielded greater improvements in tumor-to-blood ratio when imaging with directly labeled antibodies than with pretargeting.

Transient expression of human antibodies in mammalian cells.

Recombinant expression of antibody molecules in mammalian cells offers important advantages over traditionally utilized bacterial expression, including glycosylation required for antibody functionality and markedly reduced levels of endotoxin contamination. Advances in transient mammalian expression systems enable high yields (>100 mg/liter) that now allow for effective recombinant antibody production at a reasonable cost. Here, we provide step-by-step protocols for the design and recombinant expression of full-length IgG antibodies and antibody-derived constructs (including Fab, Fc-fusions and bispecifics) in mammalian cells. Antibody constructs are designed by combining antibody variable domains, generated by phage display or derived from human/humanized monoclonals, with constant regions. The constructs are then expressed from mammalian vectors, secreted into culture media, purified by affinity chromatography and characterized by biolayer interferometry. This article provides detailed protocols, sequences and strategies that allow the expression and purification of endotoxin-free antibody reagents suitable for testing in animal models within a 3-week time frame.

Going native: Direct high throughput screening of secreted full-length IgG antibodies against cell membrane proteins

ABSTRACTGel microdroplet – fluorescence activated cell sorting (GMD-FACS) is an innovative high throughput screening platform for recombinant protein libraries, and we show here that GMD-FACS can overcome many of the limitations associated with conventional screening methods for antibody libraries. For example, phage and cell surface display benefit from exceptionally high throughput, but generally require high quality, soluble antigen target and necessitate the use of anchored antibody fragments. In contrast, the GMD-FACS assay can screen for soluble, secreted, full-length IgGs at rates of several thousand clones per second, and the technique enables direct screening against membrane protein targets in their native cellular context. In proof-of-concept experiments, rare anti-EGFR antibody clones were efficiently enriched from a 10,000-fold excess of anti-CCR5 clones in just three days. Looking forward, GMD-FACS has the potential to contribute to antibody discovery and engineering for difficult targets, such as ion channels and G protein-coupled receptors.

Engineering an aglycosylated Fc variant for enhanced FcγRI engagement and pH-dependent human FcRn binding

The clinical use of therapeutic antibodies has increased sharply because of their many advantages over conventional small molecule drugs, particularly with respect to their affinity, specificity, and serum stability. Tumor or infected cells are removed by the binding of antibody Fc regions to Fc gamma receptors (FcγRs), which stimulate the activation of immune effector cells. Aglycosylated full-length IgG antibodies expressed in bacteria have different Fc conformations compared to their glycosylated counterparts produced in mammalian cells. As a result, they are unable to bind FcγRs, resulting in little to no activation of immune effector cells. In this study, we created a combinatorial library randomized at the upper CH2 loops of an aglycosylated Fc variant (Fc5: E382V/M428) and used a high-throughput flow cytometry library screening method, combined with bacterial display of homodimeric Fc domains for enhanced FcγR binding affinity. The trastuzumab Fc variant containing the identified mutations (Q295R, L328W, A330V, P331A, I332Y, E382V, M428I) not only exhibited over 120 fold higher affinity of specific binding to FcγRI than wild type aglycosylated Fc, but also retained pH-dependent FcRn binding. These results show that an aglycosylated antibody expressed in bacteria can be evolved for novel FcγR affinity and specificity.

Patient‐derived monoclonal antibodies directed towards beta2 glycoprotein‐1 display lupus anticoagulant activity

Patients with antiphospholipid syndrome (APS) display a heterogeneous population of antibodies with beta(2) glycoprotein-1 (β(2)GP1) as the major antigen. We isolated and characterized human mAbs directed against β(2)GP1 from the immune repertoire of APS patients. Variable heavy chain repertoires from B cells from two APS patients with anti-β(2)GP1 antibodies were cloned into the pHEN1-VLrep vector. Constructed full-length IgG antibodies were tested for lupus anticoagulant (LAC) activity and binding to β(2)GP1 and its domains. Two clones of each patient were selected on the basis of the reactivity of single chain Fv (scFv) fragments displayed on phages towards full-length β(2)GP1 and its isolated domain I. The affinity of selected antibodies for β(2)GP1 was lost when transforming from phages to monovalent scFvs, and was regained when antibodies were constructed as complete IgG, indicating a role for bivalency in binding to β(2)GP1. Both selected clones from patient 2 recognized domain I of β(2)GP1, and for both clones selected from patient 1, binding required the presence of both domain I and domain II. All mAbs displayed LAC activity in both activated partial thromboplastin time-based and dilute Russell's viper venom test-based clotting assays and in thrombin generation. In this study, we show successful cloning of patient-derived mAbs that require domain I of β(2)GP1 for binding, and that display LAC activity that is dependent on their affinity for β(2)GP1. These antibodies can help us to gain more insights into the pathogenesis of APS, and may facilitate standardization of APS diagnosis.

E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display

Here we describe a protocol for the selection of full-length IgG antibodies from repertoires displayed on Escherichia coli. In the method described here, full-length heavy and light chains are assembled in the periplasm into aglycosylated IgGs that are fully functional for antigen binding. Expression of an inner membrane-tethered Fc-binding protein is used to capture the IgG molecules and anchor them to the cell. Following outer-membrane permeabilization, fluorescently labeled ligand-binding library clones are selected by multiple rounds of fluorescence-activated cell sorting. Selection of a comprehensive set of IgG clones can typically be obtained within 3-4 weeks, a timescale that is comparable with most prevalent antibody display technologies. The isolated antibodies are well expressed in bacteria and exhibit affinities per binding site in the nanomolar range.

Recombinant antibody potently neutralizes types A, B, and E botulinum neurotoxins by a clearance-driven mechanism

We developed a recombinant mAb-based antitoxin to treat botulism resulting from any subtype of botulinum neurotoxins type A, B, or E. Peripheral blood lymphocytes from botulinum toxoid immunized humans were used to create single chain Fv (scFv) yeast displayed antibody libraries. BoNT/A, B, and E binding human scFv antibodies were generated by selecting the libraries on BoNTs by using flow cytometry. Lead antibodies were screened for binding to different BoNT subtypes. Affinities of antibodies cross-reactive with the different subtypes were increased by creating libraries of mutant scFv genes, displaying the gene repertoires on yeast, and selecting for higher affinity binding. For evaluation of in vivo BoNT neutralization, full-length IgG antibodies were expressed and purified. Combinations of 3–4 mAbs binding non-overlapping BoNT/A, B, and E epitopes with high affinity were engineered, which potently neutralize each of the toxins when administered as an equimolar combination. While a number of mechanisms likely contribute to the synergy observed upon combining mAbs, the most important of these is an Fc-mediated acceleration of BoNT clearance from the blood stream. The results demonstrate the feasibility of generating a highly potent human compatible recombinant antitoxin for the treatment or prevention of type A, B, or E botulism.

Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli

We describe facile isolation of full-length IgG antibodies from combinatorial libraries expressed in E. coli. Full-length heavy and light chains are secreted into the periplasm, where they assemble into aglycosylated IgGs that are captured by an Fc-binding protein that is tethered to the inner membrane. After permeabilizing the outer membrane, spheroplast clones expressing so-called E-clonal antibodies, which specifically recognize fluorescently labeled antigen, are selected using flow cytometry. Screening of a library constructed from an immunized animal yielded several antibodies with nanomolar affinities toward the protective antigen of Bacillus anthracis.