Monoclonal antibodies: technologies for early discovery and engineering

Antibodies are essential in modern life sciences biotechnology. Their architecture and diversity allow for high specificity and affinity to a wide array of biochemicals. Combining monoclonal antibody (mAb) technology with recombinant DNA and protein expression links antibody genotype with phenotype. Yet, the ability to select and screen for high affinity binders from recombinantly-displayed, combinatorial libraries unleashes the true power of mAbs and a flood of clinical applications. The identification of novel antibodies can be accomplished by a myriad of in vitro display technologies from the proven (e.g. phage) to the emerging (e.g. mammalian cell and cell-free) based on affinity binding as well as function. Lead candidates can be further engineered for increased affinity and half-life, reduced immunogenicity and/or enhanced manufacturing, and storage capabilities. This review begins with antibody biology and how the structure and genetic machinery relate to function, diversity, and in vivo affinity maturation and follows with the general requirements of (therapeutic) antibody discovery and engineering with an emphasis on in vitro display technologies. Throughout, we highlight where antibody biology inspires technology development and where high-throughput, “big data” and in silico strategies are playing an increasing role. Antibodies dominate the growing class of targeted therapeutics, alone or as bioconjugates. However, their versatility extends to research, diagnostics, and beyond.     

High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants

Developability assessment of therapeutic antibody candidates assists drug discovery by enabling early identification of undesirable instabilities. Rapid chemical stability screening of antibody variants can accelerate the identification of potential solutions. We describe here the development of a high-throughput assay to characterize asparagine deamidation. We applied the assay to identify a mutation that unexpectedly stabilizes a critical asparagine. Ninety antibody variants were incubated under thermal stress in order to induce deamidation and screened for both affinity and total binding capacity. Surprisingly, a mutation five residues downstream from the unstable asparagine greatly reduced deamidation. Detailed assessment by LC-MS analysis confirmed the predicted improvement. This work describes both a high-throughput method for antibody stability screening during the early stages of antibody discovery and highlights the value of broad searches of antibody sequence space.     

Broad epitope coverage of a human in vitro antibody library

Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad “epitope coverage” increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or “bins” and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes.     

EXPRESSION AND PURIFICATION OF HUMAN ARP1 PROTEIN AND RAPID PREPARATION OF POLYCLONAL ANTIBODY

Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms.

Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.     

Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues

Phage display is one of the most widely used technology for antibody discovery and engineering. Number of therapeutic antibodies derived from phage display increases rapidly due to its ease of use and ability to control antibody sequence information. Although there are numerous antibody candidates as promising therapeutics, most of them fail at later stages of development due to undesired biophysical properties. Antibody candidates with poor properties should be prevented or improved in early development phases to minimize enormous loss of time and resources. In this study, we showed that phage display derived therapeutic antibodies show higher self-interaction and polyspecificity compared to non-phage display derived ones. To identify molecular determinants behind this, physicochemical properties of CDR regions of 137 therapeutic antibodies were analyzed. We found multiple significant differences in both heavy and light chain CDR regions. Most profoundly, aliphatic content of HCDR3, HCDR2, and LCDR3 regions were enriched in phage display derived antibodies compared to non-phage display derived ones. Physicochemical determinants documented here seem to play important roles in polyspecific and aggregation-prone natures of antibodies which should be avoided in early development phases.     

Rapid single B cell antibody discovery using nanopens and structured light

ABSTRACT

Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today’s fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction–based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.     

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.     

木薯14-3-3蛋白家族成员MeGRF3基因的原核表达及多克隆抗体制备

14-3-3蛋白是植物体内重要的信号转导调节分子,在碳代谢、逆境胁迫响应、生长发育等过程中发挥重要调控作用。为了深入解析14-3-3蛋白家族在木薯中的生物学功能,该研究采用酶切连接方法构建木薯14-3-3蛋白家族成员MeGRF3的原核表达载体,用热激法转化大肠杆菌Rosetta(DE3)株系,诱导表达带有MeGRF3的融合蛋白;使用纯化后的MeGRF3融合蛋白免疫新西兰公兔制备多克隆抗体,并检测抗体的效价和特异性。结果显示:(1)成功构建了木薯MeGRF3的原核表达载体pET-30a-MeGRF3,并诱导表达得到带有MeGRF3和6个His标签的融合蛋白。(2)MeGRF3融合蛋白主要以包涵体的形式存在,相对分子质量约为33 kD。(3)间接酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)测定的抗体效价为1024000。(4)Western blot分析显示,木薯叶片、茎尖、茎皮和块根中均检测到与MeGRF3蛋白大小一致的条带,说明MeGRF3在这4个组织器官中均表达,但主要是在茎尖和块根中大量积累,表明该研究成功制备的MeGRF3多克隆抗体的特异性较好。     

Serum antibody profiles in individuals with latent Mycobacterium tuberculosis infection

One‐third of the world's humans has latent tuberculosis infection (LTBI), representing a large pool of potentially active TB. Recent LTBI carries a higher risk of disease progression than remote LTBI. Recent studies suggest important roles of antibodies in TB pathology, prompting us to investigate serum antibody profiles in a cohort with LTBI. In this single‐center prospective observational study, we analyzed IgG‐antibody concentrations against five major Mycobacterium tuberculosis (Mtb) antigens (including 6 kDa early secretory antigenic target (ESAT6), CFP10, and antigen 85A, which are expressed mainly in the growth phase; and mycobacterial DNA‐binding protein 1 (MDP1) and alpha‐crystallin like protein (Acr), which are expressed in the dormant phases) in individuals with recent (n=13) or remote (n=12) LTBI, no Mtb infection (n=19), or active TB (n=15). Antibody titers against ESAT6 and MDP1 were significantly higher in individuals with recent LTBI than in those with no Mtb infection or remote LTBI. All pairwise antibody titers against these five major antigens were significantly correlated throughout the stages of Mtb infection. Five individuals with recent LTBI had significantly higher antibody titers against ESAT6 (P = 0.03), Ag85A (P = 0.048), Acr (P = 0.057), and MDP1 (P = 0.0001) than in individuals with remote LTBI; they were also outside the normal range (+2 SDs). One of these individuals was diagnosed with active pulmonary TB at 18‐month follow‐up examination. These findings indicated that concentrations of antibodies against both multiplying and dormant Mtb are higher in recent LTBI and that individuals with markedly higher antibody titers may be appropriate candidates for prophylactic therapy.     

Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy

Phage display technology (PDT), a combinatorial screening approach, provides a molecular diversity tool for creating libraries of peptides/proteins and discovery of new recombinant therapeutics. Expression of proteins such as monoclonal antibodies (mAbs) on the surface of filamentous phage can permit the selection of high affinity and specificity therapeutic mAbs against virtually any target antigen. Using a number of diverse selection platforms (e.g. solid phase, solution phase, whole cell and in vivo biopannings), phage antibody libraries (PALs) from the start point provides great potential for the isolation of functional mAb fragments with diagnostic and/or therapeutic purposes. Given the pivotal role of PDT in the discovery of novel therapeutic/diagnostic mAbs, in the current review, we provide an overview on PALs and discuss their impact in the advancement of engineered mAbs.     

Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab)₂ (n=28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18;n=24, 3 mg). Serum samples were taken before injection and 2–3 and 6–14 weeks after administration. A double-antigen or bridging assay was developed to detect responses against both murine as well as chimeric antibodies. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) as well as three commercially available assays were used to study antibody response against the murine antibody OV-TL 3. With both the double-antigen (bridging) assay and the indirect ELISA 1 of the 28 patients (4%) injected with murine OV-TL 3 F(ab)₂ showed a HAMA reaction 6 weeks after injection, which was demonstrated to be a mixed anti-isotypic and anti-idiotypic response. None of the 24 patients injected with the chimeric MOv18 showed an anti-chimeric antibody response. The various commercially available assays demonstrated conflicting results. The double-antigen-or bridging assay is a reliable method to detect anti-murine and antichimeric antibodies. The assay can be easily adapted for use with human antibodies. The immunogenicity of OV-TL 3 F(ab)₂ and cMOv18 in patients is low, making both antibodies candidates for immunotherapy.This work was supported by a clinical research grant of the Netherlands Organization for Scientific Research (NWO 900-716-020) and by the Biocare Foundation (grant 92-05).     

Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics

ABSTRACT

Immunization of mice followed by hybridoma or B-cell screening is one of the most common antibody discovery methods used to generate therapeutic monoclonal antibody (mAb) candidates. There are a multitude of different immunization protocols that can generate an immune response in animals. However, an extensive analysis of the antibody repertoires that these alternative immunization protocols can generate has not been performed. In this study, we immunized mice that transgenically express human antibodies with either programmed cell death 1 protein or cytotoxic T-lymphocyte associated protein 4 using four different immunization protocols, and then utilized a single cell microfluidic platform to generate tissue-specific, natively paired immunoglobulin (Ig) repertoires from each method and enriched for target-specific binders using yeast single-chain variable fragment (scFv) display. We deep sequenced the scFv repertoires from both the pre-sort and post-sort libraries. All methods and both targets yielded similar oligoclonality, variable (V) and joining (J) gene usage, and divergence from germline of enriched libraries. However, there were differences between targets and/or immunization protocols for overall clonal counts, complementarity-determining region 3 (CDR3) length, and antibody/CDR3 sequence diversity. Our data suggest that, although different immunization protocols may generate a response to an antigen, performing multiple immunization protocols in parallel can yield greater Ig diversity. We conclude that modern microfluidic methods, followed by an extensive molecular genomic analysis of antibody repertoires, can be used to quickly analyze new immunization protocols or mouse platforms.     

Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format.

Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.     

Pharmacokinetic de-risking tools for selection of monoclonal antibody lead candidates

Pharmacokinetic studies play an important role in all stages of drug discovery and development. Recent advancements in the tools for discovery and optimization of therapeutic proteins have created an abundance of candidates that may fulfill target product profile criteria. Implementing a set of in silico, small scale in vitro and in vivo tools can help to identify a clinical lead molecule with promising properties at the early stages of drug discovery, thus reducing the labor and cost in advancing multiple candidates toward clinical development. In this review, we describe tools that should be considered during drug discovery, and discuss approaches that could be included in the pharmacokinetic screening part of the lead candidate generation process to de-risk unexpected pharmacokinetic behaviors of Fc-based therapeutic proteins, with an emphasis on monoclonal antibodies.     

Protein Array-based Approaches for Biomarker Discovery in Cancer

Biomarkers are deemed to be potential tools in early diagnosis, therapeutic monitoring, and prognosis evaluation for cancer, with simplicity as well as economic advantages compared with computed tomography and biopsy. However, most of the current cancer biomarkers present insufficient sensitivity as well as specificity. Therefore, there is urgent requirement for the discovery of biomarkers for cancer. As one of the most exciting emerging technologies, protein array provides a versatile and robust platform in cancer proteomics research because it shows tremendous advantages of miniaturized features, high throughput, and sensitive detections in last decades. Here, we will present a relatively complete picture on the characteristics and advance of different types of protein arrays in application for biomarker discovery in cancer, and give the future perspectives in this area of research.     

Advances in the development of human protein microarrays

Introduction: High-content protein microarrays in principle enable the functional interrogation of the human proteome in a broad range of applications, including biomarker discovery, profiling of immune responses, identification of enzyme substrates, and quantifying protein-small molecule, protein-protein and protein-DNA/RNA interactions. As with other microarrays, the underlying proteomic platforms are under active technological development and a range of different protein microarrays are now commercially available. However, deciphering the differences between these platforms to identify the most suitable protein microarray for the specific research question is not always straightforward.

Areas covered: This review provides an overview of the technological basis, applications and limitations of some of the most commonly used full-length, recombinant protein and protein fragment microarray platforms, including ProtoArray Human Protein Microarrays, HuProt Human Proteome Microarrays, Human Protein Atlas Protein Fragment Arrays, Nucleic Acid Programmable Arrays and Immunome Protein Arrays.

Expert commentary: The choice of appropriate protein microarray platform depends on the specific biological application in hand, with both more focused, lower density and higher density arrays having distinct advantages. Full-length protein arrays offer advantages in biomarker discovery profiling applications, although care is required in ensuring that the protein production and array fabrication methodology is compatible with the required downstream functionality.     

Trifunctional antibody ertumaxomab: Non-immunological effects on Her2 receptor activity and downstream signaling

Background: The trifunctional antibody ertumaxomab bivalently targets the human epidermal growth factor receptor 2 (Her2) on epithelial (tumor) cells and the T cell specific CD3 antigen, and its Fc region is selectively recognized by Fcγ type I/III receptor-positive immune cells. As a trifunctional immunoglobulin, ertumaxomab therefore not only targets Her2 on cancer cells, but also triggers immunological effector mechanisms mediated by T and accessory cells (e.g., macrophages, dendritic cells, natural killer cells). Whether molecular effects, however, might contribute to the cellular antitumor efficiency of ertumaxomab are largely unknown.

Methods: Potential molecular effects of ertumaxomab on Her2-overexpressing BT474 and SK-BR-3 breast cancer cells were evaluated. The dissociation constant K_d of ertumaxomab was calculated from titration curves that were recorded by flow cytometry. Treatment-induced changes in Her2 homodimerization were determined by flow cytometric fluorescence resonance energy transfer measurements on a cell-by-cell basis. Potential activation / deactivation of Her2, ERK1/2, AKT and STAT3 were analyzed by western blotting, Immunochemistry and immunofluorescent cell staining.

Results: The K_d of ertumaxomab for Her2-binding was determined at 265 nM and the ertumaxomab binding epitope was found to not overlap with that of the therapeutic anti-Her2 monoclonal antibodies trastuzumab and pertuzumab. Ertumaxomab caused an increase in Her2 phosphorylation at higher antibody concentrations, but changed neither the rate of Her2-homodimerization /-phosphorylation nor the activation state of key downstream signaling proteins analyzed.

Conclusions: The unique mode of action of ertumaxomab, which relies more on activation of immune-mediated mechanisms against tumor cells compared with currently available therapeutic antibodies for breast cancer treatment, suggests that modular or sequential treatment with the trifunctional bivalent antibody might complement the therapeutic activity of other anti-Her2/anti-ErbB receptor reagents.     

Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development

Effective translation of breakthrough discoveries into innovative products in the clinic requires proactive mitigation or elimination of several drug development challenges. These challenges can vary depending upon the type of drug molecule. In the case of therapeutic antibody candidates, a commonly encountered challenge is high viscosity of the concentrated antibody solutions. Concentration-dependent viscosity behaviors of mAbs and other biologic entities may depend on pairwise and higher-order intermolecular interactions, non-native aggregation, and concentration-dependent fluctuations of various antibody regions. This article reviews our current understanding of molecular origins of viscosity behaviors of antibody solutions. We discuss general strategies and guidelines to select low viscosity candidates or optimize lead candidates for lower viscosity at early drug discovery stages. Moreover, strategies for formulation optimization and excipient design are also presented for candidates already in advanced product development stages. Potential future directions for research in this field are also explored.     

Phage display-derived human antibodies in clinical development and therapy

Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity.

Antibody phage display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by phage display were approved for therapy. In 2002, adalimumab (Humira®) became the first phage display-derived antibody granted a marketing approval. Humira® was also the first approved human antibody, and it is currently the best-selling antibody drug on the market. Numerous phage display-derived antibodies are currently under advanced clinical investigation, and, despite the availability of other technologies such as human antibody-producing transgenic mice, phage display has not lost its importance for the discovery and engineering of therapeutic antibodies.

Here, we provide a comprehensive overview about phage display-derived antibodies that are approved for therapy or in clinical development. A selection of these antibodies is described in more detail to demonstrate different aspects of the phage display technology and its development over the last 25 years.