Molecular engineering of antibodies for therapeutic and diagnostic purposes

During the past ten years, monoclonal antibodies (mAbs) have taken center stage in the field of targeted therapy and diagnosis. This increased interest in mAbs is due to their binding accuracy (affinity and specificity) together with the original molecular and structural rules that govern interactions with their cognate antigen. In addition, the effector properties of antibodies constitute a second major advantage associated with their clinical use. The development of molecular and structural engineering and more recently of in vitro evolution of antibodies has opened up new perspectives in the de novo design of antibodies more adapted to clinical and diagnostic use. Thus, efforts are regularly made by researchers to improve or modulate antibody recognition properties, to adapt their pharmacokinetics, engineer their stability, and control their immunogenicity. This review presents the latest molecular engineering results on mAbs with therapeutic and diagnostic applications.     

Engineered Fc based antibody domains and fragments as novel scaffolds

Therapeutic monoclonal antibodies (mAbs) have been successful for the therapy of a number of diseases mostly cancer and immune disorders. However, the vast majority of mAbs approved for clinical use are full size, typically in IgG1 format. These mAbs may exhibit relatively poor tissue penetration and restricted epitope access due to their large size. A promising solution to this fundamental limitation is the engineering of smaller scaffolds based on the IgG1 Fc region. These scaffolds can be used for the generation of libraries of mutants from which high-affinity binders can be selected. Comprised of the CH2 and CH3 domains, the Fc region is important not only for the antibody effector function but also for its long half-life. This review focuses on engineered Fc based antibody fragments and domains including native (dimeric) Fc and monomeric Fc as well as CH2 and monomeric CH3, and their use as novel scaffolds and binders. The Fc based binders are promising candidate therapeutics with optimized half-life, enhanced tissue penetration and access to sterically restricted binding sites resulting in an increased therapeutic efficacy. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

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.     

High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy

The discovery of monoclonal antibodies (mAbs) that bind to a particular molecular target is now regarded a routine exercise. However, the successful development of mAbs that (1) express well, (2) elicit a desirable biological effect upon binding, and (3) remain soluble and display low viscosity at high concentrations is often far more challenging. Therefore, high throughput screening assays that assess self-association and aggregation early in the selection process are likely to yield mAbs with superior biophysical properties. Here, we report an improved version of affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) that is capable of screening large panels of antibodies for their propensity to self-associate. AC-SINS is based on concentrating mAbs from dilute solutions around gold nanoparticles pre-coated with polyclonal capture (e.g., anti-Fc) antibodies. Interactions between immobilized mAbs lead to reduced inter-particle distances and increased plasmon wavelengths (wavelengths of maximum absorbance), which can be readily measured by optical means. This method is attractive because it is compatible with dilute and unpurified mAb solutions that are typical during early antibody discovery. In addition, we have improved multiple aspects of this assay for increased throughput and reproducibility. A data set comprising over 400 mAbs suggests that our modified assay yields self-interaction measurements that are well-correlated with other lower throughput assays such as cross-interaction chromatography. We expect that the simplicity and throughput of our improved AC-SINS method will lead to improved selection of mAbs with excellent biophysical properties during early antibody discovery.     

Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application.     

High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy

The discovery of monoclonal antibodies (mAbs) that bind to a particular molecular target is now regarded a routine exercise. However, the successful development of mAbs that (1) express well, (2) elicit a desirable biological effect upon binding, and (3) remain soluble and display low viscosity at high concentrations is often far more challenging. Therefore, high throughput screening assays that assess self-association and aggregation early in the selection process are likely to yield mAbs with superior biophysical properties. Here, we report an improved version of affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) that is capable of screening large panels of antibodies for their propensity to self-associate. AC-SINS is based on concentrating mAbs from dilute solutions around gold nanoparticles pre-coated with polyclonal capture (e.g., anti-Fc) antibodies. Interactions between immobilized mAbs lead to reduced inter-particle distances and increased plasmon wavelengths (wavelengths of maximum absorbance), which can be readily measured by optical means. This method is attractive because it is compatible with dilute and unpurified mAb solutions that are typical during early antibody discovery. In addition, we have improved multiple aspects of this assay for increased throughput and reproducibility. A data set comprising over 400 mAbs suggests that our modified assay yields self-interaction measurements that are well-correlated with other lower throughput assays such as cross-interaction chromatography. We expect that the simplicity and throughput of our improved AC-SINS method will lead to improved selection of mAbs with excellent biophysical properties during early antibody discovery.     

Recent advances in the generation of human monoclonal antibody

The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies (Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently developed a method to generate human mAbs by combining the method of in vitro immunization using peripheral blood mononuclear cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs.     

The role of therapeutic antibodies in drug discovery

The last 5 years have seen a major upturn in the fortune of therapeutic monoclonal antibodies (mAbs), with nine mAbs approved for clinical use during this period and more than 70 now in clinical trials beyond phase II. Sales are expected to reach $4 billion per annum worldwide in 2002 and $15 billion by 2010. This success can be related to the engineering of mouse mAbs into mouse/human chimaeric antibodies or humanized antibodies, which have had a major effect on immunogenicity, effector function and half-life. The issue of repeated antibody dosing at high levels with limited toxicity was essential for successful clinical applications. Emerging technologies (phage display, human antibody-engineered mice) have created a vast range of novel, antibody-based therapeutics, which specifically target clinical biomarkers of disease. Modified recombinant antibodies have been designed to be more cytotoxic (toxin delivery), to enhance effector functions (bivalent mAbs) and to be fused with enzymes for prodrug therapy and cancer treatment. Antibody fragments have also been engineered to retain specificity and have increased the penetrability of solid tumours (single-chain variable fragments). Radiolabelling of antibodies has now been shown to be effective for cancer imaging and targeting. This article focuses on developments in the design and clinical use of recombinant antibodies for cancer therapy.     

Epitope specificity of two anti‐morphine monoclonal antibodies: In vitro and in silico studies

Monoclonal antibodies (mAbs) against morphine are important in the development of immunotherapeutic and diagnostic methods for the treatment and prevention of drug addiction. By the surface plasmon resonance (SPR) and enzyme immunoassay techniques, we characterized two previously obtained mAbs 3K11 and 6G1 and showed their ability to recognize free morphine and morphine‐containing antigens in different ways because of the epitope specificity thereof. Using the defined amino acid sequences, we obtained three‐dimensional models of the variable regions of Fab fragments of these antibodies and compared them with the known sequence and spatial structure of the anti‐morphine antibody 9B1. Docking simulations are performed to obtain models of the antibodies complexes with morphine. Differences in the models of 3K11 and 6G1 complexes with morphine correlate with their experimentally detected epitope specificity. The results, in particular, can be used for the structure‐based design of the corresponding humanized antibodies. According to our modeling and docking results, the very different modes of morphine binding to mAbs 3K11 and 6G1 are qualitatively similar to those previously reported for cocaine and two anti‐cocaine antibodies. Thus, the obtained structural information brings more insight into the hapten recognition diversity.     

Influence of improved FcRn binding on the subcutaneous bioavailability of monoclonal antibodies in cynomolgus monkeys

Engineering monoclonal antibodies (mAbs) with improved binding to the neonatal Fc receptor (FcRn) is a strategy that can extend their in vivo half-life and slow their systemic clearance. Published reports have predominantly characterized the pharmacokinetics of mAbs after intravenous administration. Recently, studies in mice suggest FcRn may also play a role in affecting the subcutaneous bioavailability of mAbs. Herein, we examined whether five mAbs engineered with the T250Q/M428L Fc mutations that improved their FcRn interactions, and subsequently their in vivo pharmacokinetics after intravenous administration, had improved subcutaneous bioavailability compared with their wild-type counterparts in cynomolgus monkeys. Similar to the intravenous administration findings, the pharmacokinetic profiles of our variant mAbs after subcutaneous injection showed improved half-life or clearance. In contrast, a clear effect was not observed on the subcutaneous bioavailability. We expect that while FcRn may play a role in determining mAb subcutaneous bioavailability, multiple biopharmaceutical and physiological factors are likely to influence the success of engineering strategies aimed at targeting this pathway for improving bioavailability.     

New challenges to medicare beneficiary access to mAbs

Precision binding of monoclonal antibodies (mAbs) to biological targets, their relative clinical success, and expansion of indications following initial approval, are distinctive clinical features. The relatively high cost of mAbs, together with the absence of a regulatory pathway to generics, stand out as distinctive economic features. Based on both literature review and primary data collection we enumerated mAb original approvals, supplemental indications, and off-label uses, assessed payer formulary management of mAbs, and determined new challenges to Medicare beneficiary access to mAbs. We found that the FDA has approved 22 mAbs and 30 supplemental indications pertaining to the originally approved mAbs. In addition, there are 46 off-label use citations in officially recognized pharmaceutical compendia. Across Part B carriers and Part D plans, we found considerable variation in terms of coverage and conditions of reimbursement related to on- and off-label uses of mAbs. Our results point to four major challenges facing mAb developers, health care providers, Medicare beneficiaries, payers, and policymakers. These include administrative price controls, coverage variation, projected shift from physician- to self-administered mAbs, and comparative effectiveness. We suggest more systematic use of “coverage with evidence development” as a means of optimally addressing these challenges.     

Epitope analyses of pneumococcal surface protein A: a combination of two monoclonal antibodies detects 94% of clinical isolates

Immunisation of BALB/c mice with seven heat-treated Norwegian clinical isolates of Streptococcus pneumoniae of different serotypes elicited mainly monoclonal antibodies (mAbs) to pneumococcal surface protein A (PspA). It was remarkable that the fusions resulted only in a few mAbs directed against other protein antigens. Dot blot analysis with 16 mAbs using clinical isolates representing 23 different capsular types and the uncapsulated reference strain R36A showed that some of the mAbs bound to PspA epitopes expressed by a low number of strains whereas others bound to broadly distributed epitopes. On the basis of their reactivities, seven of these mAbs could be divided into two groups recognising different subsets of pneumococci. The three mAbs in the narrow reacting group bound to epitopes found in 21-25% of the strains whereas the four mAbs in the broad reacting group detected more than 57% of the analysed strains. The epitopes for these seven antibodies were surface exposed on live exponential phase grown pneumococci as shown by flow cytometry. The finding that a combination of mAb 180,C-1 (IgG2a) from the first group and mAb 170,E-11 (IgG2a) from the second group detected 94% of the examined strains is interesting because PspA has been reported by others to be a serological highly variable protein.     

Characterization and utility of monoclonal antibodies against spike protein of transmissible gastroenteritis virus

Aims: This work aims to characterize the utility of four newly generated monoclonal antibodies (mAbs) against transmissible gastroenteritis virus (TGEV). Methods and Results: Four monoclonal antibodies (mAbs) against the N‐terminal half of spike protein (S1 protein) of TGEV were identified. Affinity constant of these mAbs was analysed. These mAbs were capable of reacting with the TGEV S1 protein analysed by ELISA and Western blot. A competition assay between the different mAbs was performed to determine whether the different antibodies mapped in the same or a different antigenic region of the protein. Investigation on the neutralizing ability of these mAbs indicated that two of these mAbs completely neutralized TGEV at an appropriate concentration. These mAbs were able to detect the TGEV‐infected cells in immunofluorescence assays and Western blot. Moreover, they differentiated TGEV S protein from other control proteins. Conclusions: The generated four mAbs are very specific, and the established immunofluorescence assays, Western blot and discrimination ELISA are useful approaches for detecting of TGEV. Significance and Impact of the Study: It is a novel report regarding the use of the S1 protein of TGEV to generate specific mAbs. Their utility and the established immunoassays contribute to the surveillance of TGE coronavirus.     

The biotechnology and applications of antibody engineering

The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However, many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering and in the successful development of new diagnostic and therapeutic antibody-based reagents.     

Fab is the most efficient format to express functional antibodies by yeast surface display

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.     

Monoclonal Antibodies Against Myelin Proteolipid Protein: Identification and Characterization of Two Major Determinants

This report describes the preparation and characterization of a panel of monoclonal antibodies (mAbs) against the myelin proteolipid protein (PLP). A Lewis rat was immunized with bovine proteolipid apoprotein and 27 mAbs were selected based on their reactivity against bovine PLP on enzyme-linked immunosorbent assays. Eleven mAbs recognized the PLP carboxyl-terminal sequence when tested against a panel of synthetic peptides in a solid-phase assay. A carboxyl-terminal pentapeptide (residues 272-276) was sufficient for antibody binding and the terminal phenylalanine residue was found particularly important. Deletion, modification, or replacement of this residue markedly reduced or obliterated antigen-antibody interaction. Nine mAbs reacted with a second antigenic determinant, residues 209-217, but these could be identified only by competitive immunoassays. This peptide was a more effective inhibitor than the longer peptides 202-217 and 205-221, suggesting that flanking residues may interfere with peptide-antibody interaction. Seven antibodies did not react with any of the synthetic peptides tested and their determinants remain unidentified. Immunoblot analysis showed that the mAbs reacted with both the PLP and the DM-20 isoforms. Twenty-three of the mAbs were of the immunoglobulin G2a or b isotype; the remaining antibodies were immunoglobulin M and all of these were specific for residues 209-217. Cultured murine oligodendrocytes were stained by most of the mAbs tested, but the most intense reactivity was observed with the carboxyl-terminus-specific mAbs. The immunocytochemical analyses demonstrate that the mAbs react with the native PLP in situ and show their potential usefulness for studies of the cell biology of myelin and oligodendrocytes.     

Monoclonal antibodies against Stx1B subunit of Escherichia coli O157:H7 distinguish the bacterium from other bacteria

Aims: The Shiga‐like toxins (Stx) are critical virulence factors of enterohaemorrhagic Escherichia coli (EHEC). Stx1B subunit plays important roles in EHEC infection. This work aims to generate and characterize monoclonal antibodies (mAbs) against the Stx1B and to investigate their utility in discrimination ELISA. Methods and Results: Two newly identified mAbs (designated 2H8 and 1B10, respectively) against the Stx1B protein were prepared via hybridoma techniques. The immunoreactivity of both mAbs to the Stx1B protein was confirmed in ELISA and Western blot. Moreover, they differentiate EHEC from Salmonella enteritis, non‐Stx1‐producing E. coli, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus agalactiae and Staphylococcus aureus. Conclusions: The anti‐STx1B mAbs are valuable diagnostic reagents for distinguishing EHEC from other bacteria. Significance and Impact of the Study: This is the first report regarding the usage of anti‐STx1B mAbs in discrimination ELISA. The established ELISA may have potential in clinical surveillance of EHEC infection.     

Practical considerations for nonclinical safety evaluation of therapeutic monoclonal antibodies

Monoclonal antibodies (mAbs) are a well established class of therapeutics as evidenced by a large number of FDA approved mAbs for the treatment of cancers and autoimmune diseases. Monoclonal antibodies that are molecularly engineered for enhanced functions and pharmacokinetic properties are routinely being considered for development by many biotechnology companies. Safety evaluation of current generation of mAbs poses new challenges due to the highly complex nature of engineering aspects and variability induced by the diverse recombinant cell systems to generate them. This review provides a basic outline for nonclinical safety evaluation of therapeutic antibodies. Important considerations for planning a preclinical program, the types of nonclinical safety studies, and a general timeline for their conduct in relation to clinical trials are described. A list of relevant regulatory documents issued by government agencies is also provided. Adoption of these principles will greatly enhance the quality and relevance of the nonclinical safety data generated and will facilitate future development of mAb therapeutics.     

Development and characterization of an anti-rituximab monoclonal antibody panel

During the development of monoclonal antibodies (mAbs) and other therapeutic proteins, immunogenicity, in particular the induction of anti-drug antibodies (ADAs), is an important concern, and thus immunogenicity assessment is a requirement for their approval. Establishment of appropriate methods for detecting and characterizing ADAs is necessary for immunogenicity assessment, but the lack of commonly available reference standards makes it difficult to compare and evaluate the methods. It is also difficult to compare the data with those obtained by other methods or facilities without reference standards. Here, we developed a panel of ADAs against anti-CD20 rituximab (Rituxan®, MabThera®); the panel consisted of eight clones of recombinant human-rat chimeric mAbs that target rituximab. The anti-rituximab mAbs showed different binding properties (specificity, epitope and affinity), and different neutralization potencies for CD20 binding, complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. The molecular size of the immune complex consisting of rituximab and the anti-rituximab mAb differed among the clones, and was well correlated with their level of Fcγ-receptor activation. These results suggest that the ADAs chosen for the newly developed panel are suitable surrogates for human ADAs, which exhibit different potential to affect the efficacy and safety of rituximab. Next, we used this panel to compare several ADA-detecting assays and revealed that the assays had different abilities to detect the ADAs with different binding characteristics. We conclude that our panel of ADAs against rituximab will be useful for the future development and characterization of assays for immunogenicity assessment.     

Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics

Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ～7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition.