ALTHEA Gold Libraries™: antibody libraries for therapeutic antibody discovery

We describe here the design, construction and validation of ALTHEA Gold Libraries?. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/J_H (H3J) fragments. One IGHV gene provided a universal V_H scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal V_H scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries? with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated K_D values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C–80°C, demonstrating that ALTHEA Gold Libraries? are a valuable source of specific, high affinity and highly stable antibodies.     

IgG cooperativity – Is there allostery? Implications for antibody functions and therapeutic antibody development

A central dogma in immunology is that an antibody's in vivo functionality is mediated by 2 independent events: antigen binding by the variable (V) region, followed by effector activation by the constant (C) region. However, this view has recently been challenged by reports suggesting allostery exists between the 2 regions, triggered by conformational changes or configurational differences. The possibility of allosteric signals propagating through the IgG domains complicates our understanding of the antibody structure-function relationship, and challenges the current subclass selection process in therapeutic antibody design. Here we review the types of cooperativity in IgG molecules by examining evidence for and against allosteric cooperativity in both Fab and Fc domains and the characteristics of associative cooperativity in effector system activation. We investigate the origin and the mechanism of allostery with an emphasis on the C-region-mediated effects on both V and C region interactions, and discuss its implications in biological functions. While available research does not support the existence of antigen-induced conformational allosteric cooperativity in IgGs, there is substantial evidence for configurational allostery due to glycosylation and sequence variations.     

Anti-TNFα domain antibody construct CEP-37247: Full antibody functionality at half the size

We report preclinical data for CEP-37247, the first human framework domain antibody construct to enter the clinic. At approximately 11–13 kDa, domain antibodies or dAbs are the smallest antibody domain able to demonstrate the antigen-recognition function of an antibody, e.g., high selectivity and affinity for target antigen. CEP-37247 is a bivalent anti-tumor necrosis factor (TNF)α domain antibody protein construct combining the antigen-recognition function of a dAb with the pharmacological advantages of an antibody Fc region. As a homodimer, with each chain comprising V_L dAb, truncated C_H1, hinge, C_H2 and C_H3 domains, CEP-37247 has a molecular mass of approximately 78 kDa, which is about half the size of a conventional IgG molecule. Surface plasmon resonance data demonstrate that CEP-37247 possesses high selectivity and affinity for TNFα. CEP-37247 is a potent neutralizer of TNFα activity in vitro in the L929 TNF mediated cytotoxicity assay. In a human TNFα-overexpressing mouse model of polyarthritis, CEP-37247 prevents development of disease and is at least as effective as the marketed product etanercept. Fc functionality is intact—CEP-37247 is capable of mediating antibody-dependent cell-mediated cytotoxicity and has a circulating half-life of approximately 4.5 days in cynomolgus macaques. Given the favorable properties outlined above and its high expression levels (approaching 7 g/L) in a CHOK1 based-expression system, CEP-37247 is progressing into the clinic where other potential advantages, such as enhanced efficacy due to improved tissue distribution and beneficial immunogenicity profile, will be evaluated.Key words: CEP-37247, ART621, domain antibody, dAbs, anti-TNFα, Fc construct     

How probable are antibody cross-reactions?

Antibodies to polypeptides are increasingly being used in research. Their specificity and tight but reversible binding make them ideal for applications such as identification of proteins, immunological quantification or purification, and peptide mapping. Antibodies are also used in medicine to deliver loads to specific sites in tissues, and in electron microscopy as heavy metal conjugates to locate antigens in thin sections. While these techniques depend on specificity of antibody binding, it is occasionally observed that cross-reactions occur. These cross-reactions are attributed to the existence of one or more antibody binding sites common to both polypeptides. It is important to know whether these cross-reactions are expected due to chance alone, or if they are improbable and likely due to some causative agent. Examples of such causative agents might include gene duplication events or convergence due to functional constraints. At the present time, good methods for predicting the probability and therefore the frequency of cross-reactions are unavailable. In this paper we apply some recently reported mathematical results to address the following questions: (1) What is the probability that polyclonal or monoclonal antibodies raised against a given polypeptide will cross-react with another polypeptide due to chance alone? (2) What is the probability that polyclonal or monoclonal antibodies raised against a given polypeptide will cross-react with one or more polypeptides in a pool of polypeptides? Approximate answers to these questions are presented for cases where amino acid compositions of linear polypeptides are known or unknown, but the amino acid sequence of one or more of the polypeptides is not known. Implications of the results for antibody use in protein research are discussed.     

Anti-TNFα domain antibody construct CEP-37247

We report preclinical data for CEP-37247, the first human framework domain antibody construct to enter the clinic. At approximately 11 – 13kDa, domain antibodies or dAbs are the smallest antibody domain able to demonstrate the antigen-recognition function of an antibody, e.g. high selectivity and affinity for target antigen. CEP-37247 is a bivalent anti-tumor necrosis factor (TNF)α domain antibody protein construct combining the antigen-recognition function of a dAb with the pharmacological advantages of an antibody Fc region. As a homodimer, with each chain comprising V_L dAb, truncated C_H1, hinge, C_H2 and C_H3 domains, CEP-37247 has a molecular mass of approximately 78kDa, which is about half the size of a conventional IgG molecule. Surface plasmon resonance data demonstrate that CEP-37247 possesses high selectivity and affinity for TNFα. CEP-37247 is a potent neutralizer of TNFα activity in vitro in the L929 TNF-mediated cytotoxicity assay. In a human TNFα-over-expressing mouse model of polyarthritis, CEP-37247 prevents development of disease, and is at least as effective as the marketed product etanercept. Fc functionality is intact – CEP-37247 is capable of mediating antibody-dependent cell-mediated cytotoxicity and has a circulating half-life of approximately 4.5 days in cynomolgus macaques. Given the favorable properties outlined above, and its high expression levels (approaching 7 g/L) in a CHOK1 based-expression system, CEP-37247 is progressing into the clinic, where other potential advantages such as enhanced efficacy due to improved tissue distribution, and beneficial immunogenicity profile, will be evaluated.     

Iodoacetyl-functionalized pullulan: A supplemental enhancer for single-domain antibody–polyclonal antibody sandwich enzyme-linked immunosorbent assay for detection of survivin

Survivin, an inhibitor of the apoptosis protein family, is a potent tumor marker for diagnosis and prognosis. The enzyme-linked immunosorbent assay (ELISA) is one of the methods that has been used for detection of survivin. However, ELISA has several disadvantages caused by the use of conventional antibodies, and we have therefore been trying to develop a novel ELISA system using camelid single-domain antibodies (VHHs) as advantageous replacements. Here we report a supplemental approach to improve the VHH-polyclonal antibody sandwich ELISA for survivin detection. Iodoacetyl-functionalized pullulan was synthesized, and its thiol reactivity was characterized by a model reaction with l-cysteine. The thiophilic pullulan was applied to an immunoassay as an additive upon coating of standard assay plates with an anti-survivin VHH fusion protein with C-terminal cysteine. The results showed that the mole ratio of the additive to VHH had a significant effect on the consequent response. Mole ratios of 0.07, 0.7, and 7 led to 90% lower, 15% higher, and 69% lower responses, respectively, than the response of a positive control in which no additive was used. The background levels observed in any additive conditions were as low as that of a negative control lacking both VHH and the additive. These results indicate the applicability of the thiol-reactive pullulan as a response enhancer to VHH-based ELISA.     

Resurrection of a clinical antibody: template proteogenomic de novo proteomic sequencing and reverse engineering of an anti-lymphotoxin-α antibody

A mouse hybridoma antibody directed against a member of the tumour necrosis factor (TNF)-superfamily, lymphotoxin-alpha (LT-α), was isolated from stored mouse ascites and purified to homogeneity. After more than a decade of storage the genetic material was not available for cloning; however, biochemical assays with the ascites showed this antibody against LT-α (LT-3F12) to be a preclinical candidate for the treatment of several inflammatory pathologies. We have successfully rescued the LT-3F12 antibody by performing MS analysis, primary amino acid sequence determination by template proteogenomics, and synthesis of the corresponding recombinant DNA by reverse engineering. The resurrected antibody was expressed, purified and shown to demonstrate the desired specificity and binding properties in a panel of immuno-biochemical tests. The work described herein demonstrates the powerful combination of high-throughput informatic proteomic de novo sequencing with reverse engineering to reestablish monoclonal antibody-expressing cells from archived protein sample, exemplifying the development of novel therapeutics from cryptic protein sources.     

Idiotype vaccine for tumor by anti-idiotypic antibody prepared against anti-(bacillus Calmette Guèrin)BCG monoclonal antibody

Summary The anti-idiotypic antibody (Ab2) prepared against the anti-BCG monoclonal antibody (mAb) (Ab1) exhibited potential vaccine activity against Meth A fibrosarcoma that shared a common antigen(s) withMycobacterium bovis strain bacillus Calmette Guèrin (BCG). Mice vaccinated with the anti-idiotypic antibody (Ab2) were protected significantly against growth of the transplanted Meth A tumor (66%), and the presence of anti-(anti-idiotypic antibody) (Ab3) was proved in the Ab2-vaccinated mice by enzyme-linked immunosorbent assay and indirect immunofluorescence analyses using unabsorbed or absorbed sera against the BCG antigen(s) and Meth A tumor cells. This indicated that the anti-idiotypic antibody (Ab2) mimicked the structures of the BCG antigen(s) and behaved as the BCG antigen(s) to induce the Abl-like antibody (Ab3) in vivo. Presumably the Ab2-induced Ab3 plays a significant role in preventing growth of the transplanted tumor in animals. By contrast, the control mice treated with normal mouse serum failed to inhibit the tumor growth. These results suggest the possible development of a tumor vaccine from the anti-idiotypic antibody (Ab2) prepared against the anti-BCG monoclonal antibody, for tumors sharing a common antigen(s) withMycobacterium bovis strain BCG.

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Idiotype vaccine for tumor by anti-idiotypic antibody prepared against anti-(bacillus Calmette Guèrin)BCG monoclonal antibody

     

Characterization of prostate-tissue-directed monoclonal antibody, α-Pro 13

Summary The -Pro 13-secreting hybridoma was produced by immunizing mice with an equal mixture of PC-3, DU145, and LNCaP established prostatic carcinoma cell lines. The specificity of -Pro 13 monoclonal antibody was evaluated by the criteria of differential binding to cultured cells; differential binding to extracts of malignant prostate, nonmalignant prostate, and malignant and nonmalignant tissues of various histiotypes in solid phase radioimmunoassay; and by immunoperoxidase staining of primary surgical tissues of varied histiotypes. The data generated by multiple assay investigation indicate that -Pro 13 exhibits preferential binding to the ductal epithelium of prostate tissue; immunoperoxidase evaluation indicates a considerable heterogeneity of staining of ductal epithelial cells. The most prevalent cross-reactivity of -Pro 13 monoclonal antibody with non-prostate tissue occurs with blood vessel endothelium of restricted tissues. Electrophoretic analysis of immunoprecipitates from radioiodinated prostatic tumor extracts indicates that the molecule recognized by -Pro 13 is of 120,000 dalton apparent nonreduced molecular weight. Under reducing conditions, the antigen (p40) consists of a major component of 40,000 dalton apparent MW and a minor component of 17,000 dalton MW. p40 has an isoelectric point of 3.5–4.5. The antigen is intrinsically stable on the PC-3 cell surface; its release into spent culture medium is negligible. p40 is also stable upon complexation with -Pro 13 antibody in that it is not shed from the cell surface as an immune complex nor is it endocytosed to any extent as an immune complex.Abbreviations MIg anti-mouse immunoglobulin - IEF isoelectric focusing - kD kilodalton - MW molecular weight - PA Staphylococcus protein A - RIA radioimmunoassay - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SCM spent culture medium     

Structural basis of the broadly neutralizing anti-interferon-α antibody rontalizumab

Interferons-alpha (IFN-α) are the expressed gene products comprising thirteen type I interferons with protein pairwise sequence similarities in the 77–96% range. Three other widely expressed human type I interferons, IFN-β, IFN-κ and IFN-ω have sequences 29–33%, 29–32% and 56–60% similar to the IFN-αs, respectively. Type I interferons act on immune cells by producing subtly different immune-modulatory effects upon binding to the extracellular domains of a heterodimeric cell-surface receptor composed of IFNAR1 and IFNAR2, most notably anti-viral effects. IFN-α has been used to treat infection by hepatitis-virus type C (HCV) and a correlation between hyperactivity of IFN-α-induced signaling and systemic lupus erythematosis (SLE), or lupus, has been noted. Anti-IFN-α antibodies including rontalizumab have been under clinical study for the treatment of lupus. To better understand the rontalizumab mechanism of action and specificity, we determined the X-ray crystal structure of the Fab fragment of rontalizumab bound to human IFN-α2 at 3Å resolution and find substantial overlap of the antibody and IFNA2 epitopes on IFN-α2.     

A rapid approach for characterization of thiol-conjugated antibody–drug conjugates and calculation of drug–antibody ratio by liquid chromatography mass spectrometry

We present the demonstration of a rapid “middle-up” liquid chromatography mass spectrometry (LC–MS)-based workflow for use in the characterization of thiol-conjugated maleimidocaproyl-monomethyl auristatin F (mcMMAF) and valine-citrulline-monomethyl auristatin E (vcMMAE) antibody–drug conjugates. Deconvoluted spectra were generated following a combination of deglycosylation, IdeS (immunoglobulin-degrading enzyme from Streptococcus pyogenes) digestion, and reduction steps that provide a visual representation of the product for rapid lot-to-lot comparison—a means to quickly assess the integrity of the antibody structure and the applied conjugation chemistry by mass. The relative abundance of the detected ions also offer information regarding differences in drug conjugation levels between samples, and the average drug–antibody ratio can be calculated. The approach requires little material (<100 μg) and, thus, is amenable to small-scale process development testing or as an early component of a complete characterization project facilitating informed decision making regarding which aspects of a molecule might need to be examined in more detail by orthogonal methodologies.     

Fluorescent antibody method Conjugation Of Fluoresceinisothiocyanate With Immune ?-Globulin

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An antiboy antibody? Re-examination of the maternal immune hypothesis

The maternal immune hypothesis (MIH) argues same sex attraction (SSA) results from maternal immune attack on fetal male-specific brain structures and involves the previous biological influence of elder brothers. One of the surveys supporting this is shown to be based on an unsuitable sample and to contain some strong contrary evidence. The hypothesis relies on at least four speculative ideas and there is evidence against each. (1) Likely immune response prevalence is too low compared with calculated SSA prevalence resulting from the fraternal birth order effect. (2) Testis immune attack would be more likely than brain attack but is not known. (3) Fetal brain structures are practically indistinguishable at birth and subsequent brain anatomical gender differentiation only occurs after birth when no attack is occurring. (4) The hypothesis also predicts unfavourable biology for late birth-order males but in fact the reverse is generally true, and neurological effects are very minor. Studies show aborted fetuses caused by likely maternal immune attack are predominantly girls rather than boys, which also argues against the theory. Studies on identical twins show that common factors such as uterine environment are only a small influence on SSA and post-natal idiosyncratic reactions and non-shared environmental factors are much larger influences.     

Purification of recombinant α-amylase with immuno-affinity chromatography using monoclonal antibody

A monoclonal antibody against recombinant thermostable α-amylase produced by Escherichia coli was isolated from serum-free medium and immobilized on Sepharose 4B. The adsorption equilibrium between α-amylase and the immobilized immuno-adsorbent showed a Langmuir type isotherm. The breakthrough curve calculated numerically using the averaged volumetric coefficient coincided well with the experimental data. More than 90% of the activity of bound α-amylase could be recovered by eluting with glycine-HCl buffer (pH 2.5). The elution profile at pH 2.5 became sharper with increasing temperature. By using an immuno-affinity column, the recombinant α-amylase produced by E. coli could be purified homogeneously from crude extract enzyme solution with two-step elution.     

Purification of recombinant α-amylase by immunoaffinity chromatography with anti-peptide antibody

Adsorption characteristics of an anti-peptide antibody, obtained by immunization of eight amino acids in the C-terminal region of chimeric α-amylase of rice α-amylase isozymes, were studied by use of the chimeric enzyme and the peptide used for immunization. This anti-peptide antibody adsorbed the enzyme, as well as the peptide antigen, with sufficient affinity for immunoaffinity purification and was used for purification of the enzyme secreted from yeast cells. Chimeric α-amylase was purified by immunoaffinity chromatography to high purity in one step from the fermentation broth. One-third of the secreted enzyme was not adsorbed by the column of anti-peptide antibody because of processing in the C-terminal region.     

Molecular insights into γδ T cell costimulation by an anti-JAML antibody

γδ T?cells bridge innate and adaptive immunity and function in immunosurveillance, immunoregulation, tumor cell recognition, and as first line of defense against microbial infection. Costimulation of epithelial γδ T?cell activation by the JAML receptor can be induced by interaction with its endogenous ligand CAR or by binding of the stimulatory antibody HL4E10. We, therefore, determined the crystal structure of the JAML-HL4E10 Fab complex at 2.95?? resolution. HL4E10 binds the membrane-proximal domain of JAML through hydrophobic interactions that account for nanomolar affinity and long half-life, contrasting with the fast kinetics and micromolar affinity of the hydrophilic CAR interaction with the membrane-distal JAML domain. Thus, despite different binding sites and mechanisms, JAML interaction with these two disparate ligands leads to the same functional outcome, namely JAML triggering and induction of cell signaling. Several characteristics of the HL4E10 antibody might then be harnessed in therapeutic applications, such as promoting healing of acute or chronic wounds.     

Humanization and characterization of an anti-human TNF-α murine monoclonal antibody

A murine monoclonal antibody, m357, showing the highly neutralizing activities for human tumor necrosis factor (TNF-α) was chosen to be humanized by a variable domain resurfacing approach. The non-conserved surface residues in the framework regions of both the heavy and light chain variable regions were identified via a molecular modeling of m357 built by computer-assisted homology modeling. By replacing these critical surface residues with the human counterparts, a humanized version, h357, was generated. The humanized h357 IgG(1) was then stably expressed in a mammalian cell line and the purified antibody maintained the high antigen binding affinity as compared with the parental m357 based on a soluble TNF-α neutralization bioassay. Furthermore, h357 IgG(1) possesses the ability to mediate antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity upon binding to cells bearing the transmembrane form of TNF-α. In a mouse model of collagen antibody-induced arthritis, h357 IgG significantly inhibited disease progression by intra-peritoneal injection of 50 μg/mouse once-daily for 9 consecutive days. These results provided a basis for the development of h357 IgG as therapeutic use.