Showing papers in "mAbs in 2012"
TL;DR: This review discusses the various dual targeting strategies for which bispecific antibodies have been developed and provides an overview of the established bispespecific antibody formats.
Abstract: Monoclonal antibodies are widely used for the treatment of cancer, inflammatory and infectious diseases and other disorders. Most of the marketed antibodies are monospecific and therefore capable of interacting and interfering with a single target. However, complex diseases are often multifactorial in nature, and involve redundant or synergistic action of disease mediators or upregulation of different receptors, including crosstalk between their signaling networks. Consequently, blockade of multiple, different pathological factors and pathways may result in improved therapeutic efficacy. This result can be achieved by combining different drugs, or use of the dual targeting strategies applying bispecific antibodies that have emerged as an alternative to combination therapy. This review discusses the various dual targeting strategies for which bispecific antibodies have been developed and provides an overview of the established bispecific antibody formats.
469 citations
TL;DR: The disulfide bond structures established decades ago for immunoglobulins have been challenged by findings from extensive characterization of recombinant and human monoclonal IgG antibodies, and variations on antibody structure, stability and biological function are discussed in this review.
Abstract: The disulfide bond structures established decades ago for immunoglobulins have been challenged by findings from extensive characterization of recombinant and human monoclonal IgG antibodies. Non-classical disulfide bond structure was first identified in IgG4 and later in IgG2 antibodies. Although, cysteine residues should be in the disulfide bonded states, free sulfhydryls have been detected in all subclasses of IgG antibodies. In addition, disulfide bonds are susceptible to chemical modifications, which can further generate structural variants such as IgG antibodies with trisulfide bond or thioether linkages. Trisulfide bond formation has also been observed for IgG of all subclasses. Degradation of disulfide bond through β-elimination generates free sulfhydryls disulfide and dehydroalanine. Further reaction between free sulfhydryl and dehydroalanine leads to the formation of a non-reducible cross-linked species. Hydrolysis of the dehydroalanine residue contributes substantially to antibody hinge region fragmentation. The effect of these disulfide bond variations on antibody structure, stability and biological function are discussed in this review.
383 citations
TL;DR: Therapeutic monoclonal antibodies (mAbs) are currently being approved for marketing in Europe and the United States, as well as other countries, on a regular basis and six therapeutic mAbs that were approved but have been withdrawn or discontinued from marketing are included.
Abstract: Therapeutic monoclonal antibodies (mAbs) are currently being approved for marketing in Europe and the United States, as well as other countries, on a regular basis As more mAbs become available to physicians and patients, keeping track of the number, types, production cell lines, antigenic targets, and dates and locations of approvals has become challenging Data are presented here for 34 mAbs that were approved in either Europe or the United States (US) as of March 2012, and nimotuzumab, which is marketed outside Europe and the US Of the 34 mAbs, 28 (abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, tositumomab-I131, cetuximab, ibrituximab tiuxetan, omalizumab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, certolizumab pegol, golimumab, canakinumab, catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab, ipilimumab, brentuximab) are currently marketed in Europe or the US Data for six therapeutic mAbs (muromonab-CD3, neb
373 citations
TL;DR: This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.
Abstract: The development of bispecific antibodies has attracted substantial interest, and many different formats have been described Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, eg, the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress
289 citations
TL;DR: This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions.
Abstract: The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.
232 citations
TL;DR: An assay based on ELISA detection of non-specific binding to baculovirus particles that can identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys is developed to increase the likelihood of obtaining a suitable drug candidate.
Abstract: A majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the clinical utility. Pharmacokinetic data in cynomolgus monkeys collected for a panel of 52 antibodies showed broad distribution of target-independent clearance values (2.4–61.3 mL/day/kg), with 15 (29%) having clearance > 10 mL/day/kg. Alteration in the interaction with the recycling FcRn receptor did not account for the faster than expected clearance observed for the antibodies; off-target binding was presumed to account for the fast clearance. We developed an assay based on ELISA detection of non-specific binding to baculovirus particles that can identify antibodies having increased risk for fast clearance. This assay can be used during lead generation or optimization to identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys, and thus increase the likelihood of obtaining a suitable drug candidate.
216 citations
TL;DR: The recent approval in Japan of mogamulizumab (POTELIGEO®), the first glyco-engineered antibody to reach the market, is a landmark in the field of therapeutic antibodies.
Abstract: Therapeutic properties of antibodies strongly depend on the composition of their glycans. Most of the currently approved antibodies are produced in mammalian cell lines, which yield mixtures of different glycoforms that are close to those of humans, but not fully identical. Glyco-engineering is being developed as a method to control the composition of carbohydrates and to enhance the pharmacological properties of mAbs. The recent approval in Japan of mogamulizumab (POTELIGEO®), the first glyco-engineered antibody to reach the market, is a landmark in the field of therapeutic antibodies. Mogamulizumab is a humanized mAb derived from Kyowa Hakko Kirin’s POTELLIGENT® technology, which produces antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. The approval was granted April 30, 2012 by the Japanese Ministry of Health, Labour and Welfare for patients with relapsed or refractory CCR4-positive adult T-cell leukemia-lymphoma.
177 citations
TL;DR: An approach to generate substantially homogeneous antibodies bearing the Man5 glycoform was developed and the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h was identified and the clearance rate was determined in a pharmacokinetics study in mice.
Abstract: The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.
169 citations
TL;DR: The results show that the HCP assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biophARMaceutical industry.
Abstract: Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a "discovery" assay, the latter as a "monitoring" assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique ("Hi3" method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.
166 citations
TL;DR: The results indicate that the quality of the IgG aggregates has more impact on the development of an immune response than their quantity or size.
Abstract: The presence of protein aggregates in biopharmaceutical formulations is of great concern for safety and efficacy reasons. The aim of this study was to correlate the type and amount of IgG monoclonal antibody aggregates with their immunogenic potential. IgG degradation was obtained by freeze-thawing cycles, pH-shift cycles, heating, shaking and metal-catalyzed oxidation. The size, amount, morphology and type of intermolecular bonds of aggregates, as well as structural changes and epitope integrity were characterized. These formulations were injected in mice transgenic (TG) for human genes for Ig heavy and light chains and their non-transgenic (NTG) counterparts. Anti-drug antibody (ADA) titers were determined by bridging ELISA. Both unstressed IgG and freeze-thawed formulation did not induce measurable ADA levels. A mild antibody response was obtained in a fairly small percentage of mice, when injected with shaken, pH-shifted and heated formulations. The metal-catalyzed oxidized IgG formulation was the most immunogenic one, in both ADA titers and number of responders. The overall titers of NTG responders were significantly higher than the ones produced by TG mice, whereas there was no significant difference between the overall number of TG and NTG responders. This study reinforces the important role of protein aggregates on immunogenicity of therapeutic proteins and provides new insight into the immunogenic potential of different types of IgG aggregates. The results indicate that the quality of the IgG aggregates has more impact on the development of an immune response than their quantity or size.
150 citations
TL;DR: The results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.
Abstract: We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG 1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.
TL;DR: Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies and hence further studies to assess effects on product efficacy may be warranted for such antibodies.
Abstract: There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary (CHO) cells or mouse myeloma cells as the expression system. Because CHO and mouse myeloma cells are mammalian cells, rIgGs produced in these cell lines are typically N-glycosylated at the conserved asparagine (Asn) residues in the CH2 domain of the Fc, which is also the case with serum IgGs. The Fc glycans present in these rIgGs are for the most part complex biantennary oligosaccharides with heterogeneity associated with the presence or the absence of several different terminal sugars. The major Fc glycans of rIgGs contain 0 or 1 or 2 (G0, G1 and G2, respectively) terminal galactose residues as non-reducing termini and their relative proportions may vary depending on the cell culture conditions in which they were expressed. Since glycosylation is strongly associated with antibody effector functions and terminal galactosylation may affect some of those functions, a panel of commercially available therapeutic rIgGs expressed in CHO cells and mouse myeloma cells were examined for their galactosylation patterns. The results suggest that the rIgGs expressed in CHO cells are generally less galactosylated compared to the rIgGs expressed in mouse myeloma cells. Accordingly, rIgGs produced in CHO cells tend to contain higher levels of G0 glycans compared with rIgGs produced in mouse myeloma cell lines. Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies. Nevertheless, galactosylation may have an effect on the mechanisms of action of some therapeutic antibodies (e.g., effector pathways) and hence further studies to assess effects on product efficacy may be warranted for such antibodies. For antibodies that do not require effector functions for biological activity, however, setting a narrow specification range for galactose content may be unnecessary.
TL;DR: Support is lent for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs after Göttingen minipigs is demonstrated.
Abstract: Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Gottingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.
TL;DR: It is concluded that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute.
Abstract: The role of Fc glycans on clearance of IgG molecule has been examined by various groups in experiments where specific glycans have been enriched or the entire spectrum of glycans was studied after administration in pre-clinical or clinical pharmacokinetic (PK) studies. The overall conclusions from these studies are inconsistent, which may result from differences in antibody structure or experimental design. In the present study a well-characterized recombinant monoclonal IgG1 molecule (mAb-1) was analyzed from serum samples obtained from a human PK study. mAb-1 was recovered from serum using its ligand cross-linked to Sepharose beads. The overall purity and recovery of all isoforms were carefully evaluated using a variety of methods. Glycans were then enzymatically cleaved, labeled using 2-aminobenzamide and analyzed by normal phase high performance liquid chromatography. The assays for recovering mAb-1 from serum and subsequent glycan analysis were rigorously qualified at a lower limit of quantitation of 15 μg/mL, thus permitting analysis to day 14 of the clinical PK study. Eight glycans were monitored and classified into two groups: (1) the oligomannose type structures (M5, M6 and M7) and (2) fucosylated biantennary oligosaccharides (FBO) structures (NGA2F, NA1F, NA2F, NA1F-GlcNAc and NGA2F-GlcNAc). We observed that the oligomannose species were cleared at a much faster rate (40%) than FBOs and conclude that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute. These observations were only possible through the application of rigorous analytical methods that we believe will need to be employed when comparing innovator and biosimilar molecules.
TL;DR: The results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion.
Abstract: The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its "regular" fucosylated counterpart and a series of mixtures containing varying proportions of "regular" and afucosylated materials. Compared with the "regular" fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC 50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.
TL;DR: Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugate.
Abstract: Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) bindin...
TL;DR: R Romiplostim, the first peptibody to be approved by the United States Food and Drug Administration and the European Medicines Agency and is indicated for the treatment of immune thrombocytopenic purpura, is being evaluated in Phase 3 clinical testing in combination with chemotherapy in women with ovarian cancer.
Abstract: Peptibodies or peptide-Fc fusions are an attractive alternative therapeutic format to monoclonal antibodies. They consist of biologically active peptides grafted onto an Fc domain. This approach retains certain desirable features of antibodies, notably an increased apparent affinity through the avidity conferred by the dimerization of two Fcs and a long plasma residency time. Peptibodies can be made in E. coli using recombinant technology. The manufacturing process involves fermentation and downstream processing, including refolding and multiple column chromatographic steps, that result in overall yields and quality suitable for commercial development. Romiplostim, marketed under the brand name Nplate®, is the first peptibody to be approved by the United States Food and Drug Administration and the European Medicines Agency and is indicated for the treatment of immune thrombocytopenic purpura. AMG 386, a peptibody antagonist to angiopoietins 1 and 2, is being evaluated in Phase 3 clinical testing in combination with chemotherapy in women with ovarian cancer. AMG 819, a peptibody targeting nerve growth factor for pain has also progressed to clinical trials. These peptibodies illustrate the versatility of the modality.
TL;DR: This data demonstrates that the ADCC-reporter gene assay has performance characteristics to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.
Abstract: Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with...
TL;DR: Efforts are regularly made by researchers to improve or modulate antibody recognition properties, to adapt their pharmacokinetics, engineer their stability, and control their immunogenicity.
Abstract: 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
TL;DR: This study demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.
Abstract: Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naive VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naive VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naive VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.
TL;DR: The data suggest that R OR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers.
Abstract: The selective cell surface expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) has made ROR1 a novel and promising target for therapeutic monoclonal antibodies (mAbs). Four mouse mAbs generated by hybridoma technology exhibited specific binding to human ROR1. Epitope mapping studies showed that two mAbs (2A2 and 2D11) recognized N-terminal epitopes in the extracellular region of ROR1 and the other two (1A1 and 1A7) recognized C-terminal epitopes. A ROR1- immunotoxin (BT-1) consisting of truncated Pseudomonas exotoxin A (PE38) and the VH and VL fragments of 2A2-IgG was made recombinantly. Both 2A2-IgG and BT-1 showed dose-dependent and selective binding to primary CLL and MCL cells and MCL cell lines. Kinetic analyses revealed 0.12-nM (2A2-IgG) to 65-nM (BT-1) avidity/affinity to hROR1, depicting bivalent and monovalent interactions, respectively. After binding to cell surface ROR1, 2A2-IgG and BT-1 were partially internalized by primary CLL cells and MCL cell lines, and BT-1 induced profound apoptosis of ROR1-expressing MCL cell lines in vitro (EC 50 = 16 pM-16 nM), but did not affect ROR1-negative cell lines. Our data suggest that ROR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers.
TL;DR: This review provides an overview and the rationale for the most advanced CD19-targeting programs in development and indicates that some promising clinical study data has already been reported.
Abstract: Despite progress in the treatment of B cell disorders, novel treatment approaches are still highly needed. CD19 is a pan-B cell marker that is recognized as a potential immunotherapy target for B cell disorders, including blood-borne malignancies and autoimmune diseases. Although initial attempts to target CD19 were unsuccessful, a new wave of investigational agents is currently in development. These agents are based on novel antibody-based technologies and formats that appear to better exploit CD19's therapeutic potential, and some promising clinical study data has already been reported. This review provides an overview and the rationale for the most advanced CD19-targeting programs in development.
TL;DR: An improved method for producing high-affinity monoclonal antibodies to cell surface tumor antigens is described and represent a general approach to isolate therapeutic antibodies against cancer.
Abstract: Isolating high-affinity antibodies against native tumor antigens on the cell surface is not straightforward using standard hybridoma procedures. Here, we describe a combination method of synthetic peptide immunization and high-throughput flow cytometry screening to efficiently isolate hybridomas for cell binding. Using this method, we identified high-affinity monoclonal antibodies specific for the native form of glypcian-3 (GPC3), a target heterogeneously expressed in hepatocellular carcinoma (HCC) and other cancers. We isolated a panel of monoclonal antibodies (YP6, YP7, YP8, YP9 and YP9.1) for cell surface binding. The antibodies were used to characterize GPC3 protein expression in human liver cancer cell lines and tissues by flow cytometry, immunoblotting and immunohistochemistry. The best antibody (YP7) bound cell surface-associated GPC3 with equilibrium dissociation constant, KD = 0.3 nmol/L and was highly specific for HCC, not normal tissues or other forms of primary liver cancers (such as cholangio...
TL;DR: Modified antibodies such as antibody-drug conjugates, bispecific antibodies, Fc or glyco-engineered antibodies and antibody fragments/domains now comprise more than half of the anticancer antibodies at Phase 1, and ~40% of those at Phase 2 and Phase 3.
Abstract: As we enter the new year of 2012, the realm of antibody therapeutics development seems full of possibilities for antibodies to watch. The commercial pipeline of antibody-based therapeutics continues to grow and now totals nearly 350 candidates. The molecular diversity of the candidates, especially those for cancer, is remarkable. Modified antibodies such as antibody-drug conjugates (ADCs), bispecific antibodies, Fc or glyco-engineered antibodies and antibody fragments/domains now comprise more than half of the anticancer antibodies at Phase 1, and ~40% of those at Phase 2 and Phase 3. These types of modified antibodies have not been developed as frequently for other disorders–by comparison, ~90% of antibodies developed for non-cancer indications are unmodified IgG–but development of the new formats for inflammatory and autoimmune disorders is expected to grow. In addition, antibody mixtures and antibodies with indirect mechanisms of action (e.g., agonism of immune activation receptors or antagonism of imm...
TL;DR: In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.
Abstract: Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.
TL;DR: The results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity, and a semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A, demonstrated that FcRN plays an important role in SC bio availability of therapeutic IgG antibodies.
Abstract: The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased...
TL;DR: Interestingly, biosimilar 2 exhibited similar affinity and bioactivity levels compared with the reference product despite the obvious difference in primary structure and partial physiochemical properties.
Abstract: Because of rapidly increasing market demand and rising cost pressure, the innovator of etanercept (Enbrel®) will inevitably face competition from biosimilar versions of the product. In this study, to elucidate the differences between the reference etanercept and its biosimilars, we characterized and compared the quality attributes of two commercially available, biosimilar TNF receptor 2-Fc fusion protein products. Biosimilar 1 showed high similarity to Enbrel® in critical quality attributes including peptide mapping, intact mass, charge variant, purity, glycosylation and bioactivity. In contrast, the intact mass and MS/MS analysis of biosimilar 2 revealed a mass difference indicative of a two amino acid residue variance in the heavy chain (Fc) sequences. Comprehensive glycosylation profiling confirmed that biosimilar 2 has significantly low sialylated N-oligosaccharides. Biosimilar 2 also displayed significant differences in charge attributes compared with the reference product. Interestingly, biosimilar 2 exhibited similar affinity and bioactivity levels compared with the reference product despite the obvious difference in primary structure and partial physiochemical properties. For a biosimilar development program, comparative analytical data can influence decisions about the type and amount of animal and clinical data needed to demonstrate biosimilarity. Because of the limited clinical experience with biosimilars at the time of their approval, a thorough knowledge surrounding biosimilars and a case-by-case approach are needed to ensure the appropriate use of these products.
TL;DR: This work shows that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display, and can be generated in high throughput.
Abstract: Described herein are methods that combine phage and yeast display to create polyclonal antibodies that are renewable, and when amplified over 100 million fold, maintain diversity without loss of representation of any of the antibodies present. The antibody representation remains essentially constant, as confirmed by deep sequencing. The provided methods allow generation, use and propagation of polyclonal antibodies, without concern that representation is lost. Furthermore, because the derivation of the polyclonal pool is carried out in vitro using phage and yeast display, it is possible in various embodiments to eliminate reactivities that are considered undesirable. Additionally, the polyclonal pool can be enriched for higher affinity antibodies.
TL;DR: The intrabody-PEST fusion approach has dual advantages of potentially solubilizing intrabodies and enhancing their functionality in parallel, and is validated with intrabODY against α-synuclein, an important target in Parkinson disease.
Abstract: Intrabodies can be powerful reagents to effect modulation of aberrant intracellular proteins that underlie a range of diseases. However, their cytoplasmic solubility can be limiting. We previously reported that overall charge and hydrophilicity can be combined to provide initial estimates of intracellular solubility, and that charge engineering via fusion can alter solubility properties experimentally. Additional studies showed that fusion of a proteasome-targeting PEST motif to the anti-huntingtin intrabody scFv-C4 can degrade mutant huntingtin proteins by directing them to the proteasome, while also increasing the negative charge. We now validate the generality of this approach with intrabodies against α-synuclein (α-syn), an important target in Parkinson disease. In this study, fusion of the PEST sequence to a set of four diverse, poorly soluble anti-α-syn intrabodies (D5E, 10H, D10 scFv, VH14 nanobody) significantly increased steady-state soluble intrabody protein levels in all cases, despite fusion with the PEST proteasomal-targeting signal. Furthermore, adding this PEST motif to the least soluble construct, VH14, significantly enhanced degradation of the target protein, α-syn~GFP. The intrabody-PEST fusion approach thus has dual advantages of potentially solubilizing intrabodies and enhancing their functionality in parallel. Empirical testing of intrabody-PEST fusions is recommended for enhancement of intrabody solubility from diverse sources.
TL;DR: The World Bispecific Antibody Summit, organized by Hanson Wade, drew over 100 participants to Boston to discuss engineering novel bispecific antibodies, generating lead candidates and clinical study and commercialization of the molecules.
Abstract: With more than 30 therapeutic monoclonal antibodies (mAbs) approved and annual global sales of the products at ~$50 billion in 2010, these products have proven to be successful in many ways. Nevertheless, there is room for improvement in performance, and substantial unmet medical needs remain. As a consequence, numerous organizations are devoting resources to engineering novel mAbs such as bispecific antibodies that have increased functionality compared with unmodified IgG molecules. The World Bispecific Antibody Summit, organized by Hanson Wade, drew over 100 participants to Boston to discuss engineering novel bispecific antibodies, generating lead candidates and clinical study and commercialization of the molecules. Approaches such as the trifunctional antibody (TRION), dual variable domain-Ig (Abbott), two-in-one (Genentech), dual affinity retargeting (MacroGenics), kappa-lambda body (NovImmune), bispecific T-cell engager (Micromet) and chemical generation (CovX/Pfizer) were discussed in detail. In addition, posters describing bispecific Affibody® molecules for targeting of EGFR and HER2 (Affibody), T-cell receptor based bi-specifics that target HLA-peptides (Immunocore), a novel mAb-Fv bispecific antibody format utilizing Fc region (Xencore), generation of a tetravalent bispecific antibody against IL4 and IL13 for the treatment of idiopathic pulmonary fibrosis (Sanofi), Combining Affibody® molecules and the Albumod™ technology to create long acting multispecific protein therapeutics (Royal Institute of Technology, Affibody) and COVA301 as a highly potent bispecific inhibitor of IL-17A and TNF-α (Covagen) were presented.