bispecific Archives - The Antibody Society

Most read from mAbs, Nov/Dec 2019

October 28, 2019 by Janice Reichert

The Antibody Society is pleased to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue.

All the articles are open access; PDFs can be freely downloaded by following the links below.

Issue 11.8 (Nov/Dec 2019)

Insights into the IgG heavy chain engineering patent landscape as applied to IgG4 antibody development

In this new Perspective, Dumet et al., present the results from their study of the patent landscape of IgG4 Fc engineering, i.e., patents claiming modifications in the heavy chain. Thirty-seven relevant patent families were identified, comprising hundreds of IgG4 Fc variants focusing on removal of residual effector functions (since IgG4s bind to FcγRI and weakly to other FcγRs), half-life enhancement and IgG4 stability. Given the number of expired or soon to expire major patents in those 3 areas, companies developing blocking antibodies now have, or will in the near future, access to free tools to design silenced, half-life extended and stable IgG4 antibodies.

Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

Parola et al. describe an antibody engineering and screening approach where complete variable light (VL) and heavy (VH) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion. By developing an improved HDR donor format that utilizes in situ linearization, they were able to achieve >15-fold improvement of genomic integration, resulting in a screening workflow that only requires a simple plasmid electroporation. This proved suitable for different applications in antibody discovery and engineering. By integrating and screening an immune library obtained from the variable gene repertoire of an immunized mouse, they isolated a diverse panel of >40 unique antigen-binding variants. They also successfully performed affinity maturation by directed evolution screening of an antibody library based on random mutagenesis, leading to the isolation of several clones with affinities in the picomolar range.

DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity

In this new Report, Sustmann et al. present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one Fc, whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the ‘Fc-load’ on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their ADCC competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.

Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for generating highly pure heterodimers in a single step are needed. Ollier et al. describe two different purification methods that exploit differences in Protein A (PA) or Protein G (PG) avidity between homo- and heterodimers. Differential elution between species was enabled by removing PA or PG binding in one of the Hcs of the bsAb. The PA method allowed the avidity purification of heterodimers based on the VH3 subclass, which naturally binds PA and interferes with separation, by using a combination of IgG3 Fc and a single amino acid change in VH3, N82aS. The PG method relied on a combination of three mutations that completely disrupts PG binding, M428G/N434A in IgG1 Fc and K213V in IgG1 CH1. Both methods achieved a high level of heterodimer purity as single-step techniques without Hc HD (93–98%). Since PA and PG have overlapping binding sites with the neonatal Fc receptor (FcRn), they investigated the effects of the engineering both in vitro and in vivo. Mild to moderate differences in FcRn binding and Fc thermal stability were observed, but these did not significantly change the serum half-lives of engineered control antibodies and heterodimers. The methods are conceptually compatible with various Hc HD platforms such as BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor), in which the PA method has already been successfully implemented.

Most read from mAbs, October 2019

September 17, 2019 by Janice Reichert

The Antibody Society is pleased to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue.

All the articles are open access; PDFs can be freely downloaded by following the links below.

Issue 11.7 (Oct 2019)

Glycoform-resolved FcɣRIIIa affinity chromatography–mass spectrometry

Lippold et al. describe a method to determination the impact of individual antibody glycoforms on FcɣRIIIa affinity, and consequently antibody-dependent cell-mediated cytotoxicity (ADCC) without performing high purity glycoengineering. They hyphenated FcɣRIIIa affinity chromatography to mass spectrometry, which allowed direct affinity comparison of glycoforms of intact monoclonal antibodies. The approach enabled reproduction and refinement of known glycosylation effects, and insights on afucosylation pairing as well as on low-abundant, unstudied glycoforms. Their method greatly improves the understanding of individual glycoform structure–function relationships, and it is highly relevant for assessing Fc-glycosylation critical quality attributes related to ADCC.

Looking for therapeutic antibodies in next-generation sequencing repositories

It is now possible to query the great diversity of natural antibody repertoires using next-generation sequencing (NGS) using methods capable of producing millions of sequences in a single experiment. In this new article, Krawczyk et al. compare clinical-stage therapeutic antibodies to the ~1b sequences from 60 independent sequencing studies in the Observed Antibody Space database, which includes antibody sequences from NGS analysis of immunoglobulin gene repertoires. Of 242 post-Phase 1 antibodies, they found 16 with sequence identity matches of 95% or better for both heavy and light chains. There were also 54 perfect matches to therapeutic CDR-H3 regions in the NGS outputs, suggesting a nontrivial amount of convergence between naturally observed sequences and those developed artificially. The authors discuss the potential implications for both the legal protection of commercial antibodies and the discovery of antibody therapeutics.

Elucidating heavy/light chain pairing preferences to facilitate the assembly of bispecific IgG in single cells

Joshi et al. report that a high yield (>65%) of bispecific IgG1 (BsIgG1) without Fab engineering can be a surprisingly common occurrence, i.e., observed for 33 of the 99 different antibody pairs evaluated. Installing charge mutations at both CH1/CL interfaces was sufficient for near quantitative yield (>90%) of BsIgG1 for most (9 of 11) antibody pairs tested with this inherent cognate chain pairing preference. Mechanistically, they demonstrate that a strong cognate pairing preference in one Fab arm can be sufficient for high BsIgG1 yield. These observed chain pairing preferences are apparently driven by variable domain sequences and can result from a few specific residues in the complementarity-determining region (CDR) L3 and H3. Transfer of these CDR residues into other antibodies increased BsIgG1 yield in most cases. Mutational analysis revealed that the disulfide bond between heavy and light chains did not affect the yield of BsIgG1. This study provides some mechanistic understanding of factors contributing to antibody heavy/light chain pairing preference and subsequently contributes to the efficient production of BsIgG in single host cells.

Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life

The neonatal Fc receptor (FcRn) promotes antibody recycling through rescue from normal lysosomal degradation. The binding interaction is pH-dependent with high affinity at low pH, but not under physiological pH conditions. In this new article, Mackness et al. describe how they combined rational design and saturation mutagenesis to generate novel antibody variants with prolonged half-life and acceptable development profiles. First, a panel of saturation point mutations was created at 11 key FcRn-interacting sites on the Fc region of an antibody. Multiple variants with slower FcRn dissociation kinetics than the wildtype (WT) antibody at pH 6.0 were successfully identified. The mutations were further combined and characterized for pH-dependent FcRn binding properties, thermal stability and the FcγRIIIa and rheumatoid factor binding. The most promising variants, YD (M252Y/T256D), DQ (T256D/T307Q) and DW (T256D/T307W), exhibited significantly improved binding to FcRn at pH 6.0 and retained similar binding properties as WT at pH 7.4. The pharmacokinetics in human FcRn transgenic mice and cynomolgus monkeys demonstrated that these properties translated to significantly prolonged plasma elimination half-life compared to the WT control. The novel variants exhibited thermal stability and binding to FcγRIIIa in the range comparable to clinically validated YTE and LS variants, and showed no enhanced binding to rheumatoid factor compared to the WT control. These engineered Fc mutants are promising new variants that are widely applicable to therapeutic antibodies, to extend their circulation half-life with obvious benefits of increased efficacy, and reduced dose and administration frequency.

Like this post but not a member? Please join!

Most read from mAbs, May-June 2019

May 23, 2019 by Janice Reichert

All the articles are open access; PDFs can be freely downloaded by following the links below.

Issue 11.4 (May-June 2019)

Combining the best of two worlds: highly flexible chimeric antigen receptor adaptor molecules (CAR-adaptors) for the recruitment of chimeric antigen receptor T cells.

In this review, Darowski et al. summarize emerging approaches that aim to further evolve CAR-T cell therapy based on combinations of so-called universal or modular CAR-(modCAR-)T cells, and their respective adaptor molecules (CAR-adaptors), which mediate the crosslinking between target and effector cells. The activity of such modCAR-T cells is entirely dependent on binding of the respective CAR-adaptor to both a tumor antigen and to the CAR-expressing T cell. Contrary to conventional CAR-T cells, where the immunological synapse is established by direct interaction of CAR and membrane-bound target, modCAR-T cells provide a highly flexible and customizable development of the CAR-T cell concept and offer an additional possibility to control T cell activity.

Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies.

Using a sequence-based discovery platform, Trinklein et al. identified new anti-CD3 antibodies from humanized rats that bind to multiple epitopes and elicit varying levels of T-cell activation. In T-BsAb format, 12 different anti-CD3 arms induce equivalent levels of tumor cell lysis by primary T-cells, but potency varies by a thousand-fold. The lead CD3-targeting arm stimulates very low levels of cytokine release, but drives robust tumor antigen-specific killing in vitro and in a mouse xenograft model. This new CD3-targeting antibody underpins a next-generation T-BsAb platform in which potent cytotoxicity is uncoupled from high levels of cytokine release, which may lead to a wider therapeutic window in the clinic.

Sym021, a promising anti-PD1 clinical candidate antibody derived from a new chicken antibody discovery platform.

In this study by Gjetting et al., the Symplex antibody discovery platform was adapted to chicken immunoglobulin genes and combined with high-throughput humanization of antibody frameworks by “mass complementarity-determining region grafting”. Wild type chickens were immunized with an immune checkpoint inhibitor programmed cell death 1 (PD1) antigen, and a repertoire of 144 antibodies was generated. The PD1 antibody repertoire was successfully humanized, and the authors found that most humanized antibodies retained affinity largely similar to that of the parental chicken antibodies. The lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding, resulting in elevated T-cell cytokine production in vitro. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to the clinically approved PD1 antibodies pembrolizumab and nivolumab. Moreover, Sym021 bound human PD1 with a stronger affinity (30 pM) compared to nivolumab and pembrolizumab, while also cross-reacting with cynomolgus and mouse PD1. This enabled direct testing of Sym021 in the syngeneic mouse in vivo cancer models and evaluation of preclinical toxicology in cynomolgus monkeys. Preclinical in vivo evaluation in various murine and human tumor models demonstrated a pronounced anti-tumor effect of Sym021, supporting its current evaluation in a Phase 1 clinical trial.

Most read from mAbs

November 21, 2018 by Janice Reichert

The Antibody Society is pleased and proud to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue. All the articles are open access; PDFs can be downloaded by following the links below.

Issue 10.8 (November/December 2018)

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination. In this new report, Resemann et al. discuss a rapid LC-MALDI-TOF/TOF workflow that can both identify the IgG2 disulfide linkages and provide a semi-quantitative assessment of the distribution of the disulfide isoforms. They established signature disulfide-bonded IgG2 hinge peptides that correspond to the A, A/B, and B disulfide isoforms, and can be applied to the fast classification of IgG2 isoforms in heterogeneous mixtures.

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis. Bailey et al. describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. They used pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. The authors demonstrated that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Based on their results, they conclude that the CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture. In this new report, Wang et al. discuss a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)–based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)–based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.

Characterization and analysis of scFv-IgG bispecific antibody size variants. Cao et al. report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, the authors developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.

Like this post but not a member? Please join!

Antibodies new to the market, and exiting

March 2, 2018 by Janice Reichert

On February 23, 2018, Kyowa Hakko Kirin Co. Ltd and its partner Ultragenyx Pharmaceutical Inc. announced that a conditional marketing authorization had been granted in the European Union (EU) for burosumab (Crysvita) as a treatment for X-linked hypophosphatemia in children 1 year of age and older, and adolescents with growing skeletons. Burosumab is a human IgG1 monoclonal antibody that binds to and inhibits the activity of fibroblast growth factor 23, thereby reducing loss of phosphate from the kidney and other metabolic abnormalities, and ameliorating bone changes that are a hallmark of the disease. The marketing approval in the EU is the first for burosumab. A biologics license application is undergoing review by the US Food and Drug Administration (FDA), and an action on the application is expected by April 17, 2018.

On February 27, 2018, Roche announced that emicizumab (Hemlibra®) had been approved in the EU for routine prophylaxis of bleeding episodes in people with hemophilia A with factor VIII inhibitors. Emicizumab, a bispecific IgG4 mAb targeting Factors IXa and X that originated at Chugai Pharmaceutical Co. Ltd., was approved by the FDA on November 16, 2017.

On March 2, 2018, Biogen and AbbVie announced the voluntary worldwide withdrawal of marketing authorizations for ZINBRYTA® (daclizumab) for relapsing multiple sclerosis (MS). The withdrawal coincides with an urgent review by the European Medicines Agency (EMA) of inflammatory brain disorder in 8 patients, and follows a 2017 review by EMA of reports of liver injury. Due to the risk of serious liver damage, EMA limited use of Zinbryta to MS patients who had tried at least two other disease modifying treatments and could not be treated with any other such treatments. ZINBRYTA® had been marketed in the EU, US, Switzerland, Canada and Australia.

Like this post but not a member? Please join! Membership is free for students and employees of the Society’s corporate sponsors.