The Antibody Society

the official website of the antibody society

The Antibody Society

An international non-profit supporting antibody-related research and development.

You are here: Home / Archives for antibody discovery

Specific Detection Reagents: What’s the Future?

by

Don’t miss the last webinar in our Antibody Validation series!

The Antibody Society has invited leaders in antibody research from industry and academia to reflect on antibody validation. They paint a uniformly alarming picture of inadequacies at many levels – which encourages users to be highly aware of the consequences of inadequate tool antibody validation, which threaten their biological endeavors.

February 19, 2020, 9am Eastern Standard Time / 3pm Central European Time, 45 min. webinar + 15 min. chat

Speaker: Dr. Andreas Plückthun 

Future recombinant binding tools may avoid antibodies, and use diversified libraries based on small and versatile protein scaffolds, for example the DARPins (Designed Ankyrin Repeat Proteins).  DARPins are single-chain, monodisperse, and non-immunogenic, and can be made rapidly and cheaply in bacteria in many formats.  They can also be expressed in and on cells as non-aggregating fusion proteins with tunable valencies, which opens a wide range of previously- inaccessible approaches in cell biology and biochemistry.

REGISTER HERE

The Importance of Antibody Reagent Validation

by

 

 

 


In case more examples are needed, two new reports describe problems associated with antibody reagents:

To educate and inform our members and the broader research community, The Antibody Society has invited leaders in antibody research to reflect on antibody validation in our 10-part webinar series on this topic.

Register now for the next installment:

WEBINAR 9: Getting to Recombinant Antibodies that Guarantee Reproducible Research

February 12, 2020, 10am Eastern Standard Time / 4pm Central European Time, 45 min. webinar + 15 min. chat

Dr. Andrew Bradbury, Specifica, suggests ways out of the validation maze by rigorous molecular identification of recombinant tool antibodies. This achievable goal could eliminate many of the shadowy issues described in this series of webinars.

Register here!

Most read from mAbs, Aug-Sep 2019

by

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 11.6 (August-September 2019)

Influence of the bispecific antibody IgG subclass on T cell redirection

Dual antigen binding is necessary for potent T cell redirection and is influenced by the structural characteristics of a bispecific antibody (BsAb), which are dependent on its IgG subclass. In this study, Kapelski and colleagues at Janssen Research and Development studied model BsAbs targeting CD19xCD3 that were generated in variants of IgG1, IgG2, and IgG4 carrying Fc mutations that reduce FcγR interaction, and two chimeric IgG subclasses termed IgG1:2 and IgG4:2, in which the IgG1- or IgG4-F(ab)2 are grafted on an IgG2 Fc. All BsAbs were shown to bind both of their target proteins (and corresponding cells) equally well, but CD19xCD3 IgG2 did not bind both antigens simultaneously, and had reduced potency in T-cell redirection assays. The activity of IgG2 BsAbs was fully restored in the chimeric subclasses IgG4:2 and IgG1:2. Their results confirmed the major contribution of the F(ab)2 region to the BsAb’s functional activity and demonstrated that the function of BsAbs can be modulated by engineering molecules combining different Fc and F(ab)2 domains.

Rapid single B cell antibody discovery using nanopens and structured light

The most read paper to date is from Winter and colleagues at Amgen. They describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Secreted mAbs from individually isolated, single antibody secreting cells (ASCs) were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction–based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.

Rapid global characterization of immunoglobulin G1 following oxidative stress

In this new report, Chen et al. describe a method for rapid and consistent global characterization of leachable metals- or peroxide-stressed Ig G1 mAbs. Using two independent protease digestions, data-independent acquisition and data-dependent acquisition liquid chromatography high-resolution mass spectrometry, they monitored 55 potential chemical modifications on trastuzumab, a humanized IgG1 mAb. Processing templates including all observed peptides were developed on Skyline to consistently monitor all modifications throughout the stress conditions for both enzymatic digestions. The Global Characterization Data Processing Site, a universal automated data processing application, was created to batch process data, plot modification trends for peptides, generate sortable and downloadable modification tables, and produce Jmol code for three-dimensional structural models of the analyzed protein. In total, 53 sites on the mAb were found to be modified. Oxidation rates generally increased with the peroxide concentration, while leachable metals alone resulted in lower rates of modifications but more oxidative degradants. Multiple chemical modifications were found on IgG1 surfaces known to interact with FcɣRIII, complement protein C1q, and FcRn, potentially affecting activity. The combination of Skyline templates and the Global Characterization Data Processing Site results in a universally applicable assay allowing users to batch process numerous modifications. Applying this new method to stability studies will promote a broader and deeper understanding of stress modifications on therapeutic proteins.

Using bispecific antibodies in forced degradation studies to analyze the structure–function relationships of symmetrically and asymmetrically modified antibodies

In another paper from Janssen Research and Development authors, Evans et al present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare 4 bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Their results demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.

Like this post but not a member? Please join!

Most read from mAbs, July 2019

by

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 11.5 (July 2019)

Connecting the sequence dots: shedding light on the genesis of antibodies reported to be designed in silico.

In this Perspective article, Vasquez et al. examine data in two publications from the same research laboratory that report on structure-based in silico design of antibodies against viral targets without sequence disclosure, and find that the antibodies align with high sequence identity to previously reported antibodies of the same specificity. They note that the lack of reproducible computational algorithms and output sequences in the initial publications obscures the relationship to previously reported antibodies, and sows doubt as to the genesis narrative described in the publications.

In their companion Perspective article, De novo discovery of antibody drugs – great promise demands scrutiny, mAbs’ Assistant Editors Jonny Finlay and Alex Lugovskoy discuss the real-world issues and the concerns raised by Vásquez et al., which suggest much more work is needed to realize the bold vision of delivering in silico designed antibody therapies to patients.

Investigation of pre-existing reactivity to biotherapeutics can uncover potential immunogenic epitopes and predict immunogenicity risk.

Bivi et al. used anti-drug antibody (ADA) assays to detect and measure pre-existing reactivity or the ability of a molecule to produce an ADA-like response in serum from treatment-naïve, healthy donors. They report that the magnitude of pre-existing reactivity evaluated pre-clinically and expressed as the 90th percentile of Tier 2 inhibition correlates with the subsequent rate of ADA emergence in the clinic. Using the components of an IgG-scFv bispecific antibody in the Tier 2 step of the ADA assay, they identified the scFv as the target of the serum pre-existing reactivity. Most importantly, the domain specificity of pre-existing ADA was the same as that of the treatment-emergent-ADA from patients treated with the molecule. Based on these data, they propose the evaluation of the magnitude and of the domain specificity of pre-existing reactivity as a powerful tool to understand the immunogenic potential of novel biotherapeutics.

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

In this new report, Asensio et al immunized mice that transgenically express human antibodies with either programmed cell death 1 protein or cytotoxic T-lymphocyte associated protein 4 using four different immunization protocols, and then utilized a single cell microfluidic platform to generate tissue-specific, natively paired immunoglobulin (Ig) repertoires from each method and enriched for target-specific binders using yeast single-chain variable fragment display. The data suggest that, although different immunization protocols may generate a response to an antigen, performing multiple immunization protocols in parallel can yield greater Ig diversity. They conclude that modern microfluidic methods, followed by an extensive molecular genomic analysis of antibody repertoires, can be used to quickly analyze new immunization protocols or mouse platforms.

A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing.

Parthiban et al report on use of their mammalian cell libraries of up to 10 million clones displaying a repertoire of IgG-formatted antibodies on the cell surface. TALE nucleases or CRISPR/Cas9 were used to direct the integration of the antibody genes into a single genomic locus, thereby rapidly achieving stable expression and transcriptional normalization. The authors used the system to affinity mature a PD-1-blocking antibody through the systematic mutation and functional survey of 4-mer variants within a 16 amino acid paratope region. Mutating VH CDR3 only, they identified a dominant “solution” involving substitution of a central tyrosine to histidine, but this variant was surpassed by a lysine substitution when light chain variants were introduced. This comprehensive and quantitative interrogation of sequence space was achieved by combining high-throughput oligonucleotide synthesis with mammalian display and flow cytometry operating at the multi-million scale.

Redirected optimized cell killing (ROCK®): A highly versatile multispecific fit-for-purpose antibody platform for engaging innate immunity.

Ellwanger et al. report on the redirected optimized cell killing (ROCK®) antibody platform, which comprises a plethora of CD16A-binding innate immune cell engagers with unique properties. In particular, they discuss the advantages of innate immune cell engagement over classical monoclonal antibodies and other engager concepts, and present details on its potential to engineer a fit-for-purpose innate immune cell engager format that can be equipped with unique CD16A domains, modules that influence pharmacokinetic properties and molecular architectures that influence the activation of immune effectors, as well as tumor targeting.

Most read from mAbs, April 2019

by

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 freely downloaded by following the links below.

Issue 11.3 (April 2019)

CH2 domain orientation of human immunoglobulin G in solution: Structural comparison of glycosylated and aglycosylated Fc regions using small-angle X-ray scattering

Yageta et al examined the small-angle X-ray scattering (SAXS) profile of the glycosylated Fc region (gFc) and aglycosylated Fc region (aFc) in solution to determine if removal of the N-linked glycan alters the CH2 domain orientation in the Fc region. For both gFc and aFc, the best-fitted SAXS profiles corresponded to ones calculated based on the crystal structure of gFc that formed a “semi-closed” CH2 domain orientation. Collectively, the data indicated that the removal of the N-linked glycan only negligibly affected the CH2 domain orientation in solution. Their findings will guide the development of methodology for the production of highly refined functional Fc variants.

Charge variants characterization and release assay development for co-formulated antibodies as a combination therapy

Characterization of co-formulated biologics can be challenging due to the high degree of similarity in the physicochemical properties of co-formulated proteins, especially at different concentrations of individual components. In this new report, Cao et al present the results of a deamidation study of one monoclonal antibody component (mAb-B) in co-formulated combination antibodies (referred to as COMBO) that contain various ratios of mAb-A and mAb-B. A single deamidation site in the complementarity-determining region of mAb-B was identified as a critical quality attribute (CQA) due to its impact on biological activity. A conventional charge-based method of monitoring mAb-B deamidation presented specificity and robustness challenges, especially when mAb-B was a minor component in the COMBO, making it unsuitable for lot release and stability testing. The authors developed and qualified a new, quality-control-friendly, single quadrupole Dalton mass detector (QDa)–based method to monitor site-specific deamidation. Their approach can be also used as a multi-attribute method for monitoring other quality attributes in COMBO. This analytical paradigm is applicable to the identification of CQAs in combination therapeutic molecules, and to the subsequent development of a highly specific, highly sensitive, and sufficiently robust method for routine monitoring CQAs for lot release test and during stability studies.

Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening

Puligedda et. al describe a system in which hybridomas specifically capture and display the mAbs they secrete. Using On-Cell mAb Screening (OCMS™), monoclonal antibodies (mAbs) displayed on the cell surface can be rapidly assayed for expression level and binding specificity using fluorescent antigens with high-content (image-based) methods or flow cytometry. OCMS™ demonstrated specific mAb binding to poliovirus and rabies virus by forming a cell surface IgG “cap”, as a universal assay for anti-viral mAbs. The authors produced and characterized OCMS™-enabled hybridomas secreting mAbs that neutralize poliovirus and used fluorescence microscopy to identify and clone a human mAb specific for the human N-methyl-D-aspartate receptor. They also used OCMS™ to assess expression and antigen binding of a recombinant mAb produced in 293T cells.

Physicochemical and functional assessments demonstrating analytical similarity between rituximab biosimilar HLX01 and the MabThera®

As discussed by Xu et al, development of bio-therapeutics has exhibited exponential growth in China over the past decade. However, no biosimilar drug has been approved in China (CN) due to the lack of a national biosimilar regulatory guidance. HLX01, a rituximab biosimilar developed in China under European Medicines Agency biosimilar guidelines and requirements, was the first such drug submitted for regulatory review in China, and it is expected to receive approval there as a biosimilar product. To demonstrate the analytical similarities of HLX01, CN-rituximab (sourced in China but manufactured in Europe) and EU-rituximab (sourced and manufactured in Europe), an extensive 3-way physicochemical and functional similarity assessment using a series of orthogonal and state-of-the-art techniques was conducted, following the similarity requirement guidelines recently published by China’s Center for Drug Evaluation. The results of the similarity study showed an identical protein amino acid sequence and highly similar primary structures between HLX01 and the reference product (RP) MabThera®, along with high similarities in higher order structures, potency, integrity, purity and impurity profiles, biological and immunological binding functions, as well as degradation behaviors under stress conditions. In addition, HLX01 presented slightly lower aggregates and better photostability compared with the RP. Despite slight changes in relative abundance of glycan moieties and heavy chain C-terminal lysine modification, no differences in biological activities and immunological properties were observed between the RP and HLX01. In conclusion, HLX01 is highly similar to CN- and EU-sourced RP in terms of physicochemical properties and biological activities, suggesting similar product quality, efficacy, and safety. The regulatory requirements interpreted and applied towards the HLX01 marketing application sets a precedent for analytical similarity assessment of biosimilar products in China.

Like this post but not a member? Please join!