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Most read from mAbs, Aug-Sep 2019

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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.

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Uptick in biosimilar antibody products approved by FDA

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The US Food and Drug Administration (FDA) began approving biosimilar products in 2015. According to FDA’s definition, a biosimilar is highly similar to, and has no clinically meaningful differences in safety, purity, and potency from, an existing FDA-approved reference product. The availability of these products can help patients by increasing the number of medication options at potentially lower costs.

 

During March 2015 to July 2019, FDA approved a total of 16 antibody therapeutics that are biosimilars of 5 reference products:

  • Trastuzumab (5 biosimilars)
  • Adalimumab (4 biosimilars)
  • Infliximab (3 biosimilars)
  • Bevacizumab (2 biosimilars)
  • Rituximab (2 biosimilars)

Notably, the rate of FDA approvals has increased in 2019. The numbers of  biosimilar antibody therapeutics approved by FDA were 0, 2, 5, and 3 for the years 2015-2018, while a total of 6 were approved in the first 7 months of 2019. The products approved in 2019 are:

Patients may soon also have access to ranibizumab and denosumab biosimilar antibody products.

  • Formycon and Bioeq IP AG recently announced that an FDA submission for FYB201, a biosimilar candidate for Lucentis®* (ranibizumab), is expected for the beginning of the fourth quarter of 2019. The submission to the European Medicines Agency (EMA) is scheduled for the first quarter of 2020. If the submissions progress as planned, marketing authorization approvals in the US and the EU are expected in 2021.
  • SB11, a proposed ranibizumab biosimilar to Lucentis is undergoing evaluation in a Phase 3 study (NCT03150589) of patients with neovascular age-related macular degeneration. Sponsored by Samsung Bioepis Co., Ltd., the study is active, but no longer recruiting patients. The estimated completion date of the study is in November 2019.
  • Sandoz recently announced the first patient was enrolled an integrated Phase 1/3 clinical study (NCT03974100) that will compare the pharmacokinetics, pharmacodynamics, efficacy, safety and immunogenicity of GP2411 (proposed biosimilar denosumab) and Prolia® (EU-authorized) in postmenopausal women with osteoporosis. The estimated primary completion date of the study is in December 2021.

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The Antibody Society maintains a comprehensive table of approved mAb therapeutics and those in regulatory review in the EU or US. Located in the ‘Web Resources’ section of our website, the list is updated regularly and can be downloaded in Excel format. Information about antibody therapeutics that may enter regulatory review in 2019 can be found in ‘Antibodies to watch in 2019’.

Four new antibody therapeutics enter regulatory review

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Marketing applications for four antibody therapeutics (crizanlizumab, enfortumab vedotin, teprotumumab, isatuximab) were recently submitted to the US Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

On July 16, 2019, Novartis announced the FDA accepted the company’s Biologics License Application (BLA) and has granted Priority Review for crizanlizumab (SEG101). Novartis submitted the application for crizanlizumab for the prevention of vaso-occlusive crises (VOCs) in patients with sickle cell disease (SCD). The FDA submission is supported by results from the Phase 2 SUSTAIN study, which showed that crizanlizumab (5 mg/kg) reduced the median annual rate of VOCs leading to health care visits by 45.3% compared with placebo (1.63 vs 2.98, P=0.010) in patients with or without hydroxyurea. Clinically significant reductions in the frequency of VOCs were observed among patients regardless of sickle cell disease genotype or hydroxyurea use. A marketing authorization application for crizanlizumab is undergoing evaluation by EMA.

  • Crizanlizumab, humanized IgG2 targeting P-selectin, was granted Breakthrough Therapy designation in December 2018

On July 16, 2019, Seattle Genetics, Inc. and Astellas Pharma Inc. announced submission of a BLA for accelerated approval to the FDA for enfortumab vedotin for the treatment of patients with locally advanced or metastatic urothelial cancer who have received a PD-1/L1 inhibitor and who have received a platinum-containing chemotherapy in the neoadjuvant/adjuvant, locally advanced or metastatic setting. The submission is based on results from the first cohort of patients in the EV-201 pivotal Phase 2 clinical trial that were presented as a late-breaking abstract at the annual meeting of the American Society of Clinical Oncology (ASCO) in June.

  • Enfortumab vedotin is a human IgG1 antibody-drug conjugate that targets Nectin-4, a protein that is highly expressed in urothelial cancers.

On July 10, 2019, Horizon Therapeutics plc announced that it has submitted a BLA to FDA for teprotumumab for the treatment of active thyroid eye disease. Teprotumumab has Breakthrough Therapy, Orphan Drug and Fast Track designations from the FDA. Horizon requested priority review for the application, which, if granted, could result in a six-month review process. The FDA has a 60-day filing review period to determine whether the BLA is complete and acceptable for filing.

  • Teprotumumab is a human IgG1 antibody that targets insulin-like growth factor 1 receptor.

On July 10, 2019, Sanofi announced that the FDA has accepted for review the BLA for isatuximab (SAR650984) for the treatment of patients with relapsed/refractory multiple myeloma (RRMM). The target action date for the FDA decision is April 30, 2020. Isatuximab received orphan designation for relapsed/refractory multiple myeloma from both the FDA and the EMA, and in the second quarter of 2019 the EMA accepted a marketing authorization application for evaluation.

  • Isatuximab is a novel IgG1 antibody that binds selectively to a specific epitope on CD38.

Like this post but not a member? Please join!

The Antibody Society maintains a comprehensive table of approved mAb therapeutics and those in regulatory review in the EU or US. Located in the ‘Web Resources’ section of our website, the list is updated regularly and can be downloaded in Excel format. Information about antibody therapeutics that may enter regulatory review in 2019 can be found in ‘Antibodies to watch in 2019’.

Most read from mAbs, July 2019

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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.

Join Us for Antibody Engineering & Therapeutics Digital Week!

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Antibody Engineering & Therapeutics Digital Week is a global 4-day series of live educational webcasts and downloadable resources providing the latest insights for accelerating next generation antibodies to commercial success.

The clinical pipeline of antibody therapeutics will be discussed in The Antibody Society’s “Antibodies to Watch” and More: Early- and Late-stage Clinical Development Trends presentation, scheduled for 11am EDT / 4pm BST / 5pm CEST Wednesday June 26, 2019.

The “Antibodies to watch” talks and papers focus on antibody therapeutics in late-stage clinical studies, as well as those is regulatory review and recently approved in the US and European Union. These topics will be discussed, along with trends observed in the burgeoning early-stage pipeline. Popular formats and mechanisms of action, as well as popular and obscure targets, for antibody therapeutics that recently entered the clinical pipeline will be included.

Click here to register for Digital Week.