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The Antibody Society

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Most read from mAbs, October 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 11.7 (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.

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Antibody Engineering & Therapeutics Student and Post-doc Poster Competition

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Attention Student and Post-doc Members!
To recognize the research activities of promising student and postdoctoral attendees of the Antibody Engineering & Therapeutics conference, The Antibody Society is sponsoring a  student/postdoc poster competition. Two winners will be selected to receive:

1) Complimentary registration to attend the conference and pre-conference sessions;

2) An opportunity to give a short oral presentation of their work in a conference session; and

3) Support for travel expenses (up to $400 for domestic or $800 for international flights, 3 nights at the hotel, ground transportation).

In order to be considered for this poster competition, you must be a student or postdoc member of The Antibody Society. If you are not already a member, you may register here for a free student or postdoc membership:

You must also check the box on the poster submission form indicating that you want your abstract to be considered for the poster competition. Your poster abstract must be submitted using the poster submission form here.

The deadline for submission of your poster abstract is October 15, 2019.
Winners will be notified by Friday, October 25, 2019.
Poster abstracts may be submitted and accepted for presentation at the conference after October 15, but any submissions received after October 15 will not be considered for the poster competition.

Interested in attending the Antibody Engineering & Therapeutics meeting? Society members save 15% on the registration fee – contact us at membership@antibodysociety.org for the code!

 

Good and bad news for antibody-drug conjugates

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On August 23, 2019 GlaxoSmithKline announced positive headline results from the pivotal DREAMM-2 study of the antibody-drug conjugate belantamab mafodotin (GSK2857916) for multiple myeloma. The two-arm study met its primary objective and demonstrated a clinically meaningful overall response rate with belantamab mafodotin in the patient population. The safety and tolerability profile was consistent with that observed in DREAMM-1, the first time in human study of belantamab mafodotin. Data from the DREAMM-2 study will be the basis for regulatory filings starting later this year.

•             Belantamab mafodotin is a humanized anti-B-cell maturation antigen monoclonal antibody that is afucosylated and conjugated to the microtubule-disrupting agent monomethyl auristatin-F.

On August 29, 2019 AbbVie announced that MERU (NCT03033511), a Phase 3 trial evaluating the antibody-drug conjugate rovalpituzumab tesirine (Rova-T) as a first-line maintenance therapy for advanced small-cell lung cancer, demonstrated no survival benefit at a pre-planned interim analysis for patients receiving Rova-T as compared with placebo. The overall safety profile was generally consistent with that observed in previous studies. The MERU trial is being closed, and the Rova-T research and development program has been terminated. AbbVie will move forward prioritizing other development programs within its oncology pipeline.

•             Rovalpituzumab tesirine is an antibody-drug conjugate composed of a humanized monoclonal antibody, dipeptide linker, and pyrrolobenzodiazepine dimer toxin with a drug-to-antibody ratio of 2. The antibody component targets cancer-stem cell-associated delta-like protein 3.

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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’.

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.

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’.