Screening of conjugated antibodies
Inventors
Belmant, Christian • Bregeon, Delphine • Dennler, Patrick • Fischer, Eliane • Romagne, François • Schibli, Roger • Gauthier, Laurent
Assignees
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Publication Number
US-10132799-B2
Publication Date
2018-11-20
Expiration Date
Abstract
Provided is a method for the functionalization of immunoglobulins through the use of transglutaminase, including methods for screening functionalized antibodies for characteristics of interest, antibody compositions comprising a plurality of functionalized antibodies, and functionalized antibodies with rodent constant regions.
Core Innovation
The invention relates to a method for evaluating antibodies by preparing a first and a second antibody sample, where each sample comprises a plurality of antibodies having at least one acceptor glutamine residue (Q) in a constant region. The first and second antibody samples differ with respect to antibody variable region sequence, while substantially all antibodies in each sample share the same sequence within that sample. The first and second antibody samples are specific for the same antigen.
Each antibody sample is reacted with a lysine-based linker comprising a reactive group (R) in the presence of transglutaminase (TGase) under conditions sufficient for conjugation. The resulting antibodies are further reacted with a compound comprising a reactive group (R') that reacts with reactive group (R) on the lysine-based linker and a moiety-of-interest (Z), thereby obtaining first and second antibody samples in which the antibodies are conjugated to the lysine-based linker comprising the moiety-of-interest (Z).
The method then evaluates antibodies of the first and second antibody samples conjugated to the lysine-based linker comprising the moiety-of-interest (Z) for a characteristic of interest. At least 90% of the antibodies in each sample have the same number of functionalized acceptor glutamine residues (Q) per antibody, with each functionalized acceptor glutamine conjugated to the lysine-based linker comprising the moiety of interest (Z). The moiety-of-interest (Z) is selected from hydrophobic organic compounds, organic compounds having an electrically negative charge, organic compounds having a molecular weight of at least 400 g/mol, or any combination thereof.
Claims Coverage
The document explicitly provides one independent claim. Across that claim, inventive features center on TGase-mediated conjugation of acceptor glutamine (Q) sites via a lysine-based linker to install a moiety-of-interest (Z), followed by comparative evaluation between antibody samples that differ in variable region sequence but bind the same antigen, while enforcing a high-uniformity functionalization constraint (≥90%) for acceptor glutamine (Q) stoichiometry.
Comparative evaluation of two antibody samples with uniform acceptor glutamine functionalization
Providing a first and a second antibody sample each comprising a plurality of antibodies comprising at least one acceptor glutamine residue (Q) in a constant region, wherein the first and second antibody-containing samples differ from one another with respect to antibody variable region sequence, wherein substantially all antibodies in each sample have the same sequence, and wherein the first and second antibody samples are specific for the same antigen, with at least 90% of the antibodies in each sample having the same number of functionalized acceptor glutamine residues (Q) per antibody.
TGase-mediated lysine-based linker conjugation to acceptor glutamine (Q) sites
Reacting each of the first and second antibody sample with a lysine-based linker comprising a reactive group (R) in the presence of transglutaminase (TGase) under conditions sufficient such that antibodies in the first and second antibody samples are conjugated to a lysine-based linker comprising the reactive group (R).
Installing moiety-of-interest (Z) using a reactive group (R') that reacts with reactive group (R)
Further reacting the resulting antibodies with a compound comprising a reactive group (R') that reacts with reactive group (R) on the lysine-based linker and a moiety-of-interest (Z), whereby a first and a second antibody sample each conjugated to a lysine-based linker comprising the moiety-of-interest (Z) is obtained.
Evaluation of conjugated antibodies for a characteristic of interest with Z constrained to specified categories
Evaluating antibodies of the first and second antibody sample each conjugated to the lysine-based linker comprising the moiety-of-interest (Z) for a characteristic of interest, wherein the moiety-of-interest (Z) is selected from a hydrophobic organic compound, an organic compound having an electrically negative charge, an organic compound having a molecular weight of at least 400 g/mol, or any combination thereof.
The claim coverage centers on a TGase-based, two-stage conjugation workflow that yields antibody conjugates with a moiety-of-interest (Z) installed via a lysine-based linker at acceptor glutamine (Q) sites, followed by comparative evaluation between two antibody samples that are specific for the same antigen but differ in variable region sequence, while requiring ≥90% of antibodies in each sample to share the same number of functionalized acceptor glutamine residues (Q) per antibody.
Stated Advantages
Provides a uniform number of functionalized acceptor glutamine residues (Q) per antibody across substantially all antibodies (at least 90%).
Enables evaluation of antibodies while maintaining comparable functionalization stoichiometry between antibody samples that differ in variable region sequence.
Documented Applications
Screening/evaluating antibody candidates by conjugating antibodies with a moiety-of-interest (Z) using TGase-mediated chemistry and comparing resulting conjugates for characteristic(s) of interest, including antigen binding, Fc receptor binding/effector functions, agonist/antagonist activity, cytotoxicity/cell death, stability, and aggregation susceptibility.
Antibody drug conjugate (ADC) screening and comparison, using moiety-of-interest (Z) that can include hydrophobic or charged organic compounds and organic compounds with specified molecular weight.
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