Enzymatic conjugation of polypeptides
Inventors
Bregeon, Delphine • Dennler, Patrick • Belmant, Christian • Gauthier, Laurent • Romagne, François • Fischer, Eliane • Schibli, Roger
Assignees
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Publication Number
US-9427478-B2
Publication Date
2016-08-30
Expiration Date
Abstract
The present application relates to methods for the enzymatic functionalization of immunoglobulins, in particular with drugs. Also disclosed herein are linking reagents, functionalized antibodies, pharmaceutical compositions, and method of treating disease and/or conditions.
Core Innovation
The invention relates to antibody or antibody fragment compositions in which a plurality of antibodies or antibody fragments share the same heavy and/or light chain amino acid sequence and specifically bind to a tumor antigen. At least 90% of the antibodies or antibody fragments have m functionalized acceptor glutamine residues per antibody or fragment, where m is an integer selected from 2 or 4.
Each functionalized acceptor glutamine residue has a defined structure that includes a pyrrolobenzodiazepine (Z) unit and associated linkage architecture. The structure is defined by a substituted framework that includes Q, C, X, L, (RR), V, and Y, where (RR) is an addition product between a reactive moiety R and a complementary reactive moiety R, V is a non-cleavable or conditionally-cleavable moiety, and Y is a spacer system.
The invention further defines pyrrolobenzodiazepine monomers and C8/C8-linked pyrrolobenzodiazepine dimers, including pyrrolo[2,1-c][1,4]benzodiazepine, as moieties of interest for functionalized acceptor glutamine residues. The disclosure describes acceptor glutamine positioning in antibody constant regions and heavy-chain constant-region acceptor glutamine availability, including variants such as N297Q and Q295/Q295N with N297Q, in compositions of antibodies sharing the same heavy and/or light chain sequences.
Claims Coverage
The provided independent claims center on tumor antigen-binding antibody or antibody fragment compositions with shared heavy/light chain sequences and at least 90% of the molecules functionalized at acceptor glutamine residues. Two inventive features are present across the claims, each requiring m to be 2 or 4 and pyrrolobenzodiazepine as Z.
Tumor-binding antibody composition with functionalized acceptor glutamine residues
A composition comprising a plurality of antibodies or antibody fragments sharing the same heavy and/or light chain amino acid sequence, specifically binding to a tumor antigen, where at least 90% have m functionalized acceptor glutamine residues per antibody or fragment, with m selected from 2 or 4, and with each functionalized acceptor glutamine residue having Formula IVc.
Tumor-binding antibody composition with lysine-based linker acceptor glutamine motif
A composition comprising a plurality of antibodies or antibody fragments sharing the same heavy and/or light chain amino acid sequence, specifically binding to a tumor antigen, where at least 90% have m functionalized acceptor glutamine residues per antibody or fragment, with m selected from 2 or 4, and with each acceptor glutamine residue having the structure (Q)-L3-RR-Y-Z, where L3 is a lysine-based linker and Z is a pyrrolobenzodiazepine.
Across the independent claims, the coverage centers on tumor antigen-binding antibody or antibody fragment compositions with shared heavy/light chain sequences and at least 90% of the antibodies bearing m functionalized acceptor glutamine residues per antibody, where m is 2 or 4. The claims further require a structural motif with an addition product (RR), a spacer system Y, and pyrrolobenzodiazepine as Z, with one claim using Formula IVc and the other specifying a lysine-based linker motif.
Stated Advantages
Improved conjugation of hydrophobic or large and charged payloads by changing the Fc/CH2 environment around glycosylation removal for access at residue 297 rather than Q295.
TGase enzymatic activity inhibition is controlled by limiting organic solvent levels to avoid reduced activity.
Use of N297S and Q295N/N297Q antibodies and/or specific linker/spacer arrangements to substantially increase mono-functional MMAF coupling and to improve functionalization outcomes for particular conjugates.
Reduced aggregation temperatures for hydrophobic DBCO-containing linkers compared to azide or thiol/acetyl-type linkers.
Comparable aggregation stability for PEG versus C6 thiol linkers.
Aggregation propensity comparable to Adcetris for trehalose and Polysorbate 80 formulations under freeze/thaw and shaking/vortex stress.
Lower soluble aggregates with fewer fragments under freeze/thaw and shake stress for the described formulation context.
Comparable or improved thermal stability by TSSA for specific SGN-35 N297 variants.
Indicated in vitro cytotoxic activity on CD30+ KARPASS-299 cells, while non-toxin controls show minimal effects.
Documented Applications
Therapeutic and diagnostic use as drug conjugates, including embodiments for solid tumors or hematological malignancies.
ADC construction.
Use of the described antibody conjugate compositions to achieve in vitro cytotoxic activity on CD30+ KARPASS-299 cells, with non-toxin controls showing minimal effects.
Assessment of the compositions as SGN-35 ADCs, including MMAE payload ADCs with PEG-linked cleavable peptide spacer (val-cit) and formulations containing trehalose and Polysorbate 80.
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