Solid phase TGase-mediated conjugation of antibodies
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Publication Number
US-10611824-B2
Publication Date
2020-04-07
Expiration Date
Abstract
Some embodiments described herein relate to solid-support based processes for the functionalization of immunoglobulins through the use of transglutaminase. Also provided are solid support compositions with bound antibody. The methods can be used, for example, in manufacturing processes or in drug screening.
Core Innovation
The invention relates to conjugating a moiety of interest (Z) to an antibody by using an acceptor glutamine residue at Kabat residue 295 and/or 297, within or appended to a constant region of the antibody, and a transglutaminase (TGase) linking step. The antibody is immobilized on a solid support via the Fc region using a protein A or G affinity medium.
The immobilized antibody is reacted with a linking reagent comprising a reactive group (R) in the presence of transglutaminase (TGase) to obtain an antibody comprising the acceptor glutamine linked to the reactive group (R) via a linker. After the reaction, a washing process removes unreacted materials.
A compound comprising the moiety Z and a reactive group (R′) reactive with the reactive group (R) is then applied to a solid support to generate an antibody-moiety-of-interest (Z) conjugate. The disclosed framework further includes recovery and reapplication of an unreacted compound comprising moiety Z and reactive group R′ to increase reaction completion, and eluting immobilized antibody conjugates to produce antibody conjugate compositions.
Claims Coverage
The document contains one independent method claim describing a TGase-mediated, immobilized-antibody conjugation workflow with complementary reactive groups R and R′ to attach a moiety of interest (Z). The claims cover eight inventive features.
Fc immobilization on protein A or G affinity medium
An antibody comprising an acceptor glutamine at Kabat residue 295 and/or 297 is immobilized on a solid support via the Fc region using a protein A or G affinity medium, with the acceptor glutamine present within or appended to a constant region of the antibody.
TGase-mediated linking to reactive group R
The immobilized antibody is reacted with a linking reagent comprising a reactive group (R) in the presence of transglutaminase (TGase) to obtain an antibody comprising the acceptor glutamine linked to the reactive group (R) via a linker.
Washing to remove unreacted materials
A washing process removes any unreacted materials after the TGase linking step.
Reactive-group coupling to generate antibody-moiety-of-interest conjugate
A compound comprising a moiety Z and a reactive group R′ reactive with reactive group (R) is applied to a solid support to generate an antibody-moiety-of-interest (Z) conjugate.
Recovery and reapplication of unreacted moiety Z compound
An unreacted compound comprising moiety Z and reactive group R′ is recovered from the solid support and reapplied to increase reaction completion.
Elution of immobilized antibody conjugates
Immobilized antibody conjugates are eluted from a solid support to produce antibody conjugate compositions.
Defined linking reagent structure
The linking reagent is provided as a compound having a structure of Formula Ib, including a pharmaceutically acceptable salt or solvate.
Functionalized acceptor glutamine conjugate defined by Formula IVb
The antibody-moiety-of-interest (Z) conjugate is an antibody or antibody fragment containing a functionalized acceptor glutamine residue defined by Formula IVb and associated structural parameters, including a pharmaceutically acceptable salt or solvate.
Overall, the claims define a conjugation method that combines Fc-region immobilization on protein A/G affinity medium, TGase-mediated attachment of a reactive group to an acceptor glutamine at Kabat residue 295 and/or 297, removal of unreacted materials, and final conjugation using complementary reactive groups to form antibody-moiety-of-interest (Z) conjugates. Dependent claim refinements include recovery and reuse of unreacted moiety-Z reactive compound, elution of conjugates, and structural constraints defined by Formula Ib and Formula IVb.
Stated Advantages
Increase reaction completion.
Documented Applications
A moiety of interest, including a therapeutic moiety and/or a diagnostic moiety, is conjugated to an antibody.
Antibody conjugate compositions are produced.
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