Modified antibody in which motif comprising cysteine residue is bound, modified antibody-drug conjugate comprising the modified antibody, and production method for same
Inventors
Park, Soon Jae • Chung, Hye-Shin • Kwon, Seonhun • Lee, Sunbae • Yoo, Sun-ah • Kim, Yong Mo
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9814782-B2
Publication Date
2017-11-14
Expiration Date
Abstract
The present invention relates to an antibody in which a motif composed of an amino acid or peptide sequence including one or more cysteine residues is bound to the terminus of a parent antibody, particularly the terminus of the heavy chain of the parent antibody. Also, the present invention relates to a modified antibody-drug conjugate (mADC) comprising a drug bound to the antibody, and a method for producing the antibody or the modified antibody-drug conjugate. The modified antibody-drug conjugate according to the invention can accurately deliver the drug to a target cell due its high specificity to antigen, and thus can increase the therapeutic effect of the drug. Also, it can increase the usability of drugs, particularly anticancer drugs, the use of which is restricted due to their toxicity, despite their high efficacy. Moreover, the invention relates to a composition for treatment of diseases, particularly cancers, which comprise the modified antibody-drug conjugate.
Core Innovation
The disclosure relates to an antibody-drug conjugate comprising a modified antibody in which a cysteine-containing motif is located at the terminus of a parent antibody. The cysteine-containing motif is a metal ion binding motif containing a cysteine residue, and the drug is conjugated to the thiol group of that cysteine residue in the metal ion binding motif. In particular, the cysteine-containing metal ion binding motif is one or two CGH or HGC motif(s) with alanine at the C-terminus and N-terminus of the motif.
The parent antibody is a modified version of an antibody comprising the cysteine-containing motif at the terminus, and the drug is conjugated directly or through a linker reagent to the thiol group of the motif cysteine residue. The disclosure characterizes the metal ion binding motif as providing metal ion binding and states that the strong metal-ion binding of a CGH motif retards cysteine oxidation and preserves thiol reactivity for conjugation.
The document further describes production of the conjugate by reacting a modified antibody comprising the cysteine-containing motif at the terminus with a linker reagent to form an antibody-linker intermediate and then reacting the intermediate with an active drug moiety so that the drug is conjugated to the thiol group of the cysteine residue. Alternatively, a drug moiety is first reacted with a linker reagent to form a drug-linker intermediate, and then reacted with the parent antibody comprising the cysteine-containing motif to produce the modified antibody-drug conjugate.
The document also describes constructing an expression vector comprising a polynucleotide sequence in which a polynucleotide sequence encoding the motif and a polynucleotide sequence encoding the parent antibody are recombinantly linked, expressing the constructed expression vector in host cells, isolating and purifying the modified antibody, and conjugating a drug to form the antibody-drug conjugate.
Claims Coverage
The claim coverage includes four independent inventive features directed to an antibody-drug conjugate and three production approaches, with a further dependent therapeutic composition.
Cysteine-containing metal ion binding motif at antibody terminus for thiol-conjugated drug
An antibody-drug conjugate comprising a modified antibody comprising a cysteine-containing motif at the terminus of a parent antibody, wherein the cysteine-containing motif is a metal ion binding motif that contains a cysteine residue; the metal ion binding motif is one or two CGH or HGC motif(s) with alanine at C-terminus and N-terminus of the motif; and the drug is conjugated to the thiol group of the cysteine residue in the metal ion binding motif.
Producing conjugate via antibody-linker intermediate then active drug conjugation to motif thiol
A method of producing a modified antibody-drug conjugate comprising reacting a modified antibody comprising a cysteine-containing motif at the terminus of a parent antibody with a linker reagent to form an antibody-linker intermediate, and reacting the intermediate with an active drug moiety to produce the modified antibody-drug conjugate wherein the drug is conjugated to the thiol group of the cysteine residue in the metal ion binding motif.
Producing conjugate via drug-linker intermediate then antibody reaction to form thiol-conjugated product
A method of producing a modified antibody-drug conjugate comprising reacting a nucleophilic group of a drug moiety with a linker reagent to form a drug-linker intermediate, and reacting the intermediate with a parent antibody comprising a cysteine-containing motif at the terminus thereof to form the modified antibody-drug conjugate wherein the drug is conjugated to the thiol group of the cysteine residue in the metal ion binding motif.
Recombinant construction of expression vector encoding motif and parent antibody followed by purification and drug conjugation
A method of producing a modified antibody comprising a motif bound to a parent antibody, and forming an antibody-drug conjugate therefrom, the method comprising constructing an expression vector in which a polynucleotide sequence encoding the motif and a polynucleotide sequence encoding the parent antibody are recombinantly linked, expressing the constructed expression vector in host cells, isolating and purifying the modified antibody, and conjugating a drug to the modified antibody to form the antibody-drug conjugate.
The claimed subject matter centers on a cysteine-containing metal ion binding motif, specifically CGH or HGC with alanine at both ends, positioned at the terminus of a parent antibody to enable drug conjugation to the motif cysteine thiol, with manufacturing routes by linker-mediated conjugation or recombinant expression followed by purification and conjugation.
Stated Advantages
In vitro functional results show reduced IC50 versus Herceptin.
In vitro functional results show increased caspase 3/7 activation indicating apoptosis mediated by released drug.
Metal-ion binding of a CGH motif retards cysteine oxidation and preserves thiol reactivity for conjugation.
Documented Applications
Cancer treatment compositions comprising the disclosed antibody-drug conjugate.
Conjugates configured for binding to HER2 and EGFR family receptors, including HER3 and HER4, with trastuzumab (Herceptin) as a parent antibody example.
In vitro evaluation using BT-474 and SK-BR-3 cells with payloads including doxorubicin (DOX) and MMAE.
Interested in licensing this patent?