US2024009319A1PendingUtilityA1
Engineered polypeptide conjugates using transglutaminase
Est. expiryJul 31, 2033(~7 yrs left)· nominal 20-yr term from priority
A61K 47/68031A61K 47/68A61K 47/6803A61K 47/6817A61K 47/6809A61K 47/6805A61P 35/00A61P 43/00
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Claims
Abstract
The present invention provides engineered polypeptide conjugates (e.g., antibody-drug-conjugates) comprising specific acyl donor glutamine-containing tags and amine donor agents. The invention also provides methods of making such engineered polypeptide conjugates using transglutaminase and methods of using thereof.
Claims
exact text as granted — not AI-modified1 - 28 . (canceled)
29 . An engineered antibody conjugate comprising the formula:
polypeptide-T-A, wherein the polypeptide comprises a full length antibody heavy chain and an antibody light chain, wherein T is an acyl donor glutamine-containing tag engineered at a specific site of the polypeptide, wherein A is an amine donor agent,
wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag through at least one of the following: a carboxyl terminus of the antibody heavy chain, a carboxyl terminus of the antibody light chain, an amino terminus of the antibody heavy chain, and an amino terminus of the antibody light chain, and
wherein the acyl donor glutamine-containing tag consists of the amino acid sequence
(SEQ ID NO: 5)
LLQGP,
(SEQ ID NO: 14)
LLQGPX,
(SEQ ID NO: 4)
LLQGPA,
or
(SEQ ID NO: 11)
LLQGPP.
30 . The engineered antibody conjugate of claim 29 , wherein the acyl donor glutamine-containing tag is located at the carboxyl terminus of the antibody heavy chain, at the carboxyl terminus of the antibody light chain, or at both the carboxyl terminus of the antibody heavy chain and the carboxyl terminus of the antibody light chain.
31 . The engineered antibody conjugate of claim 29 , comprising an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme).
32 . The engineered antibody conjugate of claim 29 , wherein the amine donor agent comprises the formula:
X—Y—Z,
wherein X is an amine donor unit; Y is a linker; and Z is an agent moiety.
33 . The engineered antibody conjugate of claim 29 , wherein the antibody is a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antigen-binding antibody fragment.
34 . The engineered antibody conjugate of claim 33 , wherein the agent moiety is a cytotoxic agent, polypeptide, radioisotope or label, or biocompatible polymer.
35 . The engineered antibody conjugate of claim 34 , wherein the cytotoxic agent is selected from the group consisting of anthracycline, an auristatin, a camptothecin, a combretastatin, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a maytansine, a puromycin, a pyrrolobenzodiazepine dimer, a taxane, a vinca alkaloid, a tubulysin, a hemiasterlin, a spliceostatin, a pladienolide, and stereoisomers, and isosteres thereof.
36 . The engineered antibody conjugate of claim 29 , wherein the acyl donor glutamine-containing tag consists of the amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO:14).
37 . The engineered antibody conjugate of claim 36 , wherein the acyl donor glutamine-containing tag consists of the amino acid sequence LLQGPA (SEQ ID NO:4).
38 . The engineered antibody conjugate of claim 36 , wherein the acyl donor glutamine-containing tag consists of the amino acid sequence LLQGPP (SEQ ID NO:11).
39 . A method for preparing an engineered polypeptide conjugate comprising the formula:
polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent,
wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, at an amino terminus, or at another site in the polypeptide, and
wherein the acyl donor glutamine-containing tag consists of the amino acid sequence
(SEQ ID NO: 5)
LLQGP,
(SEQ ID NO: 14)
LLQGPX,
(SEQ ID NO: 4)
LLQGPA,
or
(SEQ ID NO: 11)
LLQGPP,
the method comprising the steps of:
a) providing an engineered polypeptide-T molecule comprising the polypeptide and the acyl donor glutamine-containing tag;
b) contacting the amine donor agent with the engineered polypeptide-T molecule in the presence of a transglutaminase; and
c) allowing the engineered polypeptide-T molecule to covalently link to the amine donor agent to form the engineered polypeptide conjugate.
40 . The method of claim 39 , wherein the engineered polypeptide conjugate has conjugation efficiency of at least about 51%.
41 . The method of claim 39 , wherein the transglutaminase is a microbial, purified, or engineered transglutaminase.
42 . The method of claim 39 , further comprising a purification step, wherein the engineered polypeptide conjugate is purified by chromatography steps.
43 . The method of claim 39 , wherein the engineered polypeptide-T molecule is expressed in mammalian cells, optionally wherein the mammalian cells are CHO cells.
44 . The method of claim 39 , wherein the engineered polypeptide conjugate is a Fc-containing or Fab- or other types of domain binders containing polypeptide.
45 . The method of claim 39 , wherein the acyl donor glutamine-containing tag consists of the amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO:14).
46 . The method of claim 39 , wherein the acyl donor glutamine-containing tag consists of the amino acid sequence LLQGPA (SEQ ID NO:4).
47 . The method of claim 39 , wherein the acyl donor glutamine-containing tag consists of the amino acid sequence LLQGPP (SEQ ID NO:11).
48 . The method of claim 39 , wherein the polypeptide comprises a full length antibody heavy chain and an antibody light chain.Join the waitlist — get patent alerts
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