Processes for preparing glycoprotein-drug conjugates
Inventors
Tsai, Shih-Chong • Lee, Chun-Chung • LEE, MENG-SHENG • Chen, Ching-Yao • Chuang, Shih-Hsien • Chen, Yi-Jen • Wei, Win-Yin
Assignees
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Publication Number
US-11085062-B2
Publication Date
2021-08-10
Expiration Date
Abstract
A process for modifying glycoproteins is provided. The invention also provides a process for producing glycoprotein-payload conjugates, as well as the conjugates produced thereby.
Core Innovation
The invention relates to a process for preparing a glycoprotein-payload conjugate comprising a structure of formula (1). The process converts an initial glycoprotein having a glycan of formula (2) into a modified glycoprotein comprising a tri-mannosyl core of formula (3), and then builds a defined glycan moiety of formula (4). The conversion and glycan construction are controlled by specific enzyme activities and defined sugar-reactive building blocks.
In the process, the glycoprotein comprising the glycan of formula (2) is reacted with β-N-acetylglucosaminidase to produce a modified glycoprotein comprising a tri-mannosyl core of formula (3). The tri-mannosyl core of formula (3) is reacted with UDP-GlcNAc-(CH2)0-8—R, wherein R is azido, a ketone group or an aldehyde, in the presence of mannosyl (α-1,3)-glycoprotein β-1,2-N-acetylglucosaminyltransferase and mannosyl (α-1,6)-glycoprotein β-1,2-N-acetylglucosaminyltransferase.
This allows two GlcNAc-(CH2)0-8—R sugars to respectively bond to the α-1,2 position of each of Man2 and Man3 to form the glycan moiety of formula (4). Finally, two conjugator-linker-payloads, in which the payloads are the same or different, are reacted with the glycan moiety of formula (4) to produce the glycoprotein-payload conjugate comprising the structure of formula (1).
Claims Coverage
The disclosure contains one independent claim, which defines a three-step enzymatic glycoengineering and conjugation process that creates a defined glycan moiety for attachment of two conjugator-linker-payloads, with the payloads being the same or different. The independent claim includes three inventive features based on enzyme-mediated conversion, defined UDP-GlcNAc-(CH2)0-8—R chemistry, and dual conjugator-linker-payload attachment to the resulting glycan architecture.
Enzymatic conversion to a tri-mannosyl core glycoprotein
Reacting a glycoprotein comprising a glycan having formula (2) with β-N-acetylglucosaminidase to produce a modified glycoprotein comprising a tri-mannosyl core of formula (3).
Dual GlcNAc-(CH2)0-8—R installation on Man2 and Man3 via specific glycosyltransferases
Reacting the modified glycoprotein comprising the tri-mannosyl core of formula (3) with UDP-GlcNAc-(CH2)0-8—R, wherein R is azido, a ketone group or an aldehyde, in the presence of mannosyl (α-1,3)-glycoprotein β-1,2-N-acetylglucosaminyltransferase and mannosyl (α-1,6)-glycoprotein β-1,2-N-acetylglucosaminyltransferase to allow two GlcNAc-(CH2)0-8—R sugars to respectively bond to the α-1,2 position of each of Man2 and Man3, and forming a glycan moiety of formula (4).
Dual conjugator-linker-payload attachment to form the glycoprotein-payload conjugate structure
Reacting two conjugator-linker-payloads, wherein the payloads of the two conjugator-linker-payloads are the same or different, with the glycan moiety of formula (4) to produce the glycoprotein-payload conjugate comprising the structure of formula (1).
Across the independent claim, the core coverage is the combination of: (1) β-N-acetylglucosaminidase conversion to a tri-mannosyl core, (2) glycosyltransferase-mediated installation of two GlcNAc-(CH2)0-8—R functional sugars at the α-1,2 positions of Man2 and Man3 to form a defined glycan moiety, and (3) reaction of that glycan moiety with two conjugator-linker-payloads (same or different) to produce the final glycoprotein-payload conjugate structure.
Stated Advantages
Enables production of homogeneous glycoprotein-payload conjugates with defined dual-payload constructs.
Documented Applications
Preparation of antibody payload conjugates using the disclosed process framework, including generation of single- and dual-payload ADC products.
Scalable production feasibility using mammalian production for CMC scalability.
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