JP-2026514404-A - Site-directed orthogonal biocombination modalities and their applications

Abstract

A manufacturing process for an immune ligand/payload complex is provided, and in particular, a novel enzyme site-specific complexing technology using a dual enzyme orthogonal catalyst, comprising the steps of: complexing a first payload with an immune ligand via the enzymatic catalyst of a first enzyme; and complexing a second payload with an immune ligand via the enzymatic catalyst of a second enzyme, wherein the first enzyme is different from the second enzyme.

Inventors

• ギャン・チン
• メイジュン・ション
• ツァオ・エルヴィー
• リリ・シー

Assignees

• ジーンクアンタム ヘルスケア （スーチョウ） シーオー．， エルティーディー．

Dates

Publication Date

20260511

Application Date

20240329

Priority Date

20230330

Claims (20)

1. An enzymatic complexing process for preparing an immune ligand/payload complex, comprising the following steps: The steps include: conjugating the first payload to an immune ligand via enzymatic catalysis by the first enzyme; The process includes the step of conjugating a second payload to the immune ligand via an enzymatic catalyst of a second enzyme, Here, the first enzyme is different from the second enzyme. process.
2. The first enzyme catalysis step and the second enzyme catalysis step are performed simultaneously or sequentially. The process according to claim 1.
3. The method includes simultaneously conjugating the first payload and the second payload to the immune ligand via the enzymatic catalysts of the first and second enzymes, The process according to claim 1 or 2.
4. The steps include: obtaining a first complex by conjugating the first payload with the immune ligand via the enzyme catalyst of the first enzyme; The method includes conjugating the second payload to the first complex via the enzyme catalyst of the second enzyme, thereby obtaining the immune ligand/payload complex. The process according to claim 1 or 2.
5. The first enzyme comprises a ligase, and/or the second enzyme comprises an endoglycosidase. The process according to any one of claims 1 to 4.
6. The first enzyme comprises an endoglycosidase, and/or the second enzyme comprises a ligase. The process according to any one of claims 1 to 4.
7. The ligase is a transpeptidase or a variant thereof, such as saltase A, saltase B, saltase C, saltase D, saltase E, or saltase F and their variants, preferably saltase A. and/or the recognition motif of the ligated donor substrate is LPXTGJ, NPXTG, LPXTA, or LAXTG, preferably LPXTG or LPETGG. and/or the recognition motif of the ligase acceptor substrate is Gn , where G is glycine and n is an integer from 3 to 10. X is any natural or non-natural amino acid, J is either absent or an amino acid fragment containing 1 to 10 amino acids, where each amino acid is independently any natural or non-natural amino acid, preferably J is absent or G m , where m is an integer from 1 to 10. The process according to claim 5 or 6.
8. The aforementioned saltase A comprises an amino acid sequence selected from any one of Sequence IDs 1 to 27, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with them. Preferably, the saltase A contains the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. The process according to claim 7.
9. The endoglycosidase is an N-acetylglucosaminidase that covalently binds a donor containing an oxazoline oligosaccharide to an immune ligand containing a GlcNAc motif, and the donor containing the oxazoline oligosaccharide further contains a payload. The process according to claim 5 or 6.
10. The N-acetamide glucosidase is at least one selected from Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia myricola endoglycosidase-F3), Endo S2 (endoglycosidase S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo CC (endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC) or their variants. The N-acetylglucosaminidase is at least one selected from the group consisting of Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2 or their variants. Preferably, the N-acetylglucosaminidase is Endo S2 or a variant thereof. The process according to claim 9.
11. The oxazoline oligosaccharide is one or more selected from the group consisting of disaccharide oxazoline, trisaccharide oxazoline, tetrasaccharide oxazoline, pentasaccharide oxazoline, hexsaccharide oxazoline, heptasaccharide oxazoline, octsaccharide oxazoline, nonusaccharide oxazoline, decasaccharide oxazoline, and elevensaccharide oxazoline. The process according to claim 9.
12. The oxazoline oligosaccharide has the following structure: The first hexose group or its derivative - (the second hexose group or its derivative) f -β-D-glucopyranosyloxazoline, f is 0, 1, 2, 3, 4, 5, or 6, and β-D-glucopyranosyloxazoline has the following structure: The process according to claim 9.
13. The first hexose group or its derivative is selected from glucosyl, mannosyl, galactosyl, or derivatives thereof, and/or the carbon at position 6 of the first hexose group is in the -C(O)- form, and/or the second hexose group or its derivative is independently selected from glucosyl, mannosyl, galactosyl, or derivatives thereof in the presence of each, and/or each monosaccharide portion of the oligosaccharide structure is linked by a β-(1→4) glycosidic bond, and/or the first hexose group derivative and the second hexose group derivative are independently selected from uronic acid or derivatives in which the hydroxyl group of a monosaccharide is substituted with an acylamide group. The process method according to claim 12.
14. The oxazoline oligosaccharide has the following structure: The first hexose group or its derivative -β-D-glucopyranosyloxazoline, where the first hexose group or its derivative is mannose or its derivative, or the first hexose group or its derivative -β-D-glucopyranosyloxazoline, where the first hexose group or its derivative is galactose or its derivative, Preferably, the structure of the oxazoline oligosaccharide is as follows: The process according to claim 12 or 13.
15. The ligase and/or endoglycosidase covalently ligates to a self-labeled protein tag to form a ligase fusion protein and/or endoglycosidase fusion protein. The process according to claim 5 or 6.
16. The self-labeled protein tag includes a SNAP tag, CLIP tag, His tag, Halo tag, or variants thereof. The process according to claim 15.
17. In the ligase fusion protein, the ligase has an isoelectric point (pI) of approximately 7.5 to approximately 10.0, the self-labeled protein tag is a Halo tag having an isoelectric point of approximately 4.5 to approximately 5.0, and the pI of the ligase fusion protein is approximately 2.0 to approximately 4.5 pH units lower than the pI of the ligase. The process according to claim 15.
18. The Halo tag includes the amino acid sequence of Sequence ID No. 28, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. The process according to claim 17.
19. The ligase of the ligase fusion protein is a saltase A comprising an amino acid sequence selected from any one of Sequence IDs 1 to 27, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. Preferably, the saltase A contains the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. The process according to claim 18.
20. The ligase fusion protein includes the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. The process according to claim 19.

Description

This disclosure relates to a manufacturing process for immune ligand/payload complexes, and more particularly to a novel enzyme site-specific complexing technology using a dual enzyme orthogonal catalyst, and to highly homogeneous and mutually orthogonal drug-loading antibody-drug complexes, in particular dual-payload antibody-drug complexes (dpADCs), prepared thereby. Antibody-drug conjugates (ADCs) consist of three parts: an antibody, a linker, and a highly potent small-molecule drug. The small-molecule payload is linked to an antibody that specifically binds to an antigen overexpressed on the surface of tumor cells. Subsequently, the resulting ADC is internalized by the tumor cells and releases a cytotoxic payload into the tumor microenvironment (TME) or inside the tumor cells. The released payload then exerts an antitumor effect with minimal toxicity to normal tissue. Therefore, ADCs are also known as a class of targeted chemotherapy drugs with controllable side effects. By 2023, there were 14 ADC drugs approved worldwide, and more than 100 were in clinical development, but most of these ADCs are based on a single type of payload. However, tumors are highly heterogeneous and prone to drug resistance. In conventional oncological chemotherapy, combinations of two or more chemotherapy agents are used to overcome drug resistance and improve antitumor efficacy (Non-Patent Literature 1). For example, the combination of docetaxel and gemcitabine has been used in clinical trials, and the synergistic effects of various mechanisms of action and non-overlapping tumor-killing effects have been validated by enhanced patient survival in the treatment of both drug-sensitive and drug-resistant tumor models (Non-Patent Literature 2). However, the combination of chemotherapeutic agents can also increase toxicity. To take this a step further, ADC-based combination therapies are gaining interest and are being investigated in preclinical models and clinical studies. Generally, the most attractive ADC partners are those that provide synergistic or additive effects on cancer cells or TMEs without any unacceptable toxicity, and current ADC partners such as chemotherapeutic agents, molecularly targeted agents, and immunotherapeutic agents are precursors that begin to show increased antitumor efficacy with ADCs in a synergistic manner (Non-Patent Literature 3). Based on these existing research findings, the inventors hypothesize that a superior therapeutic solution for treating immunoresistant cancers with minimal off-target toxicity is a dpADC, which combines various drugs of MoA in a single modality, allowing the two drugs to reach their targets simultaneously and exert their effects in a highly synergistic manner. Currently, several types of conjugation techniques exist for constructing dual-payload antibody-drug conjugates (Non-Patent Literature 4): combinations of two chemical conjugation methods, combinations of chemical and enzymatic conjugation methods, and combinations of two enzymatic conjugation methods. While the first two methods are the most commonly available for constructing dpADCs, dual enzyme-catalyzed orthogonal specific conjugation has rarely been reported. Regarding the combination of two chemical complexing methods, for example, in 2016, Pfizer reported the first example of dual-payload complexing of a solid-phase immobilized antibody (by binding to protein A/L agarose beads) with a Fab fragment. Using a reduction-reoxidation strategy to decapsulate recombinant non-natural cysteine in the bead-bound Fab fragment, the decapsulated cysteine was then complexed with a first maleimide-functionalized linker-payload. After another washing step, the interchain disulfide bonds of Fab were then reduced with TCEP, and subsequently, a second maleimide-based linker-payload was complexed to obtain a dual-payload antibody-fragment complex. They also demonstrated solid-phase dual-payload complexing based on enzymatic and chemical complexing relays. As a proof of concept, a bicyclo[6.1.0]nonine (BCN) group was introduced into an antibody via transglutaminase-mediated conjugation, followed by the introduction of a first linker-payload via a BCN-azidocrick reaction, and then the second linker-payload was linked via cystine-maleimide chemical conjugation. Both protocols require multi-step (more than six) processes, which can cause significant problems in CMC production. (Non-Patent Literature 5). In 2017, Seagen reported a strategy for constructing dpADCs by stepwise building a trifunctional linker and chemical complexes. The researchers synthesized a trifunctional linker containing two orthogonally protected cysteine groups and one maleimide connector. The maleimide functional group of the linker was coupled to the cysteine sulfhydryl group of the antibody via a Michael addition reaction, and then the toxin was attached by two cycles of selective deprotection and Michael addition. A total of five chemical reactions were perfo