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EP-4736886-A1 - METHOD FOR PREPARING ANTIBODY-SMALL MOLECULE DRUG CONJUGATE AND USE THEREOF

EP4736886A1EP 4736886 A1EP4736886 A1EP 4736886A1EP-4736886-A1

Abstract

A method for preparing an antigen binding fragment-small molecule drug conjugate and the use thereof. A method for modifying human IgG Fc comprises modifying the N-terminal sequence of human IgG Fc. When fusing the obtained IgG Fc variant and an antigen binding fragment and expressing same, the loading capacity of the functional molecule is remarkably increased, and the polymer generation following expression is greatly reduced. Also provided are a fusion protein comprising the IgG Fc variant, and a drug conjugate.

Inventors

  • HE, Honglin
  • XU, Yanghua
  • YE, LI
  • FENG, Mengting
  • XIA, Aikun
  • ZHONG, ZIYANG

Assignees

  • SHANGHAI RUOTUO BIOSCIENCES CO., LTD.

Dates

Publication Date
20260506
Application Date
20240717

Claims (20)

  1. A method for increasing payload of a functional molecule of an antigen binding fragment-human IgG Fc fusion protein and reducing post-expression aggregate formation, comprising: (1) providing an antigen binding fragment-human IgG Fc fusion protein, wherein the fusion protein comprises a human IgG Fc comprising an engineered N-terminal sequence, wherein a method for preparing the engineered N-terminal sequence comprises: replacing 6-15 amino acid residues at N-terminus of wild-type human IgG Fc with amino acid residues set forth in SEQ ID NO: 6, and performing one or more of the following modifications on the amino acid sequence set forth in SEQ ID NO: 6: (a) inserting 1-9 amino acid residues between, upstream of, or downstream of two cysteine residues (CC) at positions 4 and 5 of the amino acid sequence set forth in SEQ ID NO: 6; and (b) mutating one of the two cysteine residues at positions 4 and 5 of the amino acid sequence set forth in SEQ ID NO: 6.
  2. The method according to claim 1, wherein in (a), 2-8 non-cysteine amino acid residues are inserted between the two cysteine residues (CC).
  3. The method according to claim 1, wherein the inserting or mutating is performed using amino acid residues selected from a group consisting of: aspartic acid, glutamic acid, alanine, glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine, and histidine.
  4. The method according to claim 3, wherein the inserting is performed using acidic amino acid residues, wherein the acidic amino acid residues are one or more of aspartic acid and glutamic acid.
  5. The method according to claim 1, wherein the method further comprises: (2) conjugating a functional molecule to the antigen binding fragment-human IgG Fc fusion protein, wherein the payload of the functional molecule is in a range of 6-8.
  6. The method according to claim 1, wherein the functional molecule comprises: a small molecule antitumor drug, a cytotoxin, a radioisotope, a bioactive protein, a molecule targeting a tumor surface marker, a tumor-inhibitory molecule, a molecule targeting a surface marker of an immune cell, a detectable label, or an extracellular hinge region, a transmembrane domain, and an intracellular signaling domain based on chimeric antigen receptor technology, or a combination thereof.
  7. The method according to claim 6, wherein the molecule targeting the tumor surface marker comprises an antibody that binds the tumor surface marker or a ligand that binds the tumor surface marker; or wherein the tumor-inhibitory molecule comprises an antitumor cytokine or an antitumor toxin; or wherein the detectable label comprises a fluorescent label or a chromogenic label.
  8. The method according to claim 7, wherein the antitumor toxin comprises a toxin acting on tubulin, a toxin acting on DNA, or a compound acting on intracellular metabolism, transcription, translation, or signal transduction; or wherein the antitumor cytokine comprises IL-2, IL-12, IL-15, IFN-beta, TNF-alpha, or variants thereof; or wherein the antibody that binds the tumor surface marker comprises an antibody that recognizes a tumor antigen.
  9. The method according to claim 8, wherein the toxin acting on tubulin comprises monomethyl auristatin, a related compound thereof, or a derivative thereof; or the toxin acting on tubulin comprises a maytansinoid, a related compound thereof, or a derivative thereof; or wherein the toxin acting on DNA comprises duocarmycin, calicheamicin, pyrrolobenzodiazepines, SN-38, DXd, a related compound thereof, or a derivative thereof.
  10. The method according to claim 1, wherein the human IgG Fc is from human IgG1, human IgG2, or human IgG4; or wherein the antigen binding fragment comprises: a heavy-chain antibody, a single-chain fragment variable (scFv), a single-domain antibody, a bispecific T-cell engager (BiTE) antibody, a dual-affinity re-targeting (DART) antibody, or an antigen-binding polypeptide of a fragment variable (Fv) or fragment d (Fd) antibody; or the antigen binding fragment comprises a variable domain of a heavy chain of an antibody, a variable domain of a light chain of an antibody, or a combination thereof.
  11. An engineered human IgG Fc variant, comprising an engineered N-terminal sequence, wherein the engineered N-terminal sequence is obtained by: replacing 6-15 amino acid residues at N-terminus of wild-type human IgG Fc with amino acid residues set forth in SEQ ID NO: 6, and performing one or more of the following modifications on the amino acid sequence set forth in SEQ ID NO: 6: (a) inserting 1-9 amino acid residues between, upstream of, or downstream of two cysteine residues (CC) at positions 4 and 5 of the amino acid sequence set forth in SEQ ID NO: 6; and (b) mutating one of the two cysteine residues at positions 4 and 5 of the amino acid sequence set forth in SEQ ID NO: 6.
  12. The engineered human IgG Fc variant according to claim 11, wherein in (a), 2-8 amino acid residues are inserted between the two cysteine residues (CC).
  13. The engineered human IgG Fc variant according to claim 11, wherein the inserting or mutating is performed using amino acid residues selected from a group consisting of: aspartic acid, glutamic acid, alanine, glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine, and histidine.
  14. The engineered human IgG Fc variant according to claim 11, wherein the inserting is performed using acidic amino acid residues, wherein the acidic amino acid residues are one or more of aspartic acid and glutamic acid.
  15. The engineered human IgG Fc variant according to claim 11, wherein the engineered N-terminal sequence of the engineered human IgG Fc variant is obtained by: mutating the amino acid sequence set forth in SEQ ID NO: 6 to an amino acid sequence set forth in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 10, SEQ ID NO: 9, SEQ ID NO: 8, or SEQ ID NO: 7.
  16. Use of the engineered human IgG Fc variant according to any one of claims 11-15 in the preparation of an antigen binding fragment-human IgG Fc fusion protein, wherein, when a functional molecule is conjugated to the antigen binding fragment-human IgG Fc fusion protein, a payload of the functional molecule is in a range of 6-8.
  17. An antigen binding fragment-human IgG Fc fusion protein, comprising a human IgG Fc, wherein the human IgG Fc is the engineered human IgG Fc variant according to any one of claims 11-15.
  18. A polynucleotide, encoding the engineered human IgG Fc variant according to any one of claims 11-15 or the fusion protein according to claim 17.
  19. An expression construct, comprising the polynucleotide according to claim 18.
  20. An expression system, comprising the expression construct according to claim 19, or comprising a genome into which the polynucleotide according to claim 18 is integrated.

Description

CROSS REFERENCE TO RELATED APPLICATION The present disclosure is an application claiming the benefit of priority to a Chinese Patent Application No. CN202310929139.3, filed on July 27, 2023, the disclosures of which are incorporated herein by reference in their entireties. FIELD OF TECHNOLOGY The present disclosure relates to the technical field of biotechnology and pharmacology, and in particular to a method for preparing an antibody-drug conjugate and a use thereof. BACKGROUND Antibody-drug conjugates (ADCs) are formed by chemically conjugating antibodies to small-molecule drugs. By leveraging the targeting specificity of antibodies, small-molecule drugs can be delivered to target cells to exert therapeutic effects. Owing to the combined advantages of the targeting specificity of antibodies and the high cytotoxic activity of small-molecule drugs in tumor tissues, ADCs can efficiently kill tumor cells, exhibit fewer side effects than conventional chemotherapeutic agents, and provide improved efficacy compared to traditional antibody-based anticancer drugs. As a result, ADCs are often referred to as "biological missiles" in the field of cancer therapy, demonstrating significant clinical value and becoming a major research focus in current oncology treatment. At present, most ADC conjugation processes involve conjugating small-molecule drugs to amino acid residues in antibodies in a random or site-specific manner, with random conjugation being predominant. Random conjugation approaches include, for example, lysine residue conjugation and cysteine residue conjugation. Lysine residue conjugation utilizes the lysine residues in antibodies. An IgG molecule contains more than 80 lysine residues, among which more than 20 sites exhibit high solvent accessibility. These lysine residues can be conjugated to small-molecule drugs via acylation reactions. However, due to the large number of potential conjugation sites, this approach readily results in substantial product heterogeneity and batch-to-batch variability of ADCs, uneven distribution of the drug-to-antibody ratio (DAR), and consequently affects the study of pharmacokinetics and metabolism of ADCs. Cysteine residue conjugation utilizes the cysteine residues in antibodies. However, antibodies generally do not contain free cysteine residues available for conjugation on their surface, as the cysteine residues typically exist in the form of disulfide bonds. Human IgG can be classified into four subclasses, namely IgG1, IgG2, IgG3, and IgG4, which differ in the positions of interchain disulfide bonds and molecular weights in their sequences. The Fc fragments of different IgG subclasses also differ in structure and function. The vast majority of ADCs that have been approved or are under development are based on conventional IgG antibodies, and among the IgG subclasses, human IgG1 is most commonly selected. Heavy-chain antibodies were initially discovered in animals such as camels and sharks, and are novel antibody molecules that naturally lack light chains and consist only of heavy chains. Compared to conventional antibodies, heavy-chain antibodies, despite lacking light chains, retain antigen-binding capability and also possess advantages such as smaller molecular weight, high expression levels, ease of engineering modification, and good stability. Consequently, heavy-chain antibodies are increasingly being applied in the development of antibody drugs. However, to date, there have been no reports of successfully marketed ADCs prepared using heavy-chain antibodies. When preparing ADCs using heavy-chain antibodies, small-molecule drugs are conjugated to antibodies via cysteine residues. If a human IgG 1 Fc is employed, the available interchain disulfide bonds are extremely limited. In the art, a single ADC molecule can generally contain at most four conjugated small-molecule drugs. If an enhanced therapeutic effect is desired, a greater number of conjugated small-molecule drugs is required, for example, six to eight small-molecule drugs. Accordingly, there is an urgent need in the art to further optimize and modify ADCs to increase the number of small-molecule drugs carried per antibody and thereby improve therapeutic effect. SUMMARY The present disclosure provides a method for preparing an antibody-drug conjugate and a use thereof. In the first aspect of the present disclosure, a method for increasing the functional molecular payload of an antigen binding fragment-human IgG Fc fusion protein and reducing post-expression aggregate formation is provided, including: (1) providing an antigen binding fragment-human IgG Fc fusion protein, wherein the fusion protein includes a human IgG Fc including an engineered N-terminal sequence, wherein a method for preparing the engineered N-terminal sequence includes: replacing 6-15 amino acid residues (preferably 7-13 residues, more preferably 8-12 residues) at the N-terminus of the wild-type human IgG Fc with amino acid residues se