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CN-122029188-A - Compositions and methods for reducing antibody immunogenicity and improving antibody stability

CN122029188ACN 122029188 ACN122029188 ACN 122029188ACN-122029188-A

Abstract

Disclosed herein are humanized antibodies having reduced immunogenicity and/or increased stability. Methods of making and using these antibodies are also disclosed herein.

Inventors

  • T.S. Nijar

Assignees

  • 普罗塞纳生物科学有限公司

Dates

Publication Date
20260512
Application Date
20240913
Priority Date
20230915

Claims (20)

  1. 1. A humanized antibody comprising a heavy chain variable domain comprising a charged amino acid at an amino acid position corresponding to Chothia canonical amino acid position 44 of a human IgG1 heavy chain and a light chain variable domain comprising a charged amino acid at an amino acid position corresponding to Chothia canonical amino acid position 100 of a human IgG1 light chain.
  2. 2. The humanized antibody of claim 1, wherein the charged amino acid at an amino acid position corresponding to Chothia canonical amino acid position 44 of the human IgG1 heavy chain is a positively charged amino acid and the charged amino acid at an amino acid position corresponding to Chothia canonical amino acid position 100 of the human IgG1 light chain is a negatively charged amino acid.
  3. 3. The humanized antibody of claim 2, wherein the positively charged amino acid at an amino acid position corresponding to Chothia canonical amino acid position 44 of the human IgG1 heavy chain forms an inter-chain salt bridge with the negatively charged amino acid at an amino acid position corresponding to Chothia canonical amino acid position 100 of the human IgG1 light chain.
  4. 4. The humanized antibody of any one of claims 1-3, wherein the positively charged amino acid is a basic amino acid.
  5. 5. The humanized antibody of any one of claims 1-4, wherein the negatively charged amino acid is an acidic amino acid.
  6. 6. The humanized antibody of any one of claims 1-5, wherein the positively charged amino acid is arginine.
  7. 7. The humanized antibody of any one of claims 1-6, wherein the negatively charged amino acid is aspartic acid.
  8. 8. The humanized antibody of any one of claims 1 to 7, wherein the humanized antibody further comprises a substituted phenylalanine derivative at an amino acid position corresponding to Chothia canonical amino acid position 91 of a human IgG1 heavy chain.
  9. 9. The humanized antibody of claim 8, wherein the substituted phenylalanine derivative at an amino acid position corresponding to Chothia canonical amino acid position 91 of the human IgG1 heavy chain forms a hydrogen bond with at least one of an amino acid at an amino acid position corresponding to Chothia canonical amino acid position 38 of the human IgG1 light chain and an amino acid at an amino acid position corresponding to Chothia canonical amino acid position 39 of the human IgG1 heavy chain.
  10. 10. The humanized antibody of claim 8 or 9, wherein the substituted phenylalanine derivative at an amino acid position corresponding to Chothia canonical amino acid position 91 of the human IgG1 heavy chain forms hydrogen bonds with both an amino acid at an amino acid position corresponding to Chothia canonical amino acid position 38 of the human IgG1 light chain and an amino acid at an amino acid position corresponding to Chothia canonical amino acid position 39 of the human IgG1 heavy chain.
  11. 11. The humanized antibody of any one of claims 8-10, wherein the substituted phenylalanine derivative comprises a hydroxy-substituted phenyl.
  12. 12. The humanized antibody of claim 11, wherein the substituted phenylalanine derivative is a tyrosine residue.
  13. 13. The humanized antibody of any one of claims 9 to 12, wherein the amino acid at an amino acid position corresponding to Chothia canonical amino acid position 38 of the human IgG1 light chain is glutamine and/or wherein the amino acid at an amino acid position corresponding to Chothia canonical amino acid position 39 of the human IgG1 heavy chain is glutamine.
  14. 14. The humanized antibody of any one of claims 1 to 13, wherein the humanized antibody further comprises at least one of: tryptophan at an amino acid position corresponding to amino acid position 103 typical of Chothia of the human IgG1 heavy chain; phenylalanine at an amino acid position corresponding to amino acid position 100C typical of Chothia of human IgG1 heavy chain, and Alanine-substituted leucine at an amino acid position corresponding to amino acid position 78 typical of Chothia of human IgG1 heavy chain.
  15. 15. The humanized antibody of any one of claims 1 to 14, wherein the humanized antibody further comprises at least one of: alanine at an amino acid position corresponding to amino acid position 49 of the Chothia canonical of the human IgG1 heavy chain, and Alanine at an amino acid position corresponding to amino acid position 74 typical of Chothia of human IgG1 heavy chain.
  16. 16. The humanized antibody of any one of claims 1 to 14, wherein the humanized antibody further comprises at least one of: Serine at an amino acid position corresponding to amino acid position 49 typical of Chothia of human IgG1 heavy chain, and Serine at an amino acid position corresponding to amino acid position 74 typical of Chothia of human IgG1 heavy chain.
  17. 17. The humanized antibody of any one of claims 1-16, wherein the humanized antibody further comprises at least one of: Glutamine at an amino acid position corresponding to amino acid position 3 typical of Chothia of human IgG1 light chain; glutamine at an amino acid position corresponding to amino acid position 17 typical of Chothia of human IgG1 light chain; tyrosine at an amino acid position corresponding to amino acid position 36 typical of Chothia of human IgG1 light chain; arginine at an amino acid position corresponding to amino acid position 39 typical of Chothia of human IgG1 light chain; alanine at an amino acid position corresponding to amino acid position 92 of the Chothia canonical of the human IgG1 light chain, and Valine at an amino acid position corresponding to amino acid position 104 typical of Chothia of human IgG1 light chain.
  18. 18. The humanized antibody of any one of claims 1 to 17, wherein the humanized antibody further comprises glutamine at an amino acid position corresponding to amino acid position 3 typical of Chothia of a human IgG1 light chain.
  19. 19. A humanized antibody comprising a heavy chain variable domain comprising a substituted phenylalanine derivative at an amino acid position corresponding to Chothia canonical amino acid position 91 of a human IgG1 heavy chain and a light chain variable domain.
  20. 20. The humanized antibody of claim 19, wherein the humanized antibody further comprises a polar uncharged amino acid at an amino acid position corresponding to Chothia canonical amino acid position 39 of a human IgG1 heavy chain and a light chain variable domain comprising a polar uncharged amino acid at an amino acid position corresponding to Chothia canonical amino acid position 38 of a human IgG1 light chain.

Description

Compositions and methods for reducing antibody immunogenicity and improving antibody stability Cross reference to related applications The present application claims priority from U.S. provisional patent application No. 63/538,598, filed on 9/15 of 2023, the contents of which are hereby incorporated by reference in their entirety. Sequence listing The present application contains a sequence table named "50887-0046WO1_ST26_SL.XML" submitted electronically as an XML file. An XML file was created at 2024, 8, 30, and size 35,169 bytes. The material in the XML file is hereby incorporated by reference in its entirety. Background Antibodies are natural proteins formed by the vertebrate immune system in response to foreign substances (antigens), primarily for the defense against infection. For over a century, antibodies have been induced in animals under artificial conditions and harvested for use in therapy or diagnosis of disease conditions, or for biological research. Each individual antibody-producing cell produces a single type of antibody having a chemically defined composition, however, antibodies obtained directly from animal serum in response to antigen vaccination actually comprise a collection of different molecules (i.e., polyclonal antibodies) made from a collection of individual antibody-producing cells. The utility of rodent (in particular murine) monoclonal antibodies in human therapy is limited by problems associated with their non-human origin, in particular their immunogenicity in human hosts. In order to minimize the human immune response to therapeutic antibody drugs, monoclonal antibody technology has evolved from full mouse antibodies to chimeric antibodies (mouse variable domains grafted onto human IgG backbones) and humanized antibodies (mouse CDRs grafted onto human IgG backbones). Humanization of mouse monoclonal antibodies was initially achieved by combining mouse variable domains with human constant domains, resulting in so-called chimeric antibodies with a human content of about 70%. Further humanization was then achieved by grafting the Complementarity Determining Regions (CDRs) of the mouse monoclonal antibody onto the human framework regions of the variable antibody domains of the human antibody. In addition, several amino acid residues present in those framework regions were identified as interacting with CDRs or antigens and were back mutated in humanized antibodies to improve binding. (Almagro et al, frontiers Bioscience13:1619-1633 (2008)). Immunogenicity is the result of a complex series of reactions to what is considered a foreign substance, and can include the production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, hypersensitivity and anaphylaxis. Several factors can contribute to protein immunogenicity, including but not limited to protein sequence, route and frequency of administration, and patient population. Several methods of humanization are known in the art, consisting essentially of introducing mutations in regions of the antibody sequence that are predicted to be immunogenic. However, these methods have drawbacks, including reduced stability of the resulting antibodies. Typically, the introduction of deimmunizing mutations during the humanization process affects antibody conformation (e.g., by disrupting molecular interaction networks that hold the antibody in the correct conformation for antigen recognition). For example, deimmunization mutations may interfere with intra-chain interactions between light and heavy chains, resulting in significant loss of molecular stability. The loss of stability in turn can lead to a number of problems including low expression yields, reduced processibility, reduced shelf life and unacceptable pharmacokinetic parameters. Because there is a need to develop improved humanized antibodies with reduced immunogenicity and/or improved stability, methods of reducing the immunogenicity and/or increasing the stability of humanized antibodies are needed. Disclosure of Invention The present disclosure relates to antibodies and antigen-binding antibody fragments that reduce antibody immunogenicity and improve antibody stability. Also disclosed herein are compositions comprising these antibodies or antigen-binding antibody fragments, and methods of using these antibodies and antigen-binding antibody fragments to reduce immunogenicity. Thus, disclosed herein are humanized antibodies comprising a heavy chain variable domain comprising a charged amino acid at an amino acid position corresponding to Chothia typical amino acid position 44 of a human IgG1 heavy chain and a light chain variable domain comprising a charged amino acid at an amino acid position corresponding to Chothia typical amino acid position 100 of a human IgG1 light chain. In some cases, the charged amino acid at an amino acid position corresponding to amino acid position 44 of the Chothia