Stable CD3 antibody binding agents and methods of use thereof

By introducing cysteine ​​residues between the VH and VL of the antibody to form a disulfide bond, the stability and manufacturing problems of bispecific antibodies in T cell recruitment were solved, achieving efficient binding to CD3ε and targeted killing of tumor cells.

CN121986117APending Publication Date: 2026-05-05JANSSEN BIOTECH INC
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
JANSSEN BIOTECH INC
Filing Date
2024-08-06
Publication Date
2026-05-05

AI Technical Summary

Technical Problem

Existing bispecific antibodies face challenges in pharmacokinetics, immunogenicity, and manufacturing when recruiting T cells to kill tumor cells, and antigen-binding fragments such as scFv have insufficient stability and aggregation tendency.

Method used

A stapled scFv (spFv) specifically binding to CD3ε was designed, which improves stability by introducing cysteine ​​residues into the linker sequence between VH and VL to form disulfide bonds and can bind to pharmaceutically acceptable carriers to form bispecific or multispecific antibodies.

Benefits of technology

It improves antibody stability and manufacturing characteristics, reduces toxicity, and enables effective recruitment of T cells and targeted killing of tumor cells.

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Abstract

Stable CD3 antigen binding agents and methods of using the same are disclosed.
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Claims

1. A binding agent comprising an antigen-binding region binding to differentiation cluster 3ε (CD3ε), wherein the antigen-binding region binding to CD3ε comprises a pinned scFv (spFv) CDR sequence selected from the group consisting of: a) HCDR1 contains the amino acid sequence of SEQ ID NO: 7, HCDR2 contains the amino acid sequence of SEQ ID NO: 8, HCDR3 contains the amino acid sequence of SEQ ID NO: 9, LCDR1 contains the amino acid sequence of SEQ ID NO: 10, LCDR2 contains the amino acid sequence of SEQ ID NO: 11, and LCDR3 contains the amino acid sequence of SEQ ID NO:

12. b) HCDR1 contains the amino acid sequence of SEQ ID NO: 13, HCDR2 contains the amino acid sequence of SEQ ID NO: 14, HCDR3 contains the amino acid sequence of SEQ ID NO: 9, LCDR1 contains the amino acid sequence of SEQ ID NO: 10, LCDR2 contains the amino acid sequence of SEQ ID NO: 11, and LCDR3 contains the amino acid sequence of SEQ ID NO:

12. c) HCDR1 contains the amino acid sequence of SEQ ID NO: 15, HCDR2 contains the amino acid sequence of SEQ ID NO: 16, HCDR3 contains the amino acid sequence of SEQ ID NO: 9, LCDR1 contains the amino acid sequence of SEQ ID NO: 10, LCDR2 contains the amino acid sequence of SEQ ID NO: 11, and LCDR3 contains the amino acid sequence of SEQ ID NO:

12. d) HCDR1 contains the amino acid sequence of SEQ ID NO: 17, HCDR2 contains the amino acid sequence of SEQ ID NO: 18, HCDR3 contains the amino acid sequence of SEQ ID NO: 19, LCDR1 contains the amino acid sequence of SEQ ID NO: 20, LCDR2 contains the amino acid sequence of SEQ ID NO: 21, and LCDR3 contains the amino acid sequence of SEQ ID NO: 22; and e) HCDR1 contains the amino acid sequence of SEQ ID NO: 23, HCDR2 contains the amino acid sequence of SEQ ID NO: 24, HCDR3 contains the amino acid sequence of SEQ ID NO: 25, LCDR1 contains the amino acid sequence of SEQ ID NO: 26, LCDR2 contains the amino acid sequence of DSS, and LCDR3 contains the amino acid sequence of SEQ ID NO:

12.

2. The binder according to claim 1, wherein the antigen-binding region bound to CD3ε comprises a VH domain as shown in SEQ ID NO: 28 and a VL domain as shown in SEQ ID NO:

29.

3. The binder according to claim 1, wherein the antigen-binding region bound to CD3ε comprises spFv as shown in SEQ ID NO:

30.

4. The binding agent according to any one of claims 1 to 3, wherein the binding agent is a bispecific antibody or a multispecific antibody.

5. The binder according to any one of claims 1 to 4, wherein the binder further comprises an immunoglobulin (Ig) constant region or a fragment of the Ig constant region, wherein optionally the fragment of the Ig constant region is an Fc region or a CH3 domain.

6. The binder of claim 5, wherein the Ig constant region, the fragment of the Ig constant region, the Fc region, or the CH3 domain comprises at least one mutation.

7. The binder according to claim 6, wherein the at least one mutation is selected from the group consisting of: L234A / L235A / D265S, F234A / L235A, L234A / L235A, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236-deletion / A 327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S and S228P / F234A / L235A / G236-deletion / G237A / P238S, where residues are numbered according to the EU index.

8. The binder according to claim 6, wherein the at least one mutation is selected from the group consisting of: T366S / L368A / Y407V, T366W, T350V, L351Y, F405A, Y407V, T366Y, T366L, F405W, T394W, K392L, T394S, Y407T, Y407A, L351Y / F405A / Y407V, T366I / K392M / T394W, F 405A / Y407V, T366L / K392M / T394W, T366L / K392L / T394W, L351Y / Y407A, L351Y / Y407V, T366A / K409F, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V and T350V / T366L / K392L / T394W, where residues are numbered according to the EU index.

9. The binder of claim 6, wherein the binder comprises a pestle-and-mortar structure mutation, wherein the pestle mutation comprises T366S / L368A / Y407V and the mortar mutation comprises T366W.

10. The binder according to any one of claims 4 to 9, wherein the binder comprises a bispecific protein, the bispecific protein comprising an antigen-binding region that binds to a second antigen other than CD3ε.

11. The binder according to claim 10, wherein the second antigen is a tumor antigen.

12. A composition comprising a binder according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. A polynucleotide comprising a nucleotide sequence encoding a VH, VL, or both VH and VL of a binding agent according to any one of claims 1 to 11.

14. A vector comprising the polynucleotide according to claim 13.

15. A cell comprising the polynucleotide according to claim 13.

16. A kit comprising a binder according to any one of claims 1 to 11.

17. A method for treating a disease or condition of a subject or for slowing the progression of said disease or condition, said method comprising administering to the subject at least one binding molecule according to any one of claims 1-11.

18. A method of directing T cells to target cells expressing a target antigen, the method comprising contacting the T cells with an effective amount of a binder according to any one of claims 10-11 or a composition comprising the binder and a pharmaceutically acceptable carrier, wherein the antigen-binding region bound to CD3ε binds the T cells, and the antigen-binding region bound to a second antigen other than CD3ε binds to the target cells.

Citation Information

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