MODIFIED ANTIGEN-BINDING POLYPEPTIDE STRUCTURES AND THEIR APPLICATION

RU2024140120APending Publication Date: 2026-06-29ZAJMVORKS BK INK

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Patent Information

Authority / Receiving Office: RU · RU
Patent Type: Applications
Current Assignee / Owner: ZAJMVORKS BK INK
Filing Date: 2024-12-27
Publication Date: 2026-06-29

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Claims

1. A set of polynucleotides for producing an antigen-binding polypeptide construct, wherein the set of polynucleotides encodes an antigen-binding polypeptide construct containing at least a first heterodimer and a second heterodimer, wherein, the first heterodimer comprises a first human or humanized polypeptide sequence (H1) of an immunoglobulin heavy chain and a first human or humanized polypeptide sequence (L1) of an immunoglobulin light chain and has a Fab region that binds to a first epitope; and the second heterodimer comprises a second human or humanized polypeptide sequence (H2) of an immunoglobulin heavy chain and a second human or humanized polypeptide sequence (L2) of an immunoglobulin light chain and has a Fab region that binds to a second epitope, wherein at least one of the H1 or L1 sequences of the first heterodimer differs from the corresponding H2 or L2 sequence of the second heterodimer, and wherein H1 and H2 each contain at least a heavy chain variable domain (Vh domain) and a heavy chain constant domain (C domain) H1 ); L1 and L2 each contain at least a light chain variable domain (V1 domain) and a light chain constant domain (C domain) l ); And H1, H2, L1 and L2 comprise at least one amino acid substitution or set of amino acid substitutions, wherein H1 preferentially pairs with L1 over L2, and H2 preferentially pairs with L2 over L1; wherein the thermal stability of the Fab region, as measured by the melting temperature (Tm) of at least the first and second heterodimers, is within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10°C of the Tm of the corresponding heterodimer without at least one amino acid substitution or set of amino acid substitutions, and where H1, L1, H2 and L2 contain the following amino acid substitutions at positions identified according to Kabat numbering: a) H1 contains amino acid substitutions 124E and 228D, L1 contains amino acid substitutions 121K, 133G, and 176R, H2 contains amino acid substitutions 124R and 125R, and L2 contains amino acid substitutions 133G and 176D; b) H1 contains amino acid substitutions 124E and 228D, L1 contains amino acid substitutions 121K, 133G, and 176R, H2 contains amino acid substitutions 124R and 125R, and L2 contains amino acid substitutions 133G, 176D, and 178D; c) H1 contains amino acid substitutions 124E, 125S, and 228D, L1 contains amino acid substitutions 121K, 133G, and 176R, H2 contains amino acid substitutions 124R and 125R, and L2 contains amino acid substitutions 133G, 176D, and 178D; d) H1 contains amino acid substitutions 124E and 172R, L1 contains amino acid substitutions 133G and 176R, H2 contains amino acid substitutions 124R and 172T, and L2 contains amino acid substitutions 133G, 174R, and 176D; e) H1 contains amino acid substitutions 124E, 125S and 228D, L1 contains amino acid substitutions 121K, 133G and 176R, H2 contains amino acid substitutions 124R and 125R, and L2 contains amino acid substitutions 133G and 176D; f) H1 contains amino acid substitutions 124E and 172R, L1 contains amino acid substitutions 133G and 176K, H2 contains amino acid substitutions 124R and 172T, and L2 contains amino acid substitutions 133G, 137K, 174R, and 176D; g) H1 contains amino acid substitutions 124E and 172R, L1 contains amino acid substitutions 133G and 176R, H2 contains amino acid substitutions 124R and 172T, and L2 contains amino acid substitutions 133G, 137K, 174R, and 176D; h) HI contains amino acid substitutions 124E, 125S, 172R and 228D, L1 contains amino acid substitutions 121K, 133G and 176R, H2 contains amino acid substitutions 124R, 125R and 172T, and L2 contains amino acid substitutions 133G, 137K, 174R and 176D; i) H1 contains amino acid substitutions 139W, 143E, 145T, and 179E, L1 contains amino acid substitutions 116A, 124R, 135V, and 178R, H2 contains amino acid substitution 179K, and L2 contains amino acid substitutions 124E, 135W, 160E, and 180E; j) H1 contains amino acid substitutions 139W, 143E, 145T and 179E, L1 contains amino acid substitutions 116A, 124R, 135V and 178R, H2 contains amino acid substitutions 139G, 179K and 190A, and L2 contains amino acid substitutions 124E, 135W, 160E and 180E; k) H1 contains amino acid substitutions 139W, 143E, 145T and 179E, L1 contains amino acid substitutions 116A, 124R and 135V, H2 contains amino acid substitutions 139G, 179L and 190A, and L2 contains amino acid substitutions 124E, 135W, 160E and 180E; 1) H1 contains amino acid substitutions 139W, 143E, 145T, and 179E, L1 contains amino acid substitutions 116A, 124R, and 135V, H2 contains amino acid substitution 179K, and L2 contains amino acid substitutions 124E, 135W, 160E, and 180E; m) H1 contains amino acid substitutions 39E, 143E, 145T, and 179E, L1 contains amino acid substitutions 38R, 124R, 160K, and 178R, H2 contains amino acid substitutions 39R, 172R, and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E, and 180E; n) H1 contains amino acid substitutions 39E, 143E, 145T, 172R, and 179E, L1 contains amino acid substitutions 38R, 124R, 160K, and 178R, H2 contains amino acid substitutions 39R, 172R, and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E, and 180E; o) H1 contains amino acid substitutions 39E, 143E, 145T and 179E, L1 contains amino acid substitutions 38R, 124R, 160K and 178R, H2 contains amino acid substitutions 39R, 146G and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E and 180E; p) H1 contains amino acid substitutions 39E, 143E, 145T, 172R, and 179E, L1 contains amino acid substitutions 38R, 124R, 160K, and 178R, H2 contains amino acid substitutions 39R, 146G, and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E, and 180E; q) H1 contains amino acid substitutions 39E, 143E, 145T and 179E, L1 contains amino acid substitutions 38R, 124R, 160K and 178R, H2 contains amino acid substitutions 39R, 146G, 172R and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E and 180E; d) HI contains amino acid substitutions 39E, 143E, 145T and 179E, L1 contains amino acid substitutions 38R, 124R, 160K and 178R, H2 contains amino acid substitutions 39R and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E and 180E; s) H1 contains amino acid substitutions 39E, 143E, 145T, 172R, and 179E, L1 contains amino acid substitutions 38R, 124R, 160K, and 178R, H2 contains amino acid substitutions 39R, 146G, 172R, and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E, and 180E; t) H1 contains amino acid substitutions 39E, 143E, 145T, 172R, and 179E, L1 contains amino acid substitutions 38R, 124R, 160K, and 178R, H2 contains amino acid substitutions 39R and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E, and 180E; u) H1 contains amino acid substitutions 39E, 143E, and 145T, L1 contains amino acid substitutions 38R, 124R, 160K, and 178R, H2 contains amino acid substitutions 39R, 172R, and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E, and 180E; v) H1 contains amino acid substitutions 39E, 143E, and 145T, L1 contains amino acid substitutions 38R, 124R, 160K, and 178R, H2 contains amino acid substitutions 39R, 146G, and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E, and 180E; w) H1 contains amino acid substitutions 39E, 143E, and 145T, L1 contains amino acid substitutions 38R, 124R, 160K, and 178R, H2 contains amino acid substitutions 39R, 146G, 172R, and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E, and 180E; x) H1 contains amino acid substitutions 39E, 143E, and 145T, L1 contains amino acid substitutions 38R, 124R, 160K, and 178R, H2 contains amino acid substitutions 39R and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E, and 180E; y) H1 contains amino acid substitutions 39E, 143E, 145T and 172R, L1 contains amino acid substitutions 38R, 124R, 160K and 178R, H2 contains amino acid substitutions 39R, 172R and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E and 180E; z) H1 contains amino acid substitutions 39E, 143E, 145T and 172R, L1 contains amino acid substitutions 38R, 124R, 160K and 178R, H2 contains amino acid substitutions 39R, 146G and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E and 180E; aa) H1 contains amino acid substitutions 39E, 143E, 145T and 172R, L1 contains amino acid substitutions 38R, 124R, 160K and 178R, H2 contains amino acid substitutions 39R, 146G, 172R and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E and 180E; bb) H1 contains amino acid substitutions 39E, 143E, 145T, and 172R, L1 contains amino acid substitutions 38R, 124R, 160K, and 178R, H2 contains amino acid substitutions 39R and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E, and 180E; cc) H1 contains amino acid substitutions 124W, 143E, 145T and 179E, L1 contains amino acid substitutions 124R, 133A, 176T and 178R, H2 contains amino acid substitutions 124A, 143F and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178L and 180E; dd) H1 contains amino acid substitutions 124W, 143E, 145T, and 179E, L1 contains amino acid substitutions 124K, 133A, 176T, and 178R, H2 contains amino acid substitutions 124A, 143F, and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178L, and 180E; ee) H1 contains amino acid substitutions 124W, 143E, 145T, and 179E, L1 contains amino acid substitutions 131K, 133A, 176T, and 178L, H2 contains amino acid substitutions 124A and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178L, and 180E; ff) H1 contains amino acid substitutions 124W, 143E, 145T and 179E, L1 contains amino acid substitutions 131K, 133A, 176T and 178L, H2 contains amino acid substitutions 124A, 143F and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178L and 180E; gg)Hl contains amino acid substitutions 124W, 143E, 145T, and 179E, L1 contains amino acid substitutions 124R, 133A, 176Ti, and 178R, H2 contains amino acid substitutions 124A, 143F, and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178E, and 180E; hh) H1 contains amino acid substitutions 124W, 143E, 145T, and 179E, L1 contains amino acid substitutions 124K, 133A, 176T, and 178R, H2 contains amino acid substitutions 124A, 143F, and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178E, and 180E; ii) H1 contains amino acid substitutions 124W, 143E, 145T, and 179E, L1 contains amino acid substitutions 131K, 133A, 176T, and 178L, H2 contains amino acid substitutions 124A, 143F, and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178E, and 180E; jj) H1 contains amino acid substitutions 124W, 143E, 145T, and 179E, L1 contains amino acid substitutions 131K, 133A, 176T, and 178L, H2 contains amino acid substitutions 124A and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178E, and 180E; kk) H1 contains amino acid substitutions 124W, 143F, 145T, and 179E, L1 contains amino acid substitutions 131K, 133A, 176T, and 178L, H2 contains amino acid substitutions 124A, 143F, and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178E, and 180E; 11) H1 contains amino acid substitutions 124W, 143F, 145T, and 179E, L1 contains amino acid substitutions 131K, 133A, 176T, and 178L, H2 contains amino acid substitutions 124A and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178E, and 180E; mm) H1 contains amino acid substitutions 124W, 143F, 145T, and 179E, L1 contains amino acid substitutions 131K, 133A, 176T, and 178L, H2 contains amino acid substitutions 124A and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178L, and 180E; or nn) H1 contains amino acid substitutions 124W, 143F, 145T, and 179E, L1 contains amino acid substitutions 131K, 133A, 176T, and 178L, H2 contains amino acid substitutions 124A, 143F, and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178L, and 180E.

2. A set of polynucleotides according to claim 1, wherein HI contains amino acid substitutions 124E and 172R, L1 contains amino acid substitutions 133G and 176R, H2 contains amino acid substitutions 124R and 172T, and L2 contains amino acid substitutions 1330, 174R and 176D.

3. A set of polynucleotides according to claim 1, wherein H1 contains amino acid substitutions 139W, 143E, 145T and 179E, L1 contains amino acid substitutions 116A, 124R, 135V and 178R, H2 contains amino acid substitution 179K, and L2 contains amino acid substitutions 124E, 135W, 160E and 180E.

4. A set of polynucleotides according to claim 1, wherein H1 contains amino acid substitutions 39E, 143E, 145T and 179E, L1 contains amino acid substitutions 38R, 124R, 160K and 178R, H2 contains amino acid substitutions 39R, 172R and 179K, and L2 contains amino acid substitutions 38E, 124E, 160E and 180E.

5. A set of polynucleotides according to claim 1, wherein H1 contains amino acid substitutions 124W, 143E, 145T and 179E, L1 contains amino acid substitutions 124R, 133A, 176T and 178R, H2 contains amino acid substitutions 124A, 143F and 179K, and L2 contains amino acid substitutions 124E, 133W, 176T, 178L and 180E.

6. A set of polynucleotides according to claim 1, wherein H1 contains amino acid substitutions 124E and 228D, L1 contains amino acid substitutions 121K, 133G and 176R, H2 contains amino acid substitutions 124R and 125R, and L2 contains amino acid substitutions 1330 and 176D.

7. A set of polynucleotides according to any one of claims 1-6, wherein L1 and L2 are polypeptide sequences of the kappa light chain.

8. The set of polynucleotides according to claim 7, wherein H1 is preferentially paired with L1 compared to L2, and H2 is preferentially paired with L2 compared to L1, in case H1, H2, L1 and L2 are co-expressed in a cell or a mammalian cell, or in case H1, H2, L1 and L2 are co-expressed in a cell-free expression system, or in case H1, H2, L1 and L2 are co-produced, or in case H1, H2, L1 and L2 are co-produced by a redox method.

9. The set of polynucleotides according to claim 8, wherein in the case where both L1 and L2 are co-expressed with at least one of H1 and H2, the pairing of H1-L1 with H1-L2 and the pairing of H2-L2 with H2-L1 is greater than the pairing of H1-L1 with H1-L2 and the pairing of H2-L2 with H2-L1 in the absence of amino acid substitutions.

10. A set of polynucleotides according to any one of claims 1 to 9, wherein the affinity of each heterodimer for the antigen to which it binds is within the range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45 or 50 times the affinity of the corresponding unmodified heterodimer for the same antigen, as measured by surface plasmon resonance (SPR) or FACS.

11. A set of polynucleotides according to any one of claims 1-10, wherein the construct further comprises an Fc comprising a first CH3 sequence and a second CH3 sequence, wherein the first CH3 sequence is connected with or without one or more linkers to a first heterodimer, and the second CH3 sequence is connected with or without one or more linkers to a second heterodimer.

12. The set of polynucleotides according to claim 11, wherein Fc is human Fc, human IgG1 Fc, human IgA Fc, human IgG Fc, human IgD Fc, human IgE Fc, human IgM Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc.

13. The set of polynucleotides according to claim 12, wherein Fc is a heterodimeric Fc.

14. The set of polynucleotides according to claim 13, wherein Fc comprises one or more modifications in at least one of the CH3 sequences.

15. The set of polynucleotides according to claim 14, wherein the Fc comprises one or more modifications in at least one of the CH3 sequences that facilitate the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc.

16. A set of polynucleotides according to claim 15, wherein Fc contains: i) a heterodimeric IgG1 Fc having the modifications L351Y F405A Y407V in a first polypeptide, and the modifications T366L K392M T394W in a second Fc polypeptide; ii) a heterodimeric IgG1 Fc having the modifications L351Y F405A Y407V in a first polypeptide, and the modifications T366L K392L T394W in a second Fc polypeptide; iii) a heterodimeric IgG1 Fc having the modifications T350V L351Y F405A Y407V in the first polypeptide and the modifications T350VT366LK392LT394W in the second Fc polypeptide; iv) a heterodimeric IgG1 Fc having the modifications T350V L351Y F405A Y407V in a first polypeptide, and the modifications T350VT366LK392MT394W in a second Fc polypeptide; or v) a heterodimeric IgG1 Fc having the modifications T350V_L351Y_S400E_F405A_Y407V in the first polypeptide, and the modifications T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide, and where the numbering of residues in the Fc polypeptide is given in accordance with the EU numbering system.

17. The set of polynucleotides according to claim 16, wherein Fc additionally comprises at least one CH2 sequence.

18. The set of polynucleotides according to claim 17, wherein the CH2 Fc sequence(s) comprises(contain) one or more modifications.

19. The set of polynucleotides according to claim 18, wherein the Fc comprises one or more modifications to promote selective binding to Fc-gamma receptors.

20. The set of polynucleotides according to claim 19, wherein Fc is connected to heterodimers via one or more linkers, or where Fc is paired with H1 and H2 via one or more linkers.

21. A set of polynucleotides according to claim 20, wherein one or more linkers comprise one or more antibody hinge regions.

22. The set of polynucleotides according to claim 21, wherein one or more linkers comprise one or more IgG1 hinge regions.

23. A set of polynucleotides according to claim 22, wherein one or more linkers contain one or more modifications.

24. A set of polynucleotides according to claim 23, wherein one or more modifications promote selective binding to Fc-gamma receptors.

25. A set of polynucleotides according to claim 1, wherein the construct is multispecific or bispecific.

26. A set of expression vectors for producing an antigen-binding polypeptide construct, wherein the set of expression vectors contains a set of polynucleotides according to any one of claims 1-25.

27. An isolated host cell for producing an antigen-binding polypeptide construct, wherein the isolated host cell contains a set of polynucleotides according to any one of claims 1-25.

28. A method for obtaining or creating an antigen-binding polypeptide construct from a host cell culture, wherein the method includes the steps of preparing: (a) obtaining a host cell culture comprising at least one host cell comprising a set of polynucleotides according to any one of claims 1 to 25; and (b) isolation of the construct from the host cell culture.

29. The method of claim 28, further comprising the step of conjugating the antigen-binding polypeptide construct to a therapeutic agent or drug.