Multispecific antibodies

Abstract

To provide a polyspecific antibody and a method for expressing a polyspecific antibody from host cells. [Solution] a) Polypeptide chain of formula (I): VH-CH1-(CH2)-(CH3)-(X)-(VI); and b) Formula (II): (V3)-(Z)-VL-C L The present disclosure relates to a polyspecific antibody comprising or comprising a polypeptide chain of formula (Y)-(V2), wherein the polypeptide chain of formula (II) comprises at least one dsscFv, dsFv, scFv, VH, or VHH; the polypeptide chain of formula (I) comprises a protein A binding domain; and the polypeptide chain of formula (II) does not bind to protein A. The present disclosure also provides a polynucleotide sequence encoding the polyspecific antibody, a vector comprising the polynucleotide, and a host cell comprising the vector and / or polynucleotide sequence. The present disclosure also provides a pharmaceutical composition comprising the same, for example, for use in treatment.

Claims

1. Equation (I): VH-CH1-(CH2) ｓ -(CH3) ｔ -X-(V1) ｐ polypeptide chains; and Formula (II): (V3)r-Z-VL-C Ｌ -Y- (V2) ｑ polypeptide chain A polyspecific antibody containing, During the ceremony: VH represents the heavy chain variable domain; CH1 represents domain 1 of the heavy chain constant region; CH2 represents domain 2 of the heavy chain constant region; CH3 represents domain 3 of the heavy chain constant region; X represents a linker or connection; V1 represents dsscFv, dsFv, scFv, VH, VL, or VHH; V3 represents dsscFv, dsFv, scFv, VH, VL, or VHH; Z represents a linker or bond; VL stands for Light Chain Variable Domain; C Ｌ represents a domain originating from the light chain constant region, such as C-kappa; Y represents a linker or bond; V2 represents dsscFv, dsFv, scFv, VH, VL, or VHH; p represents 0 or 1; q represents 0 or 1; r represents 0 or 1; s represents 0 or 1; t represents 0 or 1; If p is 0, X does not exist; if q is 0, Y does not exist; if r is 0, Z does not exist; If q is 0, then r is 1, and if r is 0, then q is 1; The polypeptide chain of formula (II) comprises at least one dsscFv, dsFv, scFv, VH, or VHH; The polypeptide chain of formula (I) comprises a protein A binding domain; A polyspecific antibody in which the polypeptide chain of formula (II) does not bind to protein A.

2. The polyspecific antibody according to claim 1, wherein the polypeptide chain of formula (I) comprises one, two, or three protein A-binding domains.

3. The polyspecific antibody according to claim 1 or 2, wherein the protein A binding domain is located in VH and / or CH2-CH3 and / or V1.

4. The polyspecific antibody according to any one of claims 1 to 3, wherein the polypeptide chain of formula (I) comprises only one protein A-binding domain present in VH or V1.

5. The polyspecific antibody according to claim 4, wherein the polypeptide chain of formula (I) comprises only one protein A-binding domain present in VH.

6. The polyspecific antibody according to claim 4, wherein the polypeptide chain of formula (I) comprises only one protein A-binding domain present in V1.

7. The polyspecific antibody according to any one of claims 1 to 6, wherein the protein A-binding domain(s) (one or more) includes or consists of a VH3 domain or a variant thereof that binds to protein A.

8. A polyspecific antibody according to any one of claims 1 to 7, wherein V2 and / or V3 do not contain the VH3 domain.

9. A polyspecific antibody according to any one of claims 1 to 7, wherein V2 and / or V3 comprises or consist of a VH3 domain or variant thereof that does not bind to protein A.

10. A polyspecific antibody according to any one of claims 1 to 9, wherein p is 1.

11. A polyspecific antibody according to any one of claims 1 to 10, wherein q is 1.

12. A polyspecific antibody according to any one of claims 1 to 11, wherein r is 1.

13. A polyspecific antibody according to any one of claims 1 to 10, wherein q is 0 and r is 1.

14. A polyspecific antibody according to any one of claims 1 to 9, wherein s is 1, t is 1, p is 0, q is 1, r is 0, and V2 is dsscFv or dsFv.

15. A polyspecific antibody according to any one of claims 1 to 11, wherein s is 0, t is 0, p is 1, q is 1, r is 0, and both V1 and V2 represent dsscFv.

16. A polyspecific antibody according to any one of claims 1 to 15, wherein V1 is bound to albumin and VH3 contains the sequence of SEQ ID NO:

78.

17. A polyspecific antibody according to any one of claims 1 to 16, wherein X and / or Y and / or Z is a peptide linker, for example, SEQ ID NOs: 1, 2, 69 and 70.

18. V1 and / or V2 and / or V3 are dsscFv or dsFv, and the light and heavy chain variable domains of V1 and / or V2 and / or V3 are linked by a disulfide bond between two genetically engineered cysteine ​​residues, where the positions of the cysteine ​​residue pairs are VH37 and VL95, VH44 and VL100, VH44 and VL105, VH45 and VL A polyspecific antibody according to any one of claims 1 to 17, comprising or selected from the group consisting of 87, VH100 and VL50, VH100b and VL49, VH98 and VL46, VH101 and VL46, VH105 and VL43, and VH106 and VL57 (numbered by Kabat), wherein the values ​​of VH and VL are independently within a given V1, V2, or V3, for example, VH44 and VL100.

19. A polynucleotide encoding a polyspecific antibody as defined in any one of claims 1 to 18.

20. A vector comprising a polynucleotide as defined in claim 19.

21. A host cell comprising the polynucleotide or vector according to claim 19 or 20, respectively.

22. A host cell comprising at least two vectors, each vector comprising a polynucleotide encoding a different polypeptide chain of a polyspecific antibody as defined in any one of claims 1 to 18.

23. A pharmaceutical composition comprising a polyspecific antibody according to any one of claims 1 to 18 and at least one excipient.

24. A polyspecific antibody according to any one of claims 1 to 18 or a pharmaceutical composition according to claim 23, for use in treatment.

25. A method for treating a patient in need, comprising the step of administering a therapeutically effective amount of a polyspecific antibody according to any one of claims 1 to 18 or a pharmaceutical composition according to claim 23.

26. A method for producing a polyspecific antibody comprising a polypeptide chain of formula (I) and a polypeptide chain of formula (II) as defined in claim 1, the following: a) A step of expressing the polypeptide chain of formula (I) and the polypeptide chain of formula (II) as defined above in a host cell, wherein the polypeptide chain of formula (II) is in excess of the polypeptide chain of formula (I); and b) a step of recovering a polypeptide composition expressed in step a), wherein the composition comprises a polyspecific antibody and an LC dimer of formula (II-II); and c) A step of purifying the polyspecific antibody, wherein if s is 1 and t is 1, the polyspecific antibody is purified as a dimer having two heavy chains of formula (I) and two associated light chains of formula (II), and if s is 0 and t is 0, the polyspecific antibody is purified as a dimer having one heavy chain of formula (I) and one associated light chain of formula (II). Includes, The polypeptide chain of formula (II) comprises at least one dsscFv, dsFv, scFv, VH, or VHH; The polypeptide chain of formula (I) comprises a protein A binding domain; The polypeptide chain of formula (II) does not bind to protein A; A method comprising the steps of: step c) subjecting the polypeptide composition recovered in step b) and optionally following at least one subsequent purification step to a protein A affinity chromatography column.

27. A method for purifying a polyspecific antibody comprising a polypeptide chain of formula (I) and a polypeptide chain of formula (II) as defined in claim 1, the following: a) A step of obtaining a composition of a polypeptide chain of formula (I) and a polypeptide chain of formula (II) as defined above, wherein the composition comprises a polyspecific antibody and a dimer (LC dimer) in which two light chains of formula (II-II) are associated together, When s is 1 and t is 1, the polyspecific antibody is a dimer having two heavy chains of formula (I) and two associated light chains of formula (II); when s is 0 and t is 0, the polyspecific antibody is a dimer having one heavy chain of formula (I) and one associated light chain of formula (II); The polypeptide chain of formula (II) comprises at least one dsscFv, dsFv, scFv, VH, or VHH; The polypeptide chain of formula (I) comprises a protein A binding domain; The polypeptide chain of formula (II) is not bound to protein A; and b) Loading the composition obtained in step a) onto a protein A affinity column such that the LC dimer does not bind to the column, but the polyspecific antibody is retained on the column; and c) the step of washing the protein A affinity column; and d) the step of eluting the polyspecific antibody; and e) Step of recovering the polyspecific antibody. Methods that include...

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