MULTISPECIFIC ANTIBODIES

• US 20170129962A1
• Filed: 09/30/2016
• Published: 05/11/2017
• Est. Priority Date: 10/02/2015
• Status: Abandoned Application

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: SCHAEFER, WOLFGANG, IMHOF-JUNG, SABINE, KLEIN, CHRISTIAN, REGULA, JOERG THOMAS, MOLHOJ, MICHAEL
• Application Number:
  US15/281,542
• Publication Number:
  US 20170129962A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 47/6845 the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/2875 against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154 against NGF C07K16/22, against TNF C07K16/241
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/522 : CH1 domain
• CPC Class Codes: C07K 2317/526 : CH3 domain
• CPC Class Codes: C07K 2317/55 : Fab or Fab'
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/66 : comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The present invention relates to multispecific antibodies, methods for their production, pharmaceutical compositions containing said antibodies and uses thereof.

Claims

1. A multispecific antibody, comprising

a) a first light chain and a first heavy chain derived from a first antibody which specifically binds to a first antigen;

and

b) a second light chain and a second heavy chain derived from a second antibody which specifically binds to a second antigen, wherein

in the second light chain the constant domain CL is replaced by the constant domain CH1 of the second heavy chain; and

in the second heavy chain the constant domain CH1 is replaced by the constant domain CL of the second light chain; and

i) wherein in the constant domain CL of the first light chain the amino acids at position 124 and 123 (numbering according to Kabat) are substituted independently from each other by an amino acid selected from K and R; and

wherein in the constant domain CH1 of the first heavy chain the amino acids at position 147 and 213 (numbering according to EU index of Kabat) are substituted independently from each other by an amino acid selected from E or D.

2. The multispecific antibody according to claim 1, wherein

in the constant domain CL of the second heavy chain the amino acid at position 124 (numbering according to Kabat) is substituted by an amino acid selected from E and D.

3. The multispecific antibody according to claim 1, wherein in the constant domain CL of the first light chain the amino acid at position 124 (numbering according to Kabat) is substituted by K.

4. The multispecific antibody according to claim 1, wherein in the constant domain CH1 of the first heavy chain the amino acid at position 213 (numbering according to EU index of Kabat) is substituted by E.

5. The multispecific antibody according to claim 4,

wherein in the constant domain CH1 of the first heavy chain the amino acid at position 147 (numbering according to EU index of Kabat) is substituted by E.

6. The multispecific antibody according to claim 1,

i) wherein in the variable domain VL of the first light chain the amino acid at position 38 (numbering according to Kabat) is substituted by an amino acid selected from K, R and H;

and

wherein in the variable domain VH of the first heavy chain the amino acid at position 39 (numbering according to Kabat) is substituted by an amino acid selected from E and D; and

ii) wherein in the variable domain VL of the second heavy chain the amino acid at position 38 (numbering according to Kabat) is substituted by an amino acid selected from E and D (in one preferred embodiment E); and

wherein in the variable domain VH of the second light chain the amino acid at position 39 (numbering according to Kabat) is substituted by an amino acid selected from K, R and H (in one preferred embodiment by K or R, in one embodiment by K).

7. The multispecific antibody according to claim 1, which comprises a first heavy chain including a CH3 domain derived from said first antibody and a second heavy chain including a CH3 domain derived from said second antibody, wherein both CH3 domains are engineered by amino acid substitutions in order to support heterodimerisation of the first heavy chain and the second heavy chain.

8. The multispecific antibody according to claim 7, wherein in the tertiary structure of the antibody the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface that is located between the respective antibody CH3 domains,

wherein the respective amino acid sequences of the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain each comprise a set of amino acids that is located within said interface in the tertiary structure of the antibody,

wherein from the set of amino acids that is located in the interface in the CH3 domain of one heavy chain at least one amino acid residue is substituted by an amino acid residue having a larger side chain volume than the original amino acid residue, thereby generating a protuberance within the interface, wherein the protuberance is located in the CH3 domain of the one heavy chain, and wherein the protuberance is positionable in a cavity located in the CH3 domain of the other heavy chain within the interface;

and

wherein from the set of amino acids that is located in the interface in the CH3 domain of the other heavy chain at least one amino acid residue is substituted by an amino acid residue having a smaller side chain volume than the original amino acid residue, thereby generating a cavity within the interface, wherein the cavity is located in the CH3 domain of the other heavy chain, and wherein in the cavity the protuberance within the interface located in the CH3 domain of the one heavy chain is positionable.

9. The multispecific antibody according to claim 8, wherein said amino acid residue having a larger side chain volume than the original amino acid residue is selected from R, F, Y and W;

and

wherein said amino acid residue having a smaller side chain volume than the original amino acid residue is selected from A, S, T and V.

10. The multispecific antibody according to claim 8, wherein from the set of amino acids that is located in the interface in the CH3 domain of the one heavy chain a first amino acid is substituted by cysteine;

and

from the set of amino acids that is located in the interface in the CH3 domain of the other heavy chain a second amino acid is substituted by cysteine, wherein the second amino acid is facing the first amino acid within the interface;

such that a disulfide bridge between the CH3 domain of the one heavy chain and the CH3 domain of the other heavy chain can be formed via the introduced cysteine residues.

11. The multispecific antibody according to claim 1 that specifically binds to human Angiopoietin-2 and human VEGF, wherein

a) the antibody comprises a variable heavy chain domain (VH) according to SEQ ID NO:

26 (<

VH Ang2>

) and a variable light chain domain (VL) according to SEQ ID NO;

27 (<

VL Ang2>

); and

b) the antibody comprises a variable heavy chain domain (VH) according to SEQ ID NO;

24 (<

VH VEGF>

) and a variable light chain domain (VL) according to SEQ ID NO;

25 (<

VL VEGF>

).

12. A method for the preparation of a multispecific antibody according to claim 1, comprising the steps of

transforming a host cell with vectors comprising nucleic acids encoding

a) the first light chain as defined in claim 1 derived from a first antibody which specifically binds to a first antigen;

b) the first heavy chain as defined in claim 1 derived from a first antibody which specifically binds to a first antigen;

c) the second light chain as defined in claim 1 derived from a second antibody which specifically binds to a second antigen; and

d) the second heavy chain as defined in claim 1 derived from a second antibody which specifically binds to a second antigen,

culturing said host cell under conditions that allow synthesis of said multispecific antibody; and

recovering said multispecific antibody from said host cell culture.

13. A host cell comprising

a) a vector comprising nucleic acids encoding the first light chain as defined in claim 1 derived from a first antibody which specifically binds to a first antigen;

b) a vector comprising nucleic acids encoding the first heavy chain as defined in claim 1 derived from a first antibody which specifically binds to a first antigen;

c) a vector comprising nucleic acids encoding the second light chain as defined in claim 1 derived from a second antibody which specifically binds to a second antigen; and

d) a vector comprising nucleic acids encoding the second heavy chain as defined in claim 1 derived from a second antibody which specifically binds to a second antigen.

14. A pharmaceutical composition comprising a multispecific antibody according to claim 1 in combination with at least one pharmaceutically acceptable carrier.

22. A nucleic acid encoding the multispecific antibody of claim 1.

23. A vector comprising the nucleic acid of claim 22.

24. A host cell comprising the vector of claim 23.

25. A method for the preparation of a multispecific antibody, comprising the step of culturing the host cell of claim 24 under conditions that allow synthesis of said multispecific antibody.

26. The method of claim 25 further comprising the step of recovering the multispecific antibody from the host cell culture.

27. A host cell comprising one or more vectors comprising nucleic acids encoding the first light chain and first heavy chain as defined in claim 1 derived from a first antibody which specifically binds to a first antigen and the second light chain and second heavy chain as defined in claim 1 derived from a second antibody which specifically binds to a second antigen.

28. A method for the preparation of a multispecific antibody, comprising the step of culturing the host cell of claim 27 under conditions that allow synthesis of said multispecific antibody.

29. The method of claim 28 further comprising the step of recovering the multispecific antibody from the host cell culture.

30. An immunoconjugate comprising the multispecific antibody according to claim 1 coupled to a cytotoxic agent.

15. (canceled)

16. A method for improving the aggregation onset temperature of a multispecific antibody comprising a first light chain and a first heavy chain derived from a first antibody which specifically binds to a first antigen;

and a second light chain and a second heavy chain derived from a second antibody which specifically binds to a second antigen, wherein in the second light chain the constant domain CL is replaced by the constant domain CH1 of the second heavy chain; and

in the second heavy chain the constant domain CH1 is replaced by the constant domain CL of the second light chain;

the method comprising the steps of;

providing a modified first light chain of the multispecific antibody, wherein in the constant domain CL the amino acids at position 124 and 123 (numbering according to Kabat) are substituted independently from each other by an amino acid selected from K and R;

providing a modified first heavy chain the multispecific antibody, wherein in the constant domain CH1 of the first heavy chain the amino acids at position 147 and 213 (numbering according to EU index of Kabat) are substituted independently from each other by an amino acid selected from E or D; and

providing a multispecific antibody with an increased aggregation onset temperature, wherein the multispecific antibody comprises the modified first light chain, the modified first heavy chain, the second light chain and the second heavy chain.

17. The method according to claim 16, further comprising the step of

providing a modified second heavy chain, wherein in the constant domain CL the amino acid at position 124 (numbering according to Kabat) is substituted by an amino acid selected from E and D, and

wherein a multispecific antibody comprising the modified first light chain, the modified first heavy chain, the second light chain and the modified second heavy chain is provided.

18. The method according to claim 16, wherein in the constant domain CL of the first light chain the amino acid at position 124 (numbering according to Kabat) is substituted by K.

19. The method according to claim 16, wherein in the constant domain CH1 of the first heavy chain the amino acid at position 213 (numbering according to EU index of Kabat) is substituted by E.

20. The method according to claim 16, wherein in the constant domain CH1 of the first heavy chain the amino acid at position 147 (numbering according to EU index of Kabat) is substituted by E.

21. A multispecific antibody obtainable by the method of claim 16.