MULTISPECIFIC ANTIBODIES

• US 20150315296A1
• Filed: 04/02/2015
• Published: 11/05/2015
• Est. Priority Date: 04/02/2014
• 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, KLOSTERMANN, STEFAN, REGULA, JOERG THOMAS, KLEIN, CHRISTIAN, MOLHOJ, MICHAEL
• Application Number:
  US14/677,824
• Publication Number:
  US 20150315296A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• 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: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/24 against cytokines, lymphokines or interferons
• 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 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• 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 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• 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, their manufacture and use.

Claims

1. A multispecific antibody, comprising:

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

b) a second light chain and a second heavy chain of a second antibody which specifically binds to a second antigen, and wherein the variable domains VL and VH in the second light chain and second heavy chain of the second antibody are replaced by each other; and

wherein

i) in the constant domain CL of the first light chain under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the first heavy chain under a) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index);

or

ii) in the constant domain CL of the second light chain under b) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the second heavy chain under b) the amino acid at positions 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

2. A multispecific antibody according to claim 1,

wherein in the constant domain CL of the first light chain under a) the amino acid at position 124 is substituted independently by lysine (K) or arginine (R) (numbering according to Kabat), and wherein in the constant domain CH1 of the first heavy chain under a) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

3. The multispecific antibody according to claim 1,

wherein in the constant domain CL of the first light chain under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the first heavy chain under a) the amino acid at position 147 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

4. The multispecific antibody according to claim 2,

wherein in the constant domain CL of the first light chain under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the first heavy chain under a) the amino acid at position 147 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

5. The multispecific antibody according to claim 1,

wherein in the constant domain CL of the second light chain under b) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the second heavy chain under b) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

6. The multispecific antibody according to claim 2,

wherein in the constant domain CL of the first light chain under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat),

and wherein in the constant domain CH1 of the first heavy chain under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

7. The multispecific antibody according to claim 1, characterized in that

a first CH3 domain of the first heavy chain of the antibody under a) and a second CH3 domain of the second heavy chain of the antibody under b) each meet at an interface which comprises an original interface between the antibody CH3 domains,

wherein said interface is altered to promote the formation of the multispecific antibody, wherein the alteration is characterized in that:

i) the CH3 domain of one heavy chain is altered,

so that within the original interface of the CH3 domain of the one heavy chain that meets the original interface of the CH3 domain of the other heavy chain within the multi specific antibody,

an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of the one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain;

and

ii) the CH3 domain of the other heavy chain is altered,

so that within the original interface of the CH3 domain of the other heavy chain that meets the original interface of the CH3 domain of the one heavy chain within the multi specific antibody,

an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the CH3 domain of the other heavy chain within which a protuberance within the interface of the CH3 domain of the one heavy chain is positionable.

8. The multispecific antibody according to claim 7, characterized in that

the said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W);

and said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

9. The multispecific antibody according to claim 7, characterized in that

both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain such that a disulfide bridge between both CH3 domains can be formed.

10. A multispecific antibody according to claim 1 that specifically binds to human TWEAK and that specifically binds to human IL17, wherein

A) the multispecific antibody comprises

a variable heavy chain domain (VH) of SEQ ID NO:

24, and a variable light chain domain (VL) of SEQ ID NO;

25; and

B) the multispecific antibody comprises

a variable heavy chain domain (VH) of SEQ ID NO;

26, and a variable light chain domain (VL) of SEQ ID NO;

27.

11. A method for the preparation of a multispecific antibody according to claim 1,

comprising the steps of

A) transforming a host cell with vectors comprising nucleic acid molecules encoding

a) the first light chain and the first heavy chain of a first antibody which specifically binds to a first antigen;

and

b) the second light chain and the second heavy chain of a second antibody which specifically binds to a second antigen, and wherein the variable domains VL and VH in the second light chain and second heavy chain of the second antibody are replaced by each other; and

wherein

i) in the constant domain CL of the first light chain under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or Histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K), arginine (R)), and wherein in the constant domain CH1 of the first heavy chain under a) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index);

or

ii) in the constant domain CL of the second light chain under b) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or Histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K), arginine (R)), and wherein in the constant domain CH1 of the second heavy chain under b) the amino acid at positions 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index);

B) culturing the host cell under conditions that allow synthesis of said antibody molecule; and

C) recovering said antibody molecule from said culture.

12. A nucleic acid encoding the amino acid sequences of a multispecific antibody according to claim 1.

13. An expression vector containing the nucleic acid according to claim 12 capable of expressing said nucleic acid in a host cell.

14. A composition comprising the multispecific antibody according to claim 1.

15. A pharmaceutical composition comprising a multispecific antibody according to claim 1 and at least one pharmaceutically acceptable excipient.

16. A host cell comprising the vector of claim 13.

17. A multispecific antibody obtained by a method according to claim 11.

18. The multispecific antibody according to claim 1, wherein the antibody comprises at least two Fab fragments, wherein the first Fab fragment comprises at least one antigen binding site specific for a first antigen;

and the second Fab fragment comprises at least one antigen binding site specific for a second antigen, wherein in the second Fab fragment the variable domains VL and VH in the second light chain and second heavy chain are replaced by each other; and

wherein the multispecific antibody is devoid of an Fc domain; and

wherein

i) in the constant domain CL of the first light chain under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the first heavy chain under a) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index);

or

ii) in the constant domain CL of the second light chain under b) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the second heavy chain under b) the amino acid at positions 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).