BISPECIFIC ANTIBODIES AGAINST CD3EPSILON AND ROR1

• US 20170306018A1
• Filed: 10/08/2015
• Published: 10/26/2017
• Est. Priority Date: 10/09/2014
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: EngMab AG (Bristol-Myers Squibb Company)
• Inventors: VU, MINH DIEM, STREIN, KLAUS, AST, OLIVER, FAUTI, TANJA, FREIMOSER-GRUNDSCHOBER, ANNE, HOSSE, RALF, KLEIN, CHRISTIAN, MOESSNER, EKKEHARD, MOSER, SAMUEL, MURR, RAMONA, UMANA, PABLO, JUNG-IMHOF, SABINE, KLOSTERMANN, STEFAN, MOLHOJ, MICHAEL, REGULA, JOERG, SCHAEFER, WOLFGANG
• Granted Patent: US 11,952,421 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/64 comprising a combination of variable region and constant region components
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/77 Internalization into the cell

Abstract

The present invention relates to bispecific antibodies against ROR1 and CD3, their manufacture and use.

Claims

1. A bispecific bi- or trivalent antibody specifically binding to human CD3ε

and extracellular domain of human ROR1, wherein variable domains VL and VH in a light chain and respective heavy chain are replaced by each other, characterized in comprising a constant domain CL wherein amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in respective constant domain CH1 amino acid at position 147 and amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat).

2. A bispecific antibody according to claim 1 characterized in comprising

a) a first light chain and a first heavy chain of a first antibody which specifically binds to the extracellular domain of human ROR1;

and

b) a second light chain and a second heavy chain of a second antibody which specifically binds to CD3ε

, and wherein 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

c) 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 and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat).

3. A bispecific antibody according to claim 1 characterized in comprising

a) a first light chain and a first heavy chain of a first antibody which specifically binds to the extracellular domain of human ROR1;

and

b) a second light chain and a second heavy chain of a second antibody which specifically binds to human CD3ε

, and wherein 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

c) 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 and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat).

4. The bispecific antibody according to claim 2, wherein said bispecific antibody comprises in addition a Fab fragment of said first antibody (further named also as “

ROR1-Fab”

) and in the constant domain CL said ROR1-Fab 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 said ROR1-Fab the amino acid at positions 147 and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat).

5. The bispecific antibody according to claim 3, wherein said bispecific antibody comprises in addition a second Fab fragment of said first antibody (“

ROR1-Fab”

).

6. The bispecific antibody according to claim 1, characterized in consisting of one Fab fragment of an antibody specifically binding to human CD3ε

(further named also as “

CD3-Fab”

), and one Fab fragment of an antibody specifically binding to the extracellular domain of human ROR1 (further named also as “

ROR1-Fab(s)”

) and a Fc part, wherein the CD3-Fab and the ROR1-Fab are linked via their C-termini to the hinge region of said Fc part and wherein either the CD3-Fab or the ROR1-Fab comprises aa substitution and the CD3-Fab comprises crossover.

7. The bispecific antibody according to claim 6, characterized in consisting of one CD3-Fab, and one ROR1-Fab and a Fc part, wherein the CD3-Fab and the ROR1-Fab are linked via their C-termini to the hinge region of said Fc part and a second ROR1-Fab, which is linked with its C-terminus to the N-terminus of the CD3-Fab and wherein the CD3-Fab comprises crossover and either the CD3-Fab or both ROR1-Fabs comprise aa substitution.

8. The bispecific antibody according to claim 7, characterized in consisting of ROR1-Fab-Fc-CD3-Fab-ROR1-Fab, wherein both ROR1-Fabs comprise aa substitution and the CD3-Fab comprises VL/VH crossover.

9. The bispecific antibody according to claim 1, characterized in consisting of two ROR1-Fabs and a Fc part, wherein the ROR1-Fabs are linked via their C-termini to the hinge region of said Fc part and a CD3-Fab, which is linked with its C-terminus to the N-terminus of one ROR1-Fab and the CD3-Fab comprises crossover and either the CD3-Fab or both ROR1-Fabs comprise aa substitution.

10. The bispecific antibody according to claim 1, characterized in consisting of one CD3-Fab, which is linked via its C-terminus to the hinge region of said Fc part and a ROR1-Fab, which is linked with its C-terminus to the N-terminus of the CD3-Fab and either the CD3-Fab or the ROR1-Fab comprise aa substitution.

11. The bispecific antibody according to claim 1, characterized in consisting of one ROR1-Fab, which is linked via its C-terminus to the hinge region of said Fc part and a CD3-Fab, which is linked with its C-terminus to the N-terminus of the ROR1-Fab and either the CD3-Fab or the ROR1-Fab comprise aa substitution.

12. The bispecific antibody according to claim 1, characterized in comprising the CDR sequences of anti-ROR1 antibody MAB1.

13. The bispecific antibody according to claim 1, characterized in comprising the VH and VL sequences of anti-ROR1 antibody MAB1, or an antibody comprising the VH, VL, CH1, and CL sequences of anti-ROR1 antibody MAB1.

14. The bispecific antibody according to claim 1, characterized by an antibody portion specifically binding to human CD3ε

, preferably a Fab fragment, comprising

a) a variable domain VH comprising the heavy chain CDRs of SEQ ID NO;

12, 13 and 14 as respectively heavy chain CDR1, CDR2 and CDR3 and a variable domain VL comprising the light chain CDRs of SEQ ID NO;

15, 16 and 17 as respectively light chain CDR1, CDR2 and CDR3 of the anti CD3ε

antibody (CDR MAB CD3 H2C), or

b) a variable domain VH comprising the heavy chain CDRs of SEQ ID NO;

23, 24 and 25 as respectively heavy chain CDR1, CDR2 and CDR3 and the variable domain VL is replaced by a variable domain VL comprising the light chain CDRs of SEQ ID NO;

26, 27 and 28 as respectively light chain CDR1, CDR2 and CDR3 of the anti CD3ε

antibody (CDR MAB CD3 CH2527).

15. The bispecific antibody according to claim 1, characterized by an antibody portion specifically binding to human CD3ε

wherein

a) variable domains are of SEQ ID NO;

10 and 11 (VHVL MAB CD3 H2C), or

b) variable domains are of SEQ ID NO;

21 and 22 (VHVL MAB CD3 CH2527).

16. The bispecific antibody according to claim 1, characterized by a Fab fragment, specifically binding to human ROR1 and a variable domain VH comprising heavy chain CDRs CDR1H of SEQ ID NO:

7, a CDR2H of SEQ ID NO;

8, a CDR3H of SEQ ID NO;

9 and a variable domain VL comprising the light chain CDRs CDR1L of SEQ ID NO;

3, a CDR2L of SEQ ID NO;

4, a CDR3L of SEQ ID NO;

5 (CDR MAB1).

17. The bispecific antibody according to claim 1, characterized by a Fab fragment, specifically binding to human ROR1 comprising a VH of SEQ ID NO:

10 and a VL of SEQ ID NO;

11 (VHVL MAB1).

18. The bispecific antibody according to claim 1, characterized in the constant domain CL wherein the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H).

19. The bispecific antibody according to claim 1, characterized in that amino acid 124 is K, amino acid 147 is E, amino acid 213 is E, and amino acid 123 is R for a kappa light chain or K for a lambda light chain.

22. An antibody according to claim 1, characterized in that a first CH3 domain of one heavy chain and a second CH3 domain of the other heavy chain each meet at an interface which comprises an original interface between the antibody CH3 domains;

wherein said interface is altered to promote formation of a bispecific antibody, wherein the alteration is characterized in that;

a) the first CH3 domain of one heavy chain is altered, so that within the original interface the first CH3 domain of one heavy chain that meets the original interface of the second CH3 domain of the other heavy chain within the bispecific 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 first CH3 domain of one heavy chain which is positionable in a cavity within the interface of the second CH3 domain of the other heavy chain and

b) the second CH3 domain of the other heavy chain is altered, so that within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the bispecific 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 second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable.

23. An antibody according to claim 1 comprising a the human IgG1 Fc part wherein Pro329 is substituted with glycine or arginine and/or Leu234 is substituted with alanine and Leu235 is substituted with alanine.

24. The antibody according to claim 23, characterized in being of construct ROR1 Fab-Fc-CD3 Fab-ROR1 Fab comprising VL/VH crossover within the CD3 Fab wherein the human IgG1 Fc part Pro329 is substituted with glycine, Leu234 is substituted with alanine and Leu235 is substituted with alanine.

25. The antibody according to claim 1, characterized in

a) being of construct ROR1 Fab-Fc-CD3 Fab-ROR1 Fab,

b) comprising VL/VH crossover within the CD3 Fab,

c) comprising a human IgG1Fc part,

d) comprising within the Fc part substitution of Pro329 with glycine and substitutions of Leu234 by alanine and Leu235 by alanine, and

e) comprising constant domain CL of both ROR1 Fabs wherein the amino acid at position 124 is substituted by lysine (K) and at position 123 by arginine (R) for a kappa light and lysine (K) for a lambda light chain, and wherein a second constant domain CH1 has amino acid at position 147 and amino acid at position 213 each substituted by glutamic acid (E).

26. An antibody according to claim 1, characterized in not internalizing in a concentration of 1 nM in primary B-CLL cells at 37°

C. during two hours.

27. An antibody according to claim 1, characterized in that the bispecific antibody does not internalize in a cell based assay at 37°

C. during 2 hrs, using ROR1-positive primary B-CLL cells and used at an antibody concentration of 1 nM, whereby not internalize means, that the mean fluorescence intensity (MFI), as detected by flow cytometry, of said bispecific antibody upon binding to ROR1-positive primary B-CLL cells measured at time 0 is not reduced more than 50%, preferably not more than 30% when re-measured after a 2 hr-incubation at 37°

C.

28. A method for the preparation of a bispecific antibody according to claim 1 comprising the steps of

a) transforming a host cell with vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody according to claim 1,

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

and

c) recovering said antibody molecule from said culture.

29. A host cell comprising vectors comprising nucleic acid molecules encoding the light chain and heavy chains of an antibody according to claim 1.

30. A pharmaceutical composition comprising an antibody according to claim 1 and a pharmaceutically acceptable excipient.

32. A method for the treatment of an ROR1-positive hematological malignancy selected from the group consisting essentially of chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B cell lymphoma (DLBCL), multiple myeloma (MM), follicular lymphoma (FL), and hematological malignancies carrying ROR1 on the surface of the tumor cells, comprising administering an antibody according to claim 1 to a subject in need thereof.

33. A method according to claim 32 for the treatment of multiple myeloma.

34. A method according to claim 32 for the treatment of chronic lymphocytic leukemia (CLL) of B-cell lineage (B-CLL).

35. A method for the treatment of an ROR1-positive solid tumor selected from the group consisting of ovarian cancer, lung cancer, breast cancer, gastric cancer, and pancreatic cancer comprising administering an antibody according to claim 1 to a subject in need thereof.

36. A method according to claim 35 for the treatment of ovarian cancer.

37. A method according to claim 35 for the treatment of breast cancer.

38. A method according to claim 35 for the treatment of lung cancer.

39. A method for the treatment of a plasma cell disorder comprising administering an antibody according to claim 1 to a subject in need thereof.

40. A method according to claim 39 wherein the plasma cell disorder is multiple myeloma or a B cell disorder expressing ROR1.

20. A bispecific antibody which specifically binds human CD3ε

and extracellular domain of human ROR1 comprising a heavy and light chain set selected from the group consisting of polypeptides SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, and SEQ ID NO;

40 or from the group consisting of polypeptides SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, and SEQ ID NO;

41.

21. An antibody according to claim 20, comprising a portion which specifically binds human CD3ε

wherein

a) variable domain VH is replaced by a variable domain VH comprising heavy chain CDRs of SEQ ID NO;

12, 13 and 14 as respectively heavy chain CDR1, CDR2 and CDR3 and variable domain VL is replaced by a variable domain VL comprising the light chain CDRs of SEQ ID NO;

15, 16 and 17 as respectively light chain CDR1, CDR2 and CDR3, or

b) variable domain VH is replaced by a variable domain VH comprising the heavy chain CDRs of SEQ ID NO;

23, 24 and 25 as respectively heavy chain CDR1, CDR2 and CDR3 and variable domain VL is replaced by a variable domain VL comprising the light chain CDRs of SEQ ID NO;

26, 27 and 28 as respectively light chain CDR1, CDR2 and CDR3.

31. (canceled)