METHOD FOR THE SELECTION AND PRODUCTION OF TAILOR-MADE, SELECTIVE AND MULTI-SPECIFIC THERAPEUTIC MOLECULES COMPRISING AT LEAST TWO DIFFERENT TARGETING ENTITIES AND USES THEREOF

• US 20150232560A1
• Filed: 12/22/2014
• Published: 08/20/2015
• Est. Priority Date: 06/27/2012
• Status: Abandoned Application

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: HEINDL, DIETER, HUELSMANN, PETER MICHAEL, KOPETZKI, ERHARD, TIEFENTHALER, GEORG, KALUZA, BRIGITTE, NIEDERFELLNER, GERHARD
• Application Number:
  US14/579,165
• Publication Number:
  US 20150232560A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• 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: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/58 involving urea or urease
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 2333/71 for growth factors; for growth regulators
• CPC Class Codes: G01N 2333/912 transferring phosphorus containing groups, e.g. kinases (2.7)
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/563 involving antibody fragments
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells

Abstract

Herein is reported a method for determining a combination of antigen binding sites comprising the steps of (i) determining the binding specificity and/or affinity and/or effector function and/or in vivo half-life of a multitude of bispecific antibodies prepared by combining each member of a first multitude of antibody Fab fragments or scFv antibody fragments with each member of a second multitude of antibody Fab fragments or scFv antibody fragments, and a linker comprising at one of its termini the second member of the first binding pair and at the respective other terminus the second member of the second binding pair, whereby the first multitude specifically binds to a first cell surface molecule and the second multitude specifically binds to a second cell surface molecule, and (ii) choosing the bispecific antibody with suitable binding specificity and/or affinity and/or effector function and/or in vivo half-life and thereby determining a combination of antigen binding sites.

Claims

1. A method for producing a bispecific antibody comprising:

incubating

(a) an antibody Fab fragment or a scFv antibody fragment conjugated to a first member of a first binding pair, wherein the Fab fragment or scFv antibody fragment specifically binds to a first surface marker or a first epitope of the first surface marker,

(b) an antibody Fab fragment or a scFv antibody fragment conjugated to a first member of a second binding pair, wherein the Fab fragment or scFv antibody fragment specifically binds to a second surface marker or a second epitope of the first surface marker, and

(c) an enantiomeric DNA polynucleotide linker comprising at one of its termini the second member of the first binding pair and at the other terminus the second member of the second binding pair,

and thereby producing the bispecific antibody.

3. The method of claim 1, wherein the bispecific antibody is a complex comprising

a) a first Fab fragment or scFv antibody fragment

i) that specifically binds to a first surface marker, and

ii) that is conjugated to a first member of a first binding pair,

b) a second Fab fragment or scFv antibody fragment

i) that specifically binds to a second surface marker, and

ii) that is conjugated to a first member of a second binding pair, and

c) an enantiomeric DNA polynucleotide linker

i) that is conjugated to the second member of the first binding pair, and

ii) that is conjugated to the second member of the second binding pair.

4. The method of claim 3, wherein the complex is a non-covalent complex.

5. The method of claim 1, wherein the first or second binding pair is a complementary pair of polynucleotides.

6. The method of claim 3, wherein the complex further comprises an effector moiety conjugated to a polynucleotide that is complementary to at least a part of the polynucleotide linker.

7. The method of claim 6, wherein the complex comprises a second effector moiety conjugated to a polynucleotide that is

i) complementary to at least a part of the polynucleotide that is conjugated to the first effector moiety and

ii) not complementary to the polynucleotide linker.

8. The method of claim 1, wherein the Fab fragments or scFv antibody fragments bind to first and second non-overlapping epitopes on the same surface marker.

9. The method of claim 1, wherein the polynucleotide linker comprises from 8 to 1000 nucleotides.

10. The method of claim 1, wherein the enantiomeric DNA is L-DNA.

11. The method according to claim 10, wherein the L-DNA is single stranded L-DNA (ss-L-DNA).

12. A pharmaceutical formulation comprising the bispecific antibody obtained by the method of claim 1 and a pharmaceutically acceptable carrier.

13. A bispecific antibody obtained by the method of claim 1.

15. A method of treating an individual having cancer comprising administering to the individual an effective amount of the bispecific antibody obtained by a method according to claim 1.

2. A method for determining a combination of antigen binding sites comprising the following steps

(i) determining the binding specificity, and/or affinity, and/or effector function, and/or in vivo half-life of a multitude of bispecific antibodies prepared by combining each member of a first multitude of antibody Fab fragments or scFv antibody fragments each linked to the same first member of a first binding pair with each member of a second multitude of antibody Fab fragments or scFv antibody fragments each linked to the same first member of a second binding pair, and an enantiomeric DNA polynucleotide linker comprising at one of its termini the second member of the first binding pair and at the respective other terminus the second member of the second binding pair,

wherein the first multitude of antibody Fab fragments or scFv antibody fragments specifically binds to a first cell surface marker or first epitope of the first surface marker and the second multitude of antibody Fab fragments or scFv antibody fragments specifically binds to a second cell surface marker or to a second epitope of the first surface marker, and

(ii) choosing the bispecific antibody with suitable binding specificity, and/or affinity, and/or effector function, and/or in vivo half-life and thereby determining a combination of antigen binding sites.

14. (canceled)

16. A bispecific antibody comprising

a) a first Fab fragment or scFv antibody fragment

i) that specifically binds to a first surface marker or a first epitope of the first surface marker, and

ii) that is conjugated to a first member of a first binding pair,

b) a second Fab fragment or scFv antibody fragment

i) that specifically binds to a second surface marker or a second epitope of the first surface marker, and

ii) that is conjugated to a first member of a second binding pair, and

c) an enantiomeric DNA polynucleotide linker

i) that is conjugated to the second member of the first binding pair at one of its termini, and

ii) that is conjugated to the second member of the second binding pair at the other terminus,

wherein the first and second Fab fragments or scFv antibody fragments and the enantiomeric DNA polynucleotide linker form a non-covalent complex.

17. A pharmaceutical composition comprising the bispecific antibody of claim 16 and a pharmaceutically acceptable carrier.

18. A method of treatment comprising administering an effective amount of the bispecific antibody of claim 16 to an individual in need thereof.

24. The bispecific antibody of claim 16, wherein the first and second members of the first binding pair comprise the nucleic acid sequences of SEQ ID NO:

05 and SEQ ID NO;

08, respectively and the first and second members of the second binding pair comprise the nucleic acid sequences of SEQ ID NO;

06 and SEQ ID NO;

07, respectively.

25. The bispecific antibody of claim 16, wherein the polynucleotide linker is 10-500 nucleotides in length.

19. A method of synthesizing a complex comprising

a) synthesizing a first binding entity that specifically binds to a first cell surface marker or its ligand and that is conjugated to a first member of a first binding pair,

b) synthesizing a second binding entity that specifically binds to a second cell surface marker or its ligand and that is conjugated to a first member of a second binding pair,

c) synthesizing a polynucleotide linker, and

d) forming the complex by combining the synthesized components of a), b) and c).

20. A multispecific binding molecule comprising

a) a first binding entity

i) that specifically binds to a first cell surface marker or its ligand, and

ii) that is conjugated to a first member of a first binding pair,

b) a second binding entity

i) that specifically binds to a second cell surface marker or its ligand, and

ii) that is conjugated to a first member of a second binding pair, and

c) a polynucleotide linker

i) that is conjugated to the second member of the first binding pair, and

ii) that is conjugated to the second member of the second binding pair.

21. A polypeptide-polynucleotide-complex of the formula:

(A-a′

;

a-S-b;

b′

-B)-X(n) or (A-a′

;

a-S-b;

b′

-B);

X(n),

wherein A as well as B is a binding entity that specifically binds to a target,

wherein a′

;

a as well as b;

b′

is a binding pair, wherein a′ and

a and do not interfere with the binding of b to b′ and

vice versa,

wherein S is a linker polynucleotide,

wherein (;

X) denotes an effector moiety bound either covalently or via a binding pair to at least one of a′

, a, b, b′

or S,

wherein (n) is an integer,

wherein - represents a covalent bond, and

wherein;

represents a non-covalent bond.

22. A Fab fragment or scFv antibody fragment conjugated to a single-stranded L-DNA moiety.

23. A mixture comprising

a) an antibody Fab fragment or a scFv antibody fragment conjugated to a first member of a first binding pair, wherein the Fab fragment or scFv specifically binds to a first surface marker or its ligand,

b) an antibody Fab fragment or a scFv antibody fragment conjugated to a first member of a second binding pair, wherein the Fab fragment or scFv antibody fragment specifically binds to a second surface marker or its ligand, and

c) an enantiomeric DNA polynucleotide linker comprising at one of its termini the second member of the first binding pair and at the other terminus the second member of the second binding pair.