WO2017009419A1

WO2017009419A1 - Bispecific antibody-like molecules having bivalency vis-à-vis each antigen

Info

Publication number: WO2017009419A1
Application number: PCT/EP2016/066763
Authority: WO
Inventors: Alessandro BERTI; Giampiero De Luca
Original assignee: Ares Life Sciences SA
Current assignee: Ares Life Sciences SA
Priority date: 2015-07-16
Filing date: 2016-07-14
Publication date: 2017-01-19
Anticipated expiration: 2018-01-16

Abstract

The invention relates to bispecific antibody-like molecules having bivalency vis-à-vis each antigen, to their uses and to methods for making them.

Description

BISPECIFIC ANTIBODY-LIKE MOLECULES HAVING BIVALENCY VIS-A-VIS EACH

ANTIGEN

Field of the invention

The present invention relates to bispecific antibody-like molecules having bivalency vis-a-vis each antigen, to their uses and to methods for making them.

Background of the invention

Multispecific antibodies, and in particular bispecific antibodies have interesting potential as therapeutic drugs. However, one of the major obstacles in the development of multi/bispecific antibodies has been the difficulty of producing the materials in sufficient quality and quantity. In addition, most of the bispecific antibodies that have been developed have the inconvenient of being monovalent; this aspect decreases the therapeutic value of such a molecule.

A wide variety of multispecific recombinant antibody formats have been developed in the recent past, e.g. tetravalent bispecific antibodies by fusion of, e.g., an IgG antibody format and single chain domains (see e.g. Coloma, M.J., et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; and Morrison, S.L, Nature Biotech. 25 (2007) 1233-1234).

Also several other new formats wherein the antibody core structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- or tetrabodies, minibodies, several single chain formats (scFv, Bis-scFv), which are capable of binding two or more antigens, have been developed (Holliger, P., et al, Nature Biotech 23 (2005) 1 126-1 136; Fischer, N., Leger O., Pathobiology 74 (2007) 3-14; Shen, J., et al, Journal of Immunological Methods 318 (2007) 65-74; Wu, C, et al, Nature Biotech. 25 (2007) 1290- 1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scFvs (Fischer N., Leger O., Pathobiology 74 (2007) 3-14).

Other new formats which do not need a linker have been also developed, but these multispecific antibodies still present several problems (such as pharmacokinetics, biological properties, stability, aggregation) which are mainly due to the size of the final molecule. In addition to these drawbacks, said multispecific antibodies or antibodies-like molecules are not easy to produce, due to low expression yield and/or side products, or are difficult to purify, due to a high percentage of contaminants. ln view of the above there is a need of improved multispecific and, in particular bispecific, antibody or antibody-like formats.

The present invention fulfils this need by providing bispecific antibody-like molecules having bivalency vis-a-vis each antigen. The format of these antibody-like molecules allows as well an efficient production since a lot of contaminants and side products are avoided.

Summary of the invention In a first aspect, the invention provides an antibody-like molecule capable of binding two different antigens A and B wherein said antibody-like molecule comprises the following amino acid chains:

a) an amino acid sequence comprising a light chain variable domain (V_LA) , a light chain constant domain (Ci_A), one or more hinge regions, an heavy chain constant domain (CHB) and an heavy chain variable domain (V_HB) , wherein V_LA and V_HB are directed respectively to antigen A and antigen B;

b) an amino acid sequence comprising an heavy chain variable domain (V_HA) and an heavy chain constant domain (CHA), which are different from V_HB and CHB of a), wherein V_HA is directed against the same antigen A as V_LA; and c) an amino acid sequence comprising a light chain variable domain (V_LB) and a light chain constant domain (Ci_B), which are different from V_LA and Ci_A of a), wherein V_LB is directed against the same antigen B as V_HB.

In another aspect, the invention provides an antibody-like molecule described herein, wherein the amino acid sequence defined in a) comprises only one hinge region. In another aspect, the invention provides an antibody-like molecule described herein, wherein the amino acid sequence defined in a) comprises two hinge regions, which can be identical or different. In another aspect, the invention provides an antibody-like molecule described herein, wherein the amino acid sequence defined in a) further comprises one or more linkers. In another aspect, the invention provides an antibody-like molecule described herein, which is bivalent vis-a-vis each of said antigens A and B and which comprises two amino acid sequences as defined in a), two amino acid sequences as defined in b) and two amino acid sequences as defined in c). In another aspect, the invention provides an antibody-like molecule described herein, which is bivalent vis-a-vis each of said antigens A and B and which consists of two amino acid sequences as defined in a), two amino acid sequences as defined in b) and two amino acid sequences as defined in c). In another aspect, the invention provides an antibody-like molecule described herein, wherein in the amino acid sequence defined in a) the one or more hinge regions connect(s) Ci_A with CHB either directly or through one or more linkers. In another aspect, the invention provides an antibody-like molecule described herein, wherein in the amino acid sequence defined in a) the one or more hinge regions connect(s) Ci_A with VHB either directly or through one or more linkers.

In still another aspect, the invention provides a nucleic acid molecule coding for one or more amino acid sequences of an antibody-like molecule described herein. In another aspect, the invention provides a nucleic acid molecule coding for the amino acid sequence defined in a). In another aspect, the invention provides a nucleic acid molecule coding for the amino acid sequence defined in b). In another aspect, the invention provides a nucleic acid molecule coding for the amino acid sequence defined in c). In another aspect, the invention provides a nucleic acid molecule coding for the amino acid sequence(s) defined in a), b) and/or c) and any combination thereof.

In still another aspect, the invention provides a vector comprising a nucleic acid molecule described herein. In another aspect, the invention provides a vector described herein, which is an expression vector. In another aspect, the invention provides a vector described herein, which comprises a first promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequence defined in a) and a second promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequence defined in b) and of a nucleic acid molecule coding for the amino acid sequence defined in c). In another aspect, the invention provides a vector described herein, which further comprises at least an internal ribosomal entry site (IRES) region between the nucleic acid molecule coding for the amino acid sequence defined in b) and of a nucleic acid molecule coding for the amino acid sequence defined in c). In another aspect, the invention provides a vector described herein, which comprises a first promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequence defined in a), a second promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequence defined in b) and a third promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequence defined in c). In another aspect, the invention provides a vector described herein, wherein the promoters present in said vector are identical or different. In another aspect, the invention provides a vector described herein, wherein the promoters present in said vector are oriented on the same or on opposite direction. In another aspect, the invention provides a vector described herein, which comprises a single promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequences defined in a), in b) and in c). In another aspect, the invention provides a vector described herein, which further comprises at least an IRES region between the nucleic acid molecule coding for the amino acid sequence defined in a) and of a nucleic acid molecule coding for the amino acid sequence defined in b) and at least an IRES region between the nucleic acid molecule coding for the amino acid sequence defined in b) and of a nucleic acid molecule coding for the amino acid sequence defined in c).

In still another aspect, the invention provides a host cell wherein a vector described herein has been inserted. In another aspect the invention provides a host cell wherein a vector described herein has been inserted by transfection, transformation or transduction. In another aspect, the invention provides a host cell described herein, which is selected from the group consisting of bacterial, yeast or mammalian cells. In another aspect, the invention provides a host cell described herein, which is an E. coli cell, a CHO cell or a HEK293 cell. In another aspect, the invention provides a CHO host cell described herein, which is a CHO-S, a CHO-DG44, a CHO- K1 , a CHO-Kl sv or a CHO Pro-5 cell. In another aspect, the invention provides a HEK293 host cell described herein, which is a HEK293 EBNA or a HEK293- F cell. The above cell lines can be either transient or stable cell lines.

In still another aspect, the invention provides a method for making an antibody-like molecule described herein. In another aspect, the invention provides a method for making an antibody- like molecule described herein, which comprises culturing a host cell described herein and isolating said antibody-like molecule.

In still another aspect, the invention provides a pharmaceutical composition comprising an antibody-like molecule described herein. In another aspect, the invention provides a pharmaceutical composition described herein for use as a medicament.

In still another aspect, the invention provides an antibody-like molecule described herein for use as a medicament.

Description of the figures Figure 1 reports a standard IgG antibody molecule. H = hinge region,— = disulfide bond

Figure 2 reports an exemplary bispecific antibody-like molecule according to the present invention. Ag = antigen, H = hinge region,— = disulfide bond Figure 3 reports an exemplary bispecific antibody-like molecule according to the present invention. Ag = antigen, H = hinge region,— = disulfide bond, » »- linker

Figure 4 reports an exemplary vector according to the present invention.

Detailed description of the invention Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms.

As used herein, the term "immunoglobulin" (Ig) refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain, resulting in the so-called "Y" shape. A light chain has two parts: the variable domain (V_L) and the constant domain (CL), which in the context of a light chain can be called constant region as well. A heavy chain has two parts as well: the variable domain (V_H) and the constant region (CH). In each pair, the light and heavy chain variable domains are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions (Figure 1 ). Full-length immunoglobulin "light chains" (about 25 Kd) are encoded by a variable domain gene at the N-terminus (about 1 10 amino acids) and a kappa or lambda constant domain (CK and CA, respectively) gene at the C-terminus. Full-length immunoglobulin "heavy chains" (about 50 Kd), are similarly encoded by a variable domain gene (about 1 16 amino acids) and one of the other constant region genes (about 330 amino acids) mentioned hereinafter. There are five types of mammalian heavy chain denoted by the Greek letters: α, δ, ε, γ, and μ. The type of heavy chain defines the antibody's isotype as IgA, IgD, IgE, IgG and IgM, respectively. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, a and δ have a constant region composed of three Ig constant domains (CH1 , CH2, and CH3), and a hinge region for added flexibility; heavy chains μ and ε have a constant region composed of four Ig constant domains (CH1 , CH2, CH3, and CH4) and a hinge region.

The "hinge region" is a specific region located between the CH1 and CH2 domains (Figure 1 ). The hinge region varies in length from 10 to more than 60 amino acids in different isotypes. Portions of this sequence assume an unfolded and flexible conformation, permitting molecular motion between the CH1 and CH2 domains. Some of the greatest differences between the constant regions of the IgG subclasses are concentrated in the hinge. Native human antibody hinge regions can be structurally defined as consisting of an upper hinge region (UH) extending from the last residue of CH1 up to but not including the first inter-heavy chain cysteine, a middle hinge region (MH) extending from the first inter-heavy chain cysteine to a proline residue adjacent to the carboxyl-end of the last MH cysteine, and a highly conserved 7-8-amino acid lower hinge (LH) (Roux, K.H, et al, The Journal of Immunology vol. 161 no. 8 (1998) 4083-4090). As used herein, the term hinge region refers to native/wild-type (e.g. hinge regions of IgA, IgD, IgE, IgG and IgM) as well as to engineered hinge regions (e.g. native/wild-type hinge regions modified in order for example to modify their flexibility, to introduce particular amino acid sequence(s) or unnatural amino acid(s)). An immunoglobulin light or heavy chain variable domain consists of a "framework" region interrupted by three hypervariable regions. Thus, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "Complementarity Determining Region" or "CDR", i.e. L-CDR1 , L-CDR2 and L-CDR3 in the light chain variable domain and H-CDR1 , H-CDR2 and H-CDR3 in the heavy chain variable domain (Kabat et al. 1991 ) and/or those residues from a "hypervariable loop" (Chothia and Lesk, 1987). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined. The sequences of the framework regions of different light (i.e. L-FR1 , L-FR2, L-FR3 and L-FR4) or heavy (i.e. H-FR1 , H-FR2, H-FR3 and H-FR4) chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen.

As used herein, the term "antibody-like molecules" refers to molecules retaining the binding capacity of an antibody but not having the classical "Y" shape.

As used herein, the term "valency" refers to the number of binding sites specific for a given antigen (e.g. TNF-alpha) that an individual antibody or antibody-like molecule has. For example, the valency of a "Y" shaped IgG antibody, such as adalimumab, is two, since each "arm" of the antibody contains a binding site specific for TNF-alpha. In the context of the present invention the expressions "having bivalency" and "being bivalent" have the same meaning and can be used interchangeably.

As used herein, the term "specificity" refers to the property of an antibody or antibody-like molecule that enable it to recognize and bind a given antigen (e.g. TNF-alpha) and not a different one (e.g. IL-1 ). For example, adalimumab is specific for TNF-alpha, i.e. it recognizes and binds only TNF-alpha. Antibodies or antibody-like molecules which recognize and bind only one given antigen are called monospecific. Antibodies or antibody-like molecules which recognize and bind two different antigens are called bispecific, and so on. In view of the above, it follows that adalimumab is a monospecific antibody having bivalency vis-a-vis TNF-alpha. The present invention is based upon the discovery that by combining specific immunoglobulin domains of different antibodies in a particular conformation it is possible to generate bispecific antibody-like molecules having bivalency vis-a-vis each antigen. This means that with a single molecule it is possible to target, in a very efficient manner (the molecule being bivalent), two different antigens. This allows e.g. to use a single molecule to treat those diseases whose etiology comprises more than one factor (for example, more than one cytokine). In addition, the particular format of these antibody-like molecules allows as well an efficient production since a lot of contaminants and side products are avoided. In fact, the specific combination of domains in the three amino acid sequences defined hereinafter as amino acid sequence a), b) and c) ensures a very low percentage of wrong light-heavy chain pairing. It should be also emphasized that the format of the antibody-like molecules described herein allows, by adding or removing the hinge region(s) in the amino acid sequence defined in a), an high number of possibilities in term of molecule flexibility. The hinge region, indeed, has a strong influence on the flexibility of the domains involved in the antigen binding. In particular, when there is the need of binding two different antigens present, e.g., on two different types of cell, the addition of one or more hinge region(s) allows a very high flexibility of the domains involved in the antigen binding. The flexibility of the antibody-like molecules can also be modulated by further adding one or more linkers. Furthermore, the presence of the hinge regions, being implicated in the formation of disulfide bonds, allows modulating the stability of the antibody-like molecules described herein.

The choice of the domains involved in the antigen binding depends on which antigens we want to target. If antibodies or fragment or derivative thereof directed against a specific antigen (e.g. TNF-alpha) are already known (e.g. adalimumab, infliximab or golimumab), it is, of course, desirable to use, in the antibody-like molecules according to the invention, the amino acid sequence of their domains involved in the antigen binding. Otherwise, if no antibodies or fragment or derivative thereof directed against a specific antigen are known, it will be necessary to identify the amino acid sequences that can be used in the antibody-like molecules according to the invention.

In a first embodiment, the invention provides an antibody-like molecule capable of binding two different antigens A and B wherein said antibody-like molecule comprises the following chains: a) an amino acid sequence comprising a light chain variable domain (V_LA) , a light chain constant domain (CLA), one or more hinge regions, an heavy chain constant domain (CHB) and an heavy chain variable domain (V_HB) wherein V_LA and V_HB are directed respectively to antigen A and antigen B;

b) an amino acid sequence comprising an heavy chain variable domain (V_HA) and an heavy chain constant domain (CHA), which are different from V_HB and CHB of a), wherein V_HA is directed against the same antigen A as V_LA; and c) an amino acid sequence comprising a light chain variable domain (V_LB) and a light chain constant domain (CLB), which are different from V_LA and CLA of a), wherein V_LB is directed against the same antigen B as V_HB.

In one embodiment, the heavy chain constant domains of the antibody-like molecule of the invention, i.e. CHA and CHB, are CH 1 domains. In one embodiment, the light chain constant domains of the antibody-like molecule of the invention, i.e. CLA and CLB, are CK or CA domains or any combination thereof. ln one embodiment, the antibody-like molecule of the invention is capable to bind TNF-alpha and IL-1 beta or VEGF and EGFR. In this case the antibody-like molecule of the invention comprises, e.g., the light chain variable domain (V_LA), the light chain constant domain (Ci_A), the heavy chain variable domain (V_HA) and the heavy chain constant domain 1 (CHA) of adalimumab or bevacizumab and it comprises, e.g., the light chain variable domain (V_LB), the light chain constant domain (Ci_B), the heavy chain variable domain (V_HB) and the heavy chain constant domain 1 (CHB) of canakinumab or cetuximab. In other embodiments, the above mentioned domains of adalimumab can be replaced by the corresponding domains of a different molecule directed against TNF-alpha (e.g. golimumab, infliximab, certolizumab pegol). By looking at pathophysiology of the diseases it is possible to decide which pair of antigens we want to target in order to improve the therapeutic efficacy of the antibody-like molecule of the invention. In addition to the embodiment reported above, wherein TNF-alpha and IL-1 beta or VEGF and EGFR are simultaneously targeted, other examples of pair of antigens A and B can be the following: TNF-alpha/CD20, TNF-alpha/IL-6R, TNF-alpha/IL-17A, BLyS/CD20, BLyS/IFN-alpha, BLyS/VLA1 , VLA4/CD20, VEGF/HER2, VEGF/DLL-4, VEGF/IL-6R, IL- 2RA/CD20, lgE/IL-33, lgE/IL-4, lgE/Eotaxin-1 , lgE/IL-5, lgE/IL-13, IgE/TNF-alpha, lgE/CCR3, lgE/CCR4, lgE/OX40L, IL13/IL-4, IL-1 -alpha/IL-1 -beta, CD33/CD47, CD33/MDR1 , IL-6/CD20, IL-6/BLyS, CD4/VEGF, TGF-beta/MCP-1 , CD52/CD20, CTLA-4/VEGF, CTLA-4/CD28, GM- CSF receptor/TNF-alpha, CD33/CD123, CD33/CD3, CD33/CD16, CD123/CD16, CD123/CD3, CD19/CD16, PD-1/PD-L1 , PD-1/CD20, PD-1 /CD16, PD-1/CD33 or PD-1/CD123.

In the following table some possible combinations of domains that can be present (with one or more hinge region(s) and optionally with one or more linkers) in the antibody-like molecules of the invention are reported:

Antibody-like V_LA, CLA, V_HA, CHA V_LB, CLB, V_HB, CHB

Antigens A and B molecule taken from: taken from:

1 adalimumab canakinumab TNF-alpha + IL-1 beta

2 golimumab canakinumab TNF-alpha + IL-1 beta

3 infliximab canakinumab TNF-alpha + IL-1 beta

4 certolizumab pegol canakinumab TNF-alpha + IL-1 beta

5 adalimumab rituximab TNF-alpha + CD20

6 golimumab rituximab TNF-alpha + CD20

7 infliximab rituximab TNF-alpha + CD20

8 certolizumab pegol rituximab TNF-alpha + CD20

9 adalimumab ofatumumab TNF-alpha + CD20 Antibody-like V_LA, CLA, V_HA, C_HA V_LB, C_LB, VHB, CHB

Antigens A and B molecule taken from: taken from:

10 golimumab ofatumumab TNF-alpha + CD20

1 1 infliximab ofatumumab TNF-alpha + CD20

12 certolizumab pegol ofatumumab TNF-alpha + CD20

13 belimumab ocrelizumab BLyS + CD20

14 tabalumab ocrelizumab BLyS + CD20

15 belimumab sifalimumab BLyS + IFN-alpha

16 tabalumab sifalimumab BLyS + IFN-alpha

17 belimumab natalizumab BLyS + VLA4

18 tabalumab natalizumab BLyS + VLA4

19 natalizumab ocrelizumab VLA4 + CD20

20 bevacizumab trastuzumab VEGF + HER2

21 bevacizumab cetuximab VEGF + EGFR

22 bevacizumab nimotuzumab VEGF + EGFR

23 bevacizumab tocilizumab VEGF + IL-6R

24 tocilizumab adalimumab IL-6R + TNF-alpha

25 tocilizumab golimumab IL-6R + TNF-alpha

26 tocilizumab infliximab IL-6R + TNF-alpha

27 tocilizumab certolizumab pegol IL-6R + TNF-alpha

28 olokizumab adalimumab IL-6 + TNF-alpha

29 olokizumab golimumab IL-6 + TNF-alpha

30 olokizumab infliximab IL-6 + TNF-alpha

31 olokizumab certolizumab pegol IL-6 + TNF-alpha

32 siltuximab rituximab IL-6 + CD20

33 sirukumab belimumab IL-6 + BLyS

34 basiliximab rituximab IL-2RA + CD20

35 daclizumab rituximab IL-2RA + CD20

36 basiliximab muromonab-CD3 IL-2RA + CD3

37 daclizumab muromonab-CD3 IL-2RA + CD3

38 omalizumab pascolizumab IgE + IL-4

39 omalizumab bertilimumab IgE + Eotaxin-1

40 zanolimumab bevacizumab CD4 + VEGF

41 fresolimumab carlumab TGF-beta + MCP-1

GM-CSF receptor + TNF-

42 mavrilimumab adalimumab

alpha

43 mavrilimumab golimumab GM-CSF receptor + TNF- Antibody-like V_LA, C_LA, VHA, CHA V_LB, C_LB, VHB, CHB

Antigens A and B molecule taken from: taken from:

alpha

GM-CSF receptor + TNF-

44 mavrilimumab infliximab

alpha

GM-CSF receptor + TNF-

45 mavrilimumab certolizumab pegol

alpha

46 alemtuzumab rituximab CD52 + CD20

47 ipilimumab bevacizumab CTLA-4 + VEGF

In one embodiment, the hinge region of the antibody-like molecule of the invention can, for example, be selected from the following list: hinge region of human lgG1 , hinge region of human lgG2, hinge region of human lgG3, hinge region of human lgG4, hinge region of human lgA1 , hinge region of human lgA2, hinge region of human IgD, hinge region of human IgE, hinge region of human IgM and any of the above hinge region comprising at least one unnatural amino acid. Preferably, the hinge region is that of human lgG3, IgD or IgE.

In one embodiment, the linker of the antibody-like molecule of the invention is a flexible or a rigid linker. Preferably the linker is not cleavable in vivo (e.g. protease resistant). The linker can, for example, be selected from the following list: "GS" linker such as (GGGGS)₂, (GGGGS)₃, (GGGGS)₄, (GGGGS)₅ and (GGGGS)₆; GSGSGS; GGGGSLVPRGSGGGGS; GGSGGHMGSGG; KESGSVSSEQLAQFRSLD; EGKSSGSGSESKST; (Gly)₄, (Gly)₆, (Gly)₈, (Gly)i₀; GSAGSAAGSGEF; (EAAAK)_n (n=1 , 2, 3, 4 or 5); A(EAAAK)_nA (n = 1 , 2, 3, 4, or 5); [A(EAAAK)_nA]m (n = 2, 3 or 4, m = 1 or 2); (Ala-Pro)₇ and PAPAP. Preferably, the linker is a "GS" linker.

A further embodiment of the present invention is an isolated nucleic acid molecule encoding any of the antibody-like molecule above or below described, or a complementary strand or degenerate sequence thereof. In this regard, the term "nucleic acid molecule" encompasses all different types of nucleic acids, including without limitation deoxyribonucleic acids (e.g., DNA, cDNA, gDNA, synthetic DNA, etc.), ribonucleic acids (e.g., RNA) and peptide nucleic acids (PNA). In a preferred embodiment, the nucleic acid molecule is a DNA molecule, such as a double-stranded DNA molecule or a cDNA molecule. The term "isolated" means nucleic acid molecules that have been identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the specific nucleic acid molecule as it exists in natural cells. A degenerate sequence designates any nucleotide sequence encoding the same amino acid sequence as a reference nucleotide sequence, but comprising a distinct nucleotide sequence as a result of the genetic code degeneracy.

In another embodiment, the invention provides a nucleic acid molecule coding for one or more amino acid sequences of an antibody-like molecule described herein. In one embodiment the nucleic acid molecule codes for the amino acid sequence defined in a) (also referred to as chain a)). In one embodiment the nucleic acid molecule codes for the amino acid sequence defined in b) (also referred to as chain b)). In one embodiment the nucleic acid molecule codes for the amino acid sequence defined in c) (also referred to as chain c)). In one embodiment the nucleic acid molecule codes for the amino acid sequence defined in a), b) and c). In one embodiment the nucleic acid molecule codes for the amino acid sequence defined in a) and b), in a) and c) or in b) and c).

A further embodiment of this invention is a vector comprising any of the nucleic acid molecules above or below described. The vector may be any cloning or expression vector, integrative or autonomously replicating, functional in any prokaryotic or eukaryotic cell. In particular, the vector may be a plasmid, cosmid, virus, phage, episome, artificial chromosome, and the like. The vector may comprise the coding sequences for the amino acid sequences defined in a), b) and c) or for only one or two of them, the remaining being present in a second vector. In one embodiment the vector comprises a first promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequence defined in a) and a second promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequence defined in b) and of a nucleic acid molecule coding for the amino acid sequence defined in c). The promoters may be the same or different and may be oriented on the on the same or on opposite directions. The nucleic acid molecules coding for the amino acid sequence defined in b) and for that defined in c), being under the control of the same promoter, may preferably be separated by an internal ribosomal entry site (IRES). In another embodiment the vector comprises a first promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequence defined in a), a second promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequence defined in b) and a third promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequence defined in c). The promoters may be the same or different and may be oriented on the on the same or on opposite directions. In another embodiment the vector comprises a single promoter which controls the expression of a nucleic acid molecule coding for the amino acid sequences defined in a), in b) and in c). The nucleic acid molecules coding for the amino acid sequence defined in a), for that defined in b), and for that defined in c), being under the control of the same promoter, may preferably be separated by an IRES. The IRES between the nucleic acid molecule coding for the amino acid sequence defined in a) and for that defined in b) and the IRES between the nucleic acid molecule coding for the amino acid sequence defined in b) and for that defined in c) may be the same or different.

Suitable promoters for eukaryotic gene expression are, for example, promoters derived from viral genes such as the murine or human cytomegalovirus (CMV), the mouse bi-directional CMV promoter or the rous sarcoma virus (RSV) promoter, which are well known to the person skilled in the art. The vector may comprise regulatory elements, such as a promoter, terminator, enhancer, selection marker, origin of replication, insulator, intron etc. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

A further embodiment of the present invention is a host cell, wherein said cell comprises a nucleic acid molecule or a vector as defined above. The nucleic acid molecule or the vector may be inserted with any of the technique which are known to the skilled artisan, e.g. transfection, transformation, transduction, electroporation, etc.

The host cell may be a prokaryotic or eukaryotic cell. Examples of prokaryotic cells include bacteria, such as E.coli. Examples of eukaryotic cells are yeast cells, plant cells, mammalian cells and insect cells including any primary cell culture or established cell line (e.g., 3T3, Vera, HEK293, TN5, etc.). Suitable host cells for the expression of glycosylated proteins are derived from multicellular organisms. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. Particularly preferred mammalian cells of the present invention are CHO cells, e.g. CHO-S, CHO-DG44, CHO-K1 , CHO-K1 sv or CHO Pro-5 cells. One of the advantages of the antibody-like molecules of this invention is that they can be expressed in E. coli since in most of their applications glycosylation is not essential. Depending on the final needs, the above host cells can be used to produce either transient or stable cell lines.

Another embodiment of this invention is therefore a method of producing an antibody-like molecule of the present invention, the method comprising culturing a host cell of the invention under conditions allowing expression of the nucleic acid molecule, and recovering/isolating the antibody-like molecule produced. The antibody-like molecules of the present invention may be produced by any technique known in the art, such as by recombinant technologies, chemical synthesis, cloning, ligations, or combinations thereof. The antibody-like molecules produced may be glycosylated or not, or may contain other post-translational modifications depending on the host cell type used. Many books and reviews provide teachings on how to clone and produce recombinant proteins using vectors and prokaryotic or eukaryotic host cells. The method of producing an antibody-like molecule of the present invention may further comprise the step of formulating the antibody into a pharmaceutical composition.

A further embodiment of the present invention is therefore a pharmaceutical composition comprising the antibody-like molecule according to the invention. Preferably, said pharmaceutical composition may further comprise additional excipients, such as buffer, stabilizer, surfactant, etc. Pharmaceutical compositions according to the invention are useful in the diagnosis, prevention, and/or treatment (local or systemic) of one and/or multiple diseases. The term "treatment" within the context of this invention refers to any beneficial effect on progression of disease, including attenuation, reduction, decrease or diminishing of the pathological development after onset of disease. The pharmaceutical compositions of the invention may be administered with a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" is meant to encompass any carrier, which does not interfere with effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered. For example, for parenteral administration, the active protein(s) may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.

In another aspect, the invention provides a pharmaceutical composition according to the invention for use as a medicament. In another aspect, the invention provides a method of treating one and/or more diseases in a patient, comprising administering to the patient a pharmaceutical composition according to the invention.

In another aspect, the invention provides an antibody-like molecule according to the invention for use as a medicament. In another aspect, the invention provides a method of treating one and/or more diseases in a patient, comprising administering to the patient an antibody-like molecule according to the invention.

In a first use according to the invention, a pharmaceutical composition according to the invention is administered pulmonary. In a second use according to the invention, a pharmaceutical composition according to the invention is administered intranasally. In a third use according to the invention, a pharmaceutical composition according to the invention is administered by inhalation. In a fourth use according to the invention, a pharmaceutical composition according to the invention is administered orally. In a fifth use according to the invention, a pharmaceutical composition according to the invention is administered intravenously or intramuscularly. In a preferred embodiment, in a use according to the invention, a pharmaceutical composition according to the invention is administered subcutaneously.

A pharmaceutical composition according to the invention is administered according to any one of the routes described above daily or every other day. For parenteral (e.g. intravenous, subcutaneous, intramuscular) administration, a pharmaceutical composition of the invention can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle (e.g. water, saline, dextrose solution) and additives that maintain isotonicity (e.g. mannitol) or chemical stability (e.g. preservatives and buffers). The formulation is sterilized by commonly used techniques.

The active ingredients of the pharmaceutical composition according to the invention can be administered to an individual in a variety of ways. The routes of administration may include intradermal, transdermal (e.g. in slow release formulations), intramuscular, intraperitoneal, intravenous, subcutaneous, epidural, topical, oral routes and by aerosol administration, intranasal route or inhaled. Any other therapeutically efficacious route of administration can be used, for example absorption through epithelial or endothelial tissues or by gene therapy wherein a DNA molecule encoding the active agent is administered to the patient (e.g. via a vector), which causes the active agent to be expressed and secreted in vivo. In addition, a pharmaceutical composition according to the invention can be administered together with other components of biologically active agents such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and vehicles.

The dosage administered to an individual will vary depending upon a variety of factors, including pharmacokinetic properties, the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.

The antibody-like molecules of the present invention can be produced, formulated, administered or used in other alternative forms that can be preferred according to the desired method of use and/or production. Useful conjugates or complexes can also be generated for improving the agents in terms of drug delivery efficacy. For this purpose, the antibody-like molecules described herein can be in the form of active conjugates or complex with molecules such as polyethylene glycol and other natural or synthetic polymers (Harris JM et al. 2003). In this regard, the present invention contemplates chemically modified antibody-like molecules, in which the antibody-like molecule is linked with a polymer. Typically, the polymer is water soluble so that the conjugate does not precipitate in an aqueous environment, such as a physiological environment. Moreover, a mixture of polymers can be used to produce the conjugates. The conjugates used for therapy can comprise pharmaceutically acceptable water- soluble polymer moieties. Suitable water-soluble polymers include polyethylene glycol (PEG), propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers. Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000. A conjugate can also comprise a mixture of such water-soluble polymers. Examples of conjugates comprise any of the antibody-like molecule disclosed here above and a polyalkyi oxide moiety attached to the N-terminus. PEG is one suitable polyalkyi oxide. As an illustration, any of the antibody-like molecules disclosed herein can be modified with PEG, a process known as "PEGylation". PEGylation can be carried out by any of the PEGylation reactions known in the art (Francis GE et al. 1998). Preferably, all these modifications do not affect significantly the ability of the antibody-like molecules to bind the antigens.

The present invention also includes antibody-like molecules that are functionally equivalent to those described above. Modified antibody-like molecules providing improved stability and/or therapeutic efficacy are also included. Examples of modified antibody-like molecules include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids which do not significantly deleteriously alter the antigen binding utility. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the therapeutic utility is maintained. Antibody-like molecules of the present invention can be modified post-translationally (e.g., acetylation, and phosphorylation) or can be modified synthetically (e.g., the attachment of a labeling group). It is understood that the antibody-like molecules designed by the present method may have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.

Antibody-like molecules of the present invention can include derivatives that are modified, for example, but not by way of limitation, the derivatives include antibody-like molecules, that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Additionally, the derivative may contain one or more non-classical and/or non-natural amino acids.

All references cited herein, including journal articles or abstracts, published or unpublished U.S. or foreign patent application, issued U.S. or foreign patents or any other references, are entirely incorporated by reference herein, including all data, tables, figures and text presented in the cited references.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various application such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning of a range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation.

Examples

The inventors have found that the format of the bispecific antibody-like molecules of the present invention allows inter alia a) obtaining molecules of a suitable size for therapeutic purposes, b) easy expression and purification and c) a very efficient production since lot of contaminants and side products are avoided. This last aspect is mainly due to the particular configuration of the chains forming the bispecific antibody-like molecules of the invention. Indeed, as shown in Figures 2 and 3, there is no possibility of mispairing, meaning that the "short" chain comprising VHA and CHA (chain b)) can only pair with that part of the "long" chain (chain a)) which comprises Vi_A and CLA and, similarly, the "short" chain comprising Vi_B and Ci_B (chain c)) can only pair with that part of the "long" chain (chain a)) comprising V_HB and CHB.

Furthermore, the inventors have surprisingly found that the bispecific antibody-like molecules of the invention having a format as that shown in Figure 2 are correctly folded and retain antigen binding specificity. This fact was unexpected since the N-C-terminal orientation, generally thought to be critical for a correct folding, of the V_HB and CHB domains is opposite to the standard one. Materials and general methods

General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991 ). Amino acids of antibody chains are numbered and referred to according to EU numbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991 )). Recombinant DNA techniques

Standard methods are used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents are used according to the manufacturer's instructions.

Gene synthesis Desired gene segments are prepared from oligonucleotides made by chemical synthesis. The gene segments, which are flanked by singular restriction endonuclease cleavage sites, are assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites e.g. Kpnl/ Sad or Ascl/Pacl into a pPCRScript (Stratagene) based pGA4 cloning vector. The DNA sequences of the subcloned gene fragments are confirmed by DNA sequencing. Gene synthesis fragments are ordered according to given specifications at Geneart (Regensburg, Germany). DNA sequence determination DNA sequences are determined by double strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH (Vaterstetten, Germany).

DNA and protein sequence analysis and sequence data management

The GCG's (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NTI Advance suite version 8.0 is used for sequence creation, mapping, analysis, annotation and illustration.

Expression vectors

For the expression of the described bispecific antibody-like molecule, variants of expression plasmids for transient expression (e.g. in HEK293 EBNA or HEK293- F) cells based either on a cDNA organization with or without a CMV-lntron A promoter or on a genomic organization with a CMV promoter are applied. Beside the expression cassettes the vectors contained an origin of replication which allows replication of this plasmid in E. coli, and a β-lactamase gene which confers ampicillin resistance in E. coli.

The nucleic acid sequences comprising the described chains as described below are generated by PCR and/or gene synthesis and assembled with known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective vectors. The subcloned nucleic acid sequences are verified by DNA sequencing. For transient transfections larger quantities of the plasmids are prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

Cell culture techniques

Standard cell culture techniques are used as described in Current Protocols in Cell Biology, Bonifacino, J. S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc. (2000).

Bispecific antibody-molecules are expressed by transient transfection of the expression vector in adherently growing HEK293-EBNA or in HEK29-F cells growing in suspension (Life Technologies) according to the manufacturer's instructions. Protein purification

Proteins are purified from filtered cell culture supernatants referring to standard protocols. In order to purify the bispecific antibody-like molecules a purification protocol based on Protein L column (GE healthcare) has been used.

Antigens binding ELISA

The binding properties of the bispecific antibody-like molecules have been evaluated in ELISA assays adapted for antibody-like molecules missing the Fc regions.

Firstly, the ability to bind the two antigens individually, e.g. VEGF or EGFR, was tested and secondly, through a bridging ELISA the ability to bind the two antigens simultaneously was also evaluated.

Example 1 : Production of bispecific antibody-like molecules format shown in Figure 2

In this example, we have prepared a bispecific antibody-like molecule directed to the two antigens

VEGF and EGFR and having bivalency vis-a-vis each of said antigen.

Specifically we have prepared the following three different nucleic acid sequences:

"chain a)": a nucleic acid sequence coding for the light chain variable domain of bevacizumab (V_LA), the light chain constant domain of bevacizumab (Ci_A), the hinge region of bevacizumab (HA), the hinge region of cetuximab (H_B), the heavy chain constant domain 1 of cetuximab (CHB) and the heavy chain variable domain of cetuximab (V_HB) ;

- "chain b)": a nucleic acid sequence coding for the heavy chain variable domain of bevacizumab (V_HA) and the heavy chain constant domain 1 of bevacizumab (CHA) ;

- "chain c)": a nucleic acid sequence coding for the light chain variable domain of cetuximab (V_LB) and the light chain constant domain of cetuximab (Ci_B).

These nucleic acid sequences have been inserted in an expression vector; the vector has been then transfected using the HEK293-F system (Life Technologies); the resulting molecules purified via a Protein L protocol (GE Healthcare) and the binding properties evaluated via ELISA according to the methods described above.

The inventors have found that there were no side products and, surprisingly, that the bispecific antibody-like molecules produced were able to bind the two antigens individually as well as simultaneously. This means that all domains of the molecules were able to correctly fold and retain their binding properties despite the "unsusal" N-C-terminal orientation, as explained above.

Example 2: Production of bispecific antibody-like molecules format shown in Figure 3

VEGF and EGFR and having bivalency vis-a-vis each of said antigen.

"chain a)": a nucleic acid sequence coding for the light chain variable domain of bevacizumab (V_LA), the light chain constant domain of bevacizumab (Ci_A), the hinge region of bevacizumab (HA), the hinge region of cetuximab (H_B), a "GS" linker (i.e. (GGGGS)4), the heavy chain variable domain of cetuximab (V_HB) and the heavy chain constant domain 1 of cetuximab (CHB) ;

- "chain c)": a nucleic acid sequence coding for the light chain variable domain of cetuximab (V_LB) and the light chain constant domain of cetuximab (Ci_B). These nucleic acid sequences have been inserted in an expression vector; the vector has been then transfected using the HEK293-F system (Life Technologies); the resulting molecules purified via a Protein L protocol (GE Healthcare) and the binding properties evaluated via ELISA according to the methods described above.

The inventors have found that there were no side products and that the bispecific antibody-like molecules produced were able to bind the two antigens individually as well as simultaneously. This means that all domains of the molecules were able to correctly fold and that the linker was able to provide enough flexibility to the molecule to bind to EGFR.

The skilled person would certainly note that the format shown in Figure 3, and produced in this example, can be easily engineered to include an Fc region or part thereof in those cases where the effector functions are desirable.

Example 3: Generation of a stable cell line for manufacturing purposes

In the examples above the cells have been transiently transfected in order to produce the molecules of interest for testing purposes. In those cases where large amount of molecules are needed, for example for manufacturing purposes, the generation of a stable cell line, e.g. CHO, is a necessary step.

The skilled person would appreciate that well-known and standard protocols are now part of the state of the art and, therefore, there is no need to describe them in details here. Just as examples, the protocols and kits commercialized by Life Technologies, Lonza or Sigma Aldrich can be used to generate stable CHO cell lines for manufacturing any bispecific antibody-like molecule of the present invention.

Claims

An antibody-like molecule capable of binding two different antigens A and B wherein said antibody-like molecule comprises the following chains:

a) an amino acid sequence comprising a light chain variable domain (V_LA), a light chain constant domain (Ci_A), one or more hinge regions, an heavy chain constant domain (CHB) and an heavy chain variable domain (V_HB) , wherein V_LA and V_HB are directed respectively to antigen A and antigen B;

b) an amino acid sequence comprising an heavy chain variable domain (V_HA) and an heavy chain constant domain (CHA), which are different from V_HB and CHB of a), wherein V_HA is directed against the same antigen A as V_LA; and

c) an amino acid sequence comprising a light chain variable domain (V_LB) and a light chain constant domain (Ci_B), which are different from V_LA and Ci_A of a), wherein V_LB is directed against the same antigen B as V_HB; and

wherein, in the amino acid sequence defined in a), the one or more hinge regions connect(s) Ci_A with CHB either directly or through one or more linkers .

The antibody-like molecule according to claim 1 , wherein the amino acid sequence defined in a) comprises only one hinge region.

The antibody-like molecule according to claim 1 , wherein the amino acid sequence defined in a) comprises two hinge regions, which are identical or different.

The antibody-like molecule according to any of the previous claims, wherein the amino acid sequence defined in a) further comprises one or more linkers.

The antibody-like molecule according to any of the previous claims, which comprises two amino acid sequences as defined in a), two amino acid sequences as defined in b) and two amino acid sequences as defined in c).

A nucleic acid molecule coding for any of the following:

a. the amino acid sequence defined in claim 1 a) or in any of claims 2-4;

b. the amino acid sequence defined in claim 1 b); or

c. the amino acid sequence defined in claim 1 c).

A vector comprising a nucleic acid molecule according to claim 6.

The vector according to claim 7, which comprises a first promoter, which controls the expression of the nucleic acid molecule according to claim 6a, and a second promoter, which controls the expression of the nucleic acid molecules according to claims 6b and

6c.

A host cell wherein the vector according to claim 7 or 8 has been inserted.

10. The host cell according to claim 9, wherein said host cell is selected from the group consisting of bacterial, yeast or mammalian cells.

1 1 . The host cell according to claim 9, wherein said host cell is an E. coli cell, a CHO cell or a HEK293 cell.

12. A method for making the antibody-like molecule according to any of claims 1 to 5, wherein said method comprises culturing the host cell according to any of claims 9 to 1 1 and isolating said antibody-like molecule.

13. A pharmaceutical composition comprising the antibody-like molecule according to any of claims 1 to 5 or the antibody-like molecule produced according to the method of claim 12.

14. The antibody-like molecule according to any of claims 1 to 5, the antibody-like molecule produced according to the method of claim 12, or the pharmaceutical composition according to claim 13 for use as a medicament.