The present invention relates to multivalent F.sub.vantibody construct having at least four variable domains which are linked with each over via the peptide linkers 1, 2 and 3. The invention also concerns expression plasmids which code for such an F.sub.vantibody construct and a method of producing the F.sub.vantibody constructs as well as their use.

SUMMARY OF THE INVENTION

The present invention relates to a multivalent F.sub.vantibody construct having at least four variable domains which are linked with each over via the peptide linkers 1, 2 and 3. The invention also concerns expression plasmids which code for such an F.sub.vantibody construct and a method of producing the F.sub.vantibody constructs as well as their use.

DETAILED DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide an antibody by means of which undesired immune responses can be avoided. Furthermore, it shall have a stability which makes it usable for therapeutic uses.

Therefore, the subject matter of the present invention relates to a multivalent F.sub.v antibody construct which has great stability. Such a construct is suitable for diagnostic and therapeutic purposes.

The present invention is based on the applicant's insights that the stability of an F.sub.v antibody construct can be increased if it is present in the form of a single-chain dimer where the four variable domains are linked with one another via three peptide linkers. The applicant also recognized that the F.sub.v antibody construct folds with itself when the middle peptide linker has a length of about 10 to 30 amino acids. The applicant also recognized that the F.sub.v antibody construct folds with other F.sub.v antibody constructs when the middle peptide linker has a length of about up to 10 amino acids so as to obtain a multimeric, i.e., multivalent, F.sub.v antibody construct. The applicant also realized that the F.sub.v antibody construct can be multispecific.

According to the invention the applicant's insights are utilized to provide a multivalent F.sub.v antibody construct which comprises at least four variable domains which are linked with one another via peptide linkers 1, 2 and 3.

The expression "F.sub.v antibody construct" refers to an antibody which has variable domains but no constant domains.

The expression "multivalent F.sub.v antibody construct" refers to an F.sub.Vantibody which has several, but at least four, variable domains. This is achieved when the single-chain F.sub.v antibody construct folds with itself so as to give four variable domains, or folds with other single-chain F.sub.v antibody constructs. In the latter case, an F.sub.v antibody construct is given which has 8, 12, 16, etc., variable domains. It is favorable for the F.sub.v antibody construct to have four or eight variable domains, i.e., it is bivalent or tetravalent (FIG. 1). Furthermore, the variable domains may be equal or differ from one another, so that he antibody construct recognizes one or several antigens. The antibody construct preferably recognizes one or two antigens, i.e., it is monospecific and bispecific, respectively. Examples of such antigens are proteins CD19 and CD3.

The expression "peptide linkers 1, 3" refers to a peptide linker adapted to link variable domains of an F.sub.v antibody construct with one another. The peptide linker may contain any amino acids, the amino acids glycine (G), serine (S) and proline (P) being preferred. The peptide linkers 1 and 3 may be equal or differ from each other. Furthermore, the peptide linker may have a length of about 0 to 10 amino acids. In the former case, the peptide linker is only a peptide bond from the COOH residue of one of the variable domains and the NH.sub.2 residue of another of the variable domains. The peptide linker preferably comprises the amino acid sequence GG.

The expression "peptide linker 2" refers to a peptide linker adapted to link variable domains of an F.sub.v antibody construct with one another. The peptide linker may contain any amino acids, the amino acids glycine (G), serine (S) and proline (P) being preferred. The peptide linker may also have a length of about 3 to 10 amino acids, in particular 5 amino acids, and most particularly the amino acid sequence GGPGS, which serves for achieving that the single-chain F.sub.v antibody constructs. The peptide linker can also have a length of about 11 to 20 amino acids, in particular 15 to 20 amino acids, and most particularly the amino acid sequence (G.sub.4S).sub.4, which serves for achieving that the single-chain F.sub.v antibody construct folds with itself.

An F.sub.v antibody constructs according to the invention can be produced by common methods. A method is favorable in which DNAs coding for the peptide linkers 1, 2 and 3 are ligated with DNAs coding for the four variable domains of an F.sub.v antibody construct such that the peptide linkers link the variable domains with one another and the resulting DNA molecule is expressed in an expression plasmid. Reference is made to Example 1 to 6. As to the expressions "F.sub.v antibody construct" and "peptide linker" reference is made to the above explanations and, by way of supplement, to Maniatis, T. et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, 1982.

DNAs which code for an F.sub.v antibody construct according to the invention also represent a subject matter of the present invention. Furthermore, expression plasmids which contain such DNAs also represent a subject matter of the present invention. Preferred expression plasmids are pDISC3x19-LL, pDISC3x19-SL, pPIC-DISC-LL, pPIC-DISC-SL, pDISC5-LL and pDISC6-SL. The first four were deposited with the DSMZ (Deutsche Sammlung fur Mikroorganismen und Zellen) [German-type collection for micro-organisms and cells] on Apr. 30, 1998 under DSM 12150, DSM 12149, DSM 12152 and DSM 12151, respectively.

Another subject matter of the present invention relates to a kit, comprising:

(a) an F.sub.v antibody construct according to the invention, and/or

(b) an expression plasmid according to the invention, and

(c) conventional auxiliary agents, such as buffers, solvents and controls.

One or several representatives of the individual components may be present.

The present invention provides a multivalent F.sub.v antibody construct where the variable domains are linked with one another via peptide linkers. Such an antibody construct distinguishes itself in that it contains no parts which can lead to undesired immune reactions. Furthermore, it has great stability. It also enables to bind several antigens simultaneously. Therefore, the F.sub.v antibody construct according to the invention is perfectly adapted to be used not only for diagnostic but also for therapeutic purposes. Such purposes can be seen as regards any disease, in particular a viral, bacterial or tumoral disease.
 

Claim 1 of 4 Claims

1. A bispecific tetravalent homodimeric F.sub.v antibody formed by two single-chain F.sub.v monomers, each of said F.sub.v monomers having at least four variable domains, wherein said four variable domains are V.sub.H-A, V.sub.L-A, V.sub.H-B and V.sub.L-B, wherein V.sub.H-A and V.sub.L-A are V.sub.H and V.sub.L domains of an antibody specific for antigen A, respectively, and V.sub.H-B and V.sub.L-B are V.sub.H and V.sub.L domains of an antibody specific for antigen B, respectively; V.sub.H-A is linked to V.sub.L-B by peptide linker 1, V.sub.L-B is linked to V.sub.H-B by peptide linker 2, V.sub.H-B is linked to V.sub.L-A by peptide linker 3; and said peptide linker 1 and said peptide linker 3 are a peptide bond or have about 1 to about 10 amino acids; and said peptide linker 2 has 3 to about 10 amino acids.
 

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