Multivalent and multispecific binding proteins and their use

Details Multivalent and multispecific binding proteins and their use Multivalent and multispecific binding proteins and their use

1998-09-03

2002-12-10

Huff, Sheela (Department: 1642)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C530S387100, C530S387300, C424S135100

Reexamination Certificate

active

06492123

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to binding proteins, methods of making the proteins, and uses thereof. More particularly, the present invention relates to synthetic binding proteins which comprise immunoglobulin heavy and light chain variable domain binding regions. Polypeptides are provided which form multimers, eg dimers, which may have more than one binding specificity.
Because of the length of the specification, it is appropriate to include here a list of contents to help the reader find passages of interest. In addition to claims, figures and an abstract, the specification includes the following, in order:
Statement of the field of the invention;
Contents;
Brief discussion of prior art and introduction to the invention;
(Background)
Discussion of the invention;
Summary of the Invention
Brief description of the figures;
Discussion of bivalent and bispecific antibodies, preparation and uses;
Preparation of “diabodies”, discussion of structure and various utilities;
Discussion of construction of repertoires of diabodies and their display on bacteriophage;
Listing of the experimental examples;
Experimental examples and discussion;
All documents mentioned in this text are incorporated herein by reference.
BACKGROUND
Natural antibodies are multivalent, for example Ig G antibodies have two binding sites and IgM antibodies have five binding sites for antigen. The multivalency means that the antibodies can take advantage of multiple interactions in binding to solid phase antigen, and therefore increasing the avidity of binding to the antigen. It is possible to make recombinant bivalent IgG and pentameric decavalent IgM antibodies by expression in mammalian cells. To date, of the various possibilities for multivalency bivalent antibodies have been of greatest interest.
Of further interest are antibodies which are able to bind to two or more different epitopes, those which have multispecificity. Bispecific antibodies have many proven and expected utilities, discussed infra.
Structurally, the simplest antibody (IgG) comprises four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulphide bonds. The light chains exist in two distinct forms called kappa (K) and lambda (&lgr;). Each chain has a constant region (C) and a variable region (V). Each chain is organised into a series of domains. The light chains have two domains, corresponding to the C region and to the V region. The heavy chains have four domains, one corresponding to the V region and three domains (1, 2 and 3) in the C region.
The antibody has two arms (each arm being a Fab region), each of which has a VL and a VH region associated with each other. It is this pair of V regions that differs from one antibody to another (owing to amino acid sequence variations), and which together are responsible for recognising the antigen and providing an antigen binding site. In even more detail, each V region is made up from three complementarity determining regions (CDR) separated by four framework regions (FR). The CDRs are the most variable part of the variable regions, and they perform the critical antigen binding function.
It has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Example binding fragments are (i) the Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) the dAb fragment which consists of a VH domain; (v) isolated CDR regions; and (vi) F(ab′)
2
fragments, a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region.
The fragments largely represent portions of complete antibodies. However, the term “fragment” is also applied to synthetic molecules which comprise antibody heavy and light chain variable domains, or binding portions of these domains, associated so as to have specific antigen binding capability. A good example of an antibody fragment which is of this type is the “single chain Fv” (scFv) fragment which consists of an antibody heavy chain variable domain linked to an antibody light chain variable domain by a peptide linker which allows the two domains to associate to form a functional antigen binding site (see, for example U.S. Pat. No. 4,946,778, Ladner et al., (Genex); WO 88/09344, Creative Biomolecules, Inc/Huston et al.). WO 92/01047, Cambridge Antibody Technology et al./McCafferty et al., describes the display of scFv fragments on the surface of soluble recombinant genetic display packages, such as bacteriophage.
Some experimental work has been described wherein the length of the peptide linker of scFv molecules was varied. The range of linker lengths has in general been from 12 to 18 amino acids, with 15 amino acids being the most usual, see e.g. M C Whitlow et al.,
Protein Engineering
6: 989-995, 1993. In all cases the linker was long enough for the two domains, one VL, the other VH, to associate to form an antigen binding site, this being a characterising feature of scFv's. Condra et al., for example (
The Journal of Biological Chemistry
, 256:2292-2295, 1990) varied the length of the linker of scFv fragments able to block human rhinovirus attachment to cellular receptors. What the authors describe in this paper as a VL domain includes amino acids from the C domain (as can be seen from reviewing Kabat et al.,
Sequences of proteins of Immunological Interest
, US Government Printing Office), and these therefore molecules have “long” linkers between the variable domains, allowing the VL and VH domains in each molecule to associate to form an antigen binding site.
SUMMARY OF THE INVENTION
The present invention contributes further to the field of antibody fragments by providing polypeptides which are able to associate to form multivalent or multispecific multimers, particularly dimers. As discussed in greater detail below, in one aspect the present invention provides a polypeptide which comprises a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the domains being linked but incapable of associating with each other to form an antigen binding site. Such polypeptides are able to associate to form multimers, such as dimers, the heavy chain binding region of one polypeptide associating with the light chain binding region of another polypeptide to form an antigen binding site. In the case of dimerisation two antigen binding sites are formed.
The term “diabody” has been coined to describe dimers according to the present invention. This term has already gained some recognition in the art. When used in the present application, the term is not intended to be construed in general discussion as applying only to dimers, except where context demands so. Trimerisation of the polypeptides is feasible, three antigen binding sites then being formed. Also, the term is used in relation to both multivalent and multispecific multimers.
Each domain of the polypeptide may be a complete immunoglobulin heavy or light chain (as the case may be) variable domain, or it may be a functional equivalent or a mutant or derivative of a naturally occuring domain, or a synthetic domain constructed , for example, in vitro using a technique such as one described in WO 93/11236 (Medical Research Council et al./Griffiths et al.). For instance, it is possible to join together domains corresponding to antibody variable domains which are missing at least one amino acid. The important characterising feature is the ability of each domain to associate with a complementary domain to form an antigen binding site. Accordingly, the terms “immunoglobulin heavy/light chain variable region” should not be construed to exclude variants which do not have a material effect on how the invention works.
A “derivative” is a substance which is related to a polypeptide. The derivative may differ by the ad

Affiliated with

Also associated with

Rating

Multivalent and multispecific binding proteins and their use does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Multivalent and multispecific binding proteins and their use, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Multivalent and multispecific binding proteins and their use will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2976586