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Title:  V-like domain binding molecules
United States Patent: 
January 23, 2007

Galanis; Maria (Waverley, AU), Hudson; Peter John (Blackburn, AU), Irving; Robert Alexander (Mulgrave, AU), Nuttall; Stewart Douglas (Ivanhoe, AU)
Diatech Pty. Ltd. (Queensland, AU)
Appl. No.:  09/623,611
March 5, 1999
PCT Filed: 
March 05, 1999
PCT No.: 
371(c)(1),(2),(4) Date: 
October 06, 2000
PCT Pub. No.: 
PCT Pub. Date: 
September 10, 1999


Executive MBA in Pharmaceutical Management, U. Colorado


The present invention relates to binding moieties comprising at least one monomeric V-like domain (VLD) derived from a non-antibody ligand, the at least one monomeric V-like domain being characterized in that at least one CDR loop structure or part thereof is modified or replaced such that the solubility of the modified VLD is improved when compared with the unmodified VLD.


The present invention relates to the design of novel soluble VLD binding molecules derived from the V-like domain of immunoglobulin superfamily members, such as the human CTLA-4 molecule. The preferred binding molecules of the present invention provide the following advantages (i) use of a native human protein obviates the need for subsequent humanisation of the recombinant molecule, a step often required to protect against immune system response if used in human treatment; (ii) the domain is naturally monomeric as described above (incorporation of residue Cys120 in a C-terminal tail results in production of a dimeric molecule); and (iii) structural modifications have resulted in improved E. coli expression levels.

Prior to publication of the first CTLA-4 structure determination, available sequence data and mutational analyses of both this molecule and CD28 were analysed. This allowed modelling and prediction of the regions corresponding to antibody CDR1, 2 and 3 regions. It was hypothesised that such areas would be susceptible to mutation or substitution without substantial effect upon the molecular framework and hence would allow expression of a correctly folded molecule. The subsequently published structure (Metzler et al. 1997) showed these predictions to be accurate, despite the unexpected separation of CDR1 from the ligand-binding site, and the extensive bending of CDR3 to form a planar surface contiguous with the ligand binding face.

In an initial set of experiments the V-like domain of the human CTLA-4 molecule was modified by replacement of CDR loop structures with either of two defined polypeptides. The two polypeptides were human somatostatin (Som) and a portion of the human influenza virus haemagglutinin protein (HA-tag). Somatostatin (SRIF: somatotropin release-inhibiting factor) is a 14 residue polypeptide comprising a disulphide bond that forces the central 10 residues into a loop. Human somatostatin is biologically widespread within the body and mediates a number of diverse physiological functions such as regulation of growth hormone secretion etc (Reisne, 1995). Human somatostatin binds a number of specific receptors (there are at least five subtypes) which have differing tissue specificities and affinities (Schonbrunn et al. 1995). These aspects of binding and activation are as yet poorly understood, but one salient feature is the high density of somatostatin receptors present on a number of cancerous cell lines, for example cancers of the neuro-endocrine system and small lung cancers (Reubi 1997). Artificial analogues of somatostatin have been produced for imaging of such tumours which are resistant to degradation compared with the highly labile somatostatin polypeptide.

The haemagglutinin epitope sequence consists of the 9 residues YPYDVPDYA (SEQ ID NO: 63). A commercially produced antibody is available which specifically recognises this sequence. The epitope tag can be detected when randomly or directionally incorporated within the structure of proteins (Canfield et al. 1996).

Replacement of one or more CDR loop structures in the CTLA-4 V-like domain with somatostatin or the HA-tag resulted in the production of soluble, monomeric, unglycosylated binding molecules using different bacterial expression systems. This surprising finding shows that V-like domains provide a basic framework for constructing soluble, single domain molecules, where the binding specificity of the molecule may be engineered by modifications of the CDR loop structures.

The basic framework residues of the V-like domain may be modified in accordance with structural features present in camelid antibodies. The camel heavy chain immunoglobulins differ from "conventional" antibody structures by consisting of only a single VH domain (Hamers-Casterman et al. 1993). Several unique features allow these antibodies to overcome the dual problems of solubility and inability to present a sufficiently large antigen binding surface.

First, several non-conventional substitutions (predominantly hydrophobic to polar in nature) at exposed framework residues reduce the hydrophobic surface, while maintaining the internal beta-sheet framework structure (Desmyter et al. 1996). Further, within the three CDR loops several structural features compensate for the loss of antigen binding-surface usually provided by the VL domain. While the CDR2 loop does not differ extensively from other VH domains, the CDR-1 and -3 loops adopt non-canonical conformations which are extremely heterologous in length. For example, the H1 loop may contain anywhere between 2 8 residues compared to the usual five in Ig molecules. However, it is the CDR3 loop which exhibits greatest variation: in 17 camel antibody sequences reported, the length of this region varies between 7 and 21 residues (Muyldermans et al. 1994). Thirdly, many camelid VH domains possess a disulphide linkage interconnecting CDRs-1 and -3 in the case of camels and interconnecting CDRs-1 and -2 in the case of llamas (Vu et al. 1997). The function of this structural feature appears to be maintenance of loop stability and providing a more contoured, as distinct from planar, loop conformation which both allows binding to pockets within the antigen and gives an increased surface area. However, not all camelid antibodies possess this disulphide bond suggesting that it is not an absolute structural requirement.

These foregoing features have enabled camelid V-domains to present as soluble molecules in vivo and with sufficiently high affinity to form an effective immune response against a wide variety of target antigens. In contrast, cell surface receptors of the Ig superfamily (such as CD4 and CD2) comprise V-like binding domains and appear to bind cognate receptors with surface features other than the CDR loops. These V-like domains bind to cognate receptors with very low affinity. Physiological signalling between two cells are mediated by the avidity of multi-point binding, when two cell surfaces connect and each contains multiple receptors. CD2 is a well-characterised example: binding to CD58 is mediated by a highly constrained set of minor electrostatic interactions generated by charged and polar residues located in the GFCC'C''(SEQ ID NO: 141) face (not the antibody type CDR-1, CDR-2 or CDR-3 loops). This results in a low affinity but highly specific interaction (Bodian et al 1994).

The present invention also relates to a method for generating and selecting single VLD molecules with novel binding affinities for target molecules. This method involves the application of well known molecular evolution techniques to V-like domains derived from members of the immunoglobulin superfamily. The method may involve the production of phage or ribosomal display libraries for screening large numbers of mutated V-like domains.

Filamentous fd-bacteriophage genomes are engineered such that the phage display, on their surface, proteins such as the Ig-like proteins (scFv, Fabs) which are encoded by the DNA that is contained within the phage (Smith, 1985; Huse et al., 1989; McCafferty et al., 1990; Hoogenboom et al., 1991). Protein molecules can be displayed on the surface of Fd bacteriophage, covalently coupled to phage coat proteins encoded by gene III, or less commonly gene VIII. Insertion of antibody genes into the gene III coat protein give expression of 3 5 recombinant protein molecules per phage, situated at the ends. In contrast, insertion of antibody genes into gene VIII has the potential to display about 2000 copies of the recombinant protein per phage particle, however this is a multivalent system which could mask the affinity of a single displayed protein. Fd phagemid vectors are also used, since they can be easily switched from the display of functional Ig-like fragments on the surface of Fd-bacteriophage to secreting soluble Ig-like fragments in E. coli. Phage-displayed recombinant protein fusions with the N-terminus of the gene III coat protein are made possible by an amber codon strategically positioned between the two protein genes. In amber suppressor strains of E. coli, the resulting Ig domain-gene III fusions become anchored in the phage coat.

A selection process based on protein affinity can be applied to any high-affinity binding reagents such as antibodies, antigens, receptors and ligands (see, for example, Winter and Milstein, 1991, the entire contents of which are incorporated herein by reference). Thus the selection of the highest affinity binding protein displayed on bacteriophage is coupled to the recovery of the gene encoding that protein. Ig-displaying phage can be affinity selected by binding to cognate binding partners covalently coupled to beads or adsorbed to plastic surfaces in a manner similar to ELISA or solid phase radioimmunoassays. While almost any plastic surface will adsorb protein antigens, some commercial products are especially formulated for this purpose, such as Nunc Immunotubes.

Ribosomal display libraries involve polypeptides synthesized de novo in cell-free translation systems and displayed on the surface of ribosomes for selection purposes (Hanes and Pluckthun, 1997; He and Taussig, 1997). The "cell-free translation system" comprises ribosomes, soluble enzymes required for protein synthesis (usually from the same cell as the ribosomes), transfer RNAs, adenosine triphosphate, guanosine triphosphate, a ribonucleoside triphosphate regenerating system (such as phosphoenol pyruvate and pyruvate kinase), and the salts and buffer required to synthesize a protein encoded by an exogenous mRNA. The translation of polypeptides can be made to occur under conditions which maintain intact polysomes, i.e. where ribosomes, mRNA molecule and translated polypeptides are associated in a single complex. This effectively leads to "ribosome display" of the translated polypeptide.

For selection, the translated polypeptides, in association with the corresponding ribosome complex, are mixed with a target molecule which is bound to a matrix (e.g. Dynabeads). The target molecule may be any compound of interest (or a portion thereof) such as a DNA molecule, a protein, a receptor, a cell surface molecule, a metabolite, an antibody, a hormone or a virus. The ribosomes displaying the translated polypeptides will bind the target molecule and these complexes can be selected and the mRNA re-amplified using RT-PCR.

Although there are several alternative approaches to modify binding molecules the general approach for all displayed proteins conforms to a pattern in which individual binding reagents are selected from display libraries by affinity to their cognate receptor. The genes encoding these reagents are modified by any one or combination of a number of in vivo and in vitro mutation strategies and constructed as a new gene pool for display and selection of the highest affinity binding molecules.

Claim 1 of 22 Claims

1. A modified monomeric non-antibody ligand V-like domain (VLD) comprising within the VLD at least one CDR loop structure or part thereof that is modified or replaced such that (i) the size of the CDR loop structure or part thereof is increased by at least one amino acid residue when compared with the corresponding CDR loop structure or part thereof in an unmodified VLD; and/or (ii) the modification or replacement results in formation of a disulphide bond within or between one or more of the CDR loop structures, wherein the CDR loop structure is a surface polypeptide loop structure corresponding to a complementarity determining region of an antibody V-domain, and wherein the non-antibody ligand is selected from the group consisting of CTLA-4, CD28 and ICOS.


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