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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/26—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to the preparation of multivalent and multispecific binding proteins.
• the invention relates to the preparation of antigen binding proteins comprising a plurality of binding units linked in series by means of intervening polypeptide linker groups, the amino acid sequence of which linker groups confer restricted conformational flexibility.
• Antigen binding proteins which are multivalent that is, comprise more than one antigen binding site
• those which are also multispecific where the antigen binding sites have differing antigen specificities
• Other uses for which multivalent and multispecific binding proteins have been proposed include assays, such as immunoassays and agglutination assays, and purification processes.
• constructs comprising two or more polypeptide chains are described in WO 94/09131 (Scotgen Limited) and WO 97/14719 (Unilever) and WO 97/38102 (Unilever) ; multivalent molecules comprising two or more single chain Fv molecules linked together are described in WO 93/11161 (Enzon Inc.) and WO 94/13806 (Dow Chemical Co. ) .
• WO 94/04678 (Casterman et al) describes immunoglobulins capable of exhibiting the functional properties of classical, four chain, immunoglobulins but which comprise two heavy polypeptide chains only and are naturally devoid of light polypeptide chains. Fragments corresponding to isolated VH domains or to VH dimers linked by the hinge disulphide are also disclosed. These immunoglobulins, which may be isolated from Camelids, do not rely on the association of heavy and light chain variable domains for the formation of the antigen-binding site; instead, the heavy chain variable domain (hereinafter VHH) alone forms the complete antigen binding site, constituting a single domain binding site.
• VHH heavy chain variable domain
• WO 96/34103 Casterman et al disclose multivalent, multispecific constructs comprising VHH fragments combined with a linker sequence.
• Suitable linker sequences disclosed and exemplified are derived from sequences corresponding to the hinge domain of an immunoglobulin devoid of light chains.
• multivalent, multispecific antigen-binding proteins comprising a polypeptide comprising in series two or more single domain binding units which are preferably variable domains of a heavy chain derived from an immunoglobulin naturally devoid of light chains.
• the individual single domain binding units may suitably be linked by means of peptide linkers, preferably flexible peptide linkers, which allow the variable domains to flex in relation to each other with the aim of ensuring that they can bind to multiple antigenic determinants simultaneously.
• the invention provides the use of a polypeptide group, the amino acid sequence of which group confers restricted conformational flexibility, as a linking group to link binding units in a multivalent binding protein.
• the invention also provides a multivalent binding protein comprising a plurality of binding units linked by means of intervening polypeptide linker groups, the amino acid sequence of which linker group confers restricted conformational flexibility.
• the invention further provides a nucleotide sequence encoding a multivalent antigen binding protein according to the invention and cloning and expression vectors comprising such nucleotide sequences. Also provided are host cells transformed with vectors comprising such nucleotide sequences.
• a ⁇ multivalent binding protein' is a protein which has more than one binding units which allow for specific binding with a molecule partner in a binding pair. Included within this are bivalent, trivalent and so on. Examples of suitable binding units include antigen binding domains of antibodies, binding domains of receptors such as hormone receptors, lectins, enzymes, and cell adhesion molecules.
• a multivalent antigen binding protein' is a protein which has more than one antigen binding unit.
• An ⁇ antigen binding unit' is any structure which exhibits antigen-binding activity. This may be an antibody or an immunologically active fragment thereof.
• An ⁇ antibody' refers to an immunoglobulin which may be derived from natural sources or synthetically produced. Unless indicated otherwise, ⁇ antibody' and ⁇ immunoglobulin' are used synonymously throughout this specification.
• An antibody fragment is a portion of a whole antibody which retains the ability to exhibit antigen-binding activity.
• the antigen binding site may be formed through association of antibody light and heavy chain variable domains or may comprise individual antibody variable domains, constituting a single domain binding site.
• Suitable fragments include Fab (comprising an antibody light chain associated with the V H and C H ⁇ domains of an antibody heavy chain) , Fv (comprising the variable domains of antibody heavy and light chains associated with each other) and scFv (comprising an antibody V H domain linked to a VL domain by a flexible peptide linker) fragments.
• the antigen binding site comprises a single variable domain, this may be a heavy chain variable domain, most suitably a heavy chain variable domain derived from an immunoglobulin naturally devoid of light chains .
• Restricted conformational flexibility relates to restriction of movement of the antigen binding units about the backbone of the intervening polypeptide linker group.
• an antigen binding protein comprising two single binding units is hereinafter referred to as a ⁇ bi-head' .
• Figure 1 shows a nucleotide sequence of the Pstl-BstEII insert of plasmid pUR4640, encoding the heavy chain variable domain of an anti-RR6 antibody (denoted R9) from a llama.
• Figure 2 shows the nucleotide sequence of the Pstl-BstEII insert of plasmid pUR4601, encoding the heavy chain variable domain of an anti-hCG antibody (denoted H14) from a llama.
• Figure 3 shows a map of plasmid pUR4619.
• Figure 4 shows the nucleotide sequence within plasmid pUR4619 which encodes an anti-hCG-anti-RR6 bispecific biheaded antigen-binding protein (denoted HI4-R9) , missing the first 4 and last 3 amino acids.
• Figure 5 shows the A405 signals of an ELISA to determine bispecificity of various HI4-R9 biheads.
• Figure 6 shows the scores achieved in a rapid assay technology (RAT) format assay following the detection of 1 IU/ml hCG (human chorionic gonadotrophin protein) with various anti-hCG-anti-RR6 bihead antigen binding proteins derived from a llama wherein the anti-hCG and anti-RR6 fragments are linked as follows (see Example 1.5) : Number Linker
• Figure 7 shows a comparison of the sensitivity of detection of hCG in a RAT assay using various biheads (see Example 1.5) .
• the invention is based on the unexpected finding that by using a polypeptide linking group conferring restricted conformational flexibility to link together antigen binding units, multivalent antigen binding proteins having advantageous binding affinity, as demonstrated by their increased sensitivity of diagnosis and detection, are obtained. Furthermore, constructs according to the invention may conveniently be produced at high yields economically and efficiently on a scale appropriate for industrial use.
• the linking means has been provided by flexible peptide groups. Flexibility of conformation in the linker group has been considered desirable in order to allow the multivalent construct to assume the correct orientation to allow simultaneous binding of multiple antigens. Surprisingly, the present inventors have found that by restricting the conformational flexibility of the linking polypeptide group, multivalent antigen binding constructs having improved binding affinity may be obtained. This is entirely contrary to the teaching in the art that the linking group should desirably be flexible.
• the invention is applicable to the preparation of multivalent antigen binding constructs comprising antigen binding units where the antigen binding site is formed through association of antibody light and heavy chain variable domains.
• the constructs prepared according to the invention comprise a plurality of single domain binding units, more particularly a plurality of heavy chain variable domains derived from an immunoglobulin naturally devoid of light chains such as may be obtained from lymphoid cells, especially peripheral blood lymphocytes, bone marrow cells or spleen cells derived from Camelids as described in WO 94/04678 (Casterman et al) discussed above.
• heavy chain variable domains derived from other immunoglobulins modified ( ⁇ camelised' ) to enable them to function as single binding domains in the same way as the heavy chain variable domains derived from Camelids may also suitably be used according to the invention.
• Bivalent forms, that is having two antigen binding sites, of the multivalent antigen binding proteins prepared according to the invention are preferred but it will be appreciated that higher multivalent forms, which are also encompassed in the present invention, may find application under suitable circumstances, for example where more than two antigens are required to bind, for example in processes for scavenging molecules from solution or processes where close proximity of molecules form the basis of an assay.
• the linker group preferably comprises one or more proline residues.
• Linker groups comprising other sequence features which promote the formation of a beta-turn configuration in the peptide backbone, such as peptide linkers containing valine residues or constrained residues such as 8-bicyclic and 5,9-bicyclic tripeptide units
• peptide linker groups derived from naturally occurring proteins such as cell wall proteins (CWP) , in particular, CWP1, or cellobishydrolases (CBH) , such as CBH1P, which serve to restrict conformational flexibility or linker groups showing at least 50% homology thereto as determined by the ALIGN program of Dayhoff et al (1983), Methods Enzymol., 91, 524-545, may also suitably be used according to the invention.
• CWP cell wall proteins
• CBH cellobishydrolases
• Peptide linker groups which encode a glycosylation binding site and/or are resistant to proteolytic attack may also suitably be employed.
• the presence of a carbohydrate attached to the amino acid residues has the effect of restricting the flexibility of the peptide backbone.
• the polypeptide linking group according to the invention comprises from 4 to 30 amino acid residues, preferably from 5 to 15 amino acid residues.
• Preferred polypeptide linking groups according to the invention comprise an amino acid sequence selected from:
• A-T-T-T-G-S-S-P-G-P-T (SEQ. ID NO. 4)
• Multivalent antigen binding proteins according to the invention may be prepared by transforming a host by incorporating a gene encoding the polypeptide as set forth above and expressing said gene in said host.
• the host or hosts may be selected from prokaryotic bacteria, such as Gram-negative bacteria, for example E. coli, and Gram-positive bacteria, for example B. subtilis or lactic acid bacteria, lower eukaryotes such as yeasts, for example belonging to the genera Saccharomyces, Kluyveromyces , Hansenula or Pichia, or moulds such as those belonging to the genera Aspergillus or Trichoderma .
• prokaryotic bacteria such as Gram-negative bacteria, for example E. coli
• Gram-positive bacteria for example B. subtilis or lactic acid bacteria
• lower eukaryotes such as yeasts, for example belonging to the genera Saccharomyces, Kluyveromyces , Hansenula or Pichia
• moulds such as those belonging to the genera Aspergillus or Trichoderma .
• Preferred hosts for use in connection with the present invention are the lower eukaryotic moulds and yeasts.
• Proteins according to the invention may be recovered and purified using conventional techniques such as affinity chromatography, ion exchange chromatography or gel filtration chromatography.
• the activity of the multivalent binding proteins according to the invention may conveniently be measured by standard techniques known in the art such as enzyme-linked immunoadsorbant assay (ELISA) , radioimmune assay (RIA) or by using biosensors.
• ELISA enzyme-linked immunoadsorbant assay
• RIA radioimmune assay
• the anti-RR-6 response was measured by using Nunc Covalink plates, which where coated with the azo-dye. After incubation with (diluted) serum samples, the bound llama antibodies were detected via a incubation with poly-clonal rabbit-anti-llama antiserum (obtained via immunising rabbits with llama immunoglobulins which were purified via ProtA and ProtG columns; ID-DLO) , followed by an incubation with swine-anti-rabbit immunoglobulins (Dako) conjugated with alkaline phosphatase.
• acid guanidium thiocyanate extraction e.g. via the method described by Chomczynnski and Sacchi, Analytical Biochemistry, 162: 156-159 (1987).
• DNA fragments encoding HC-V fragments and part of the long or short hinge region were amplified by PCR using specific primers :
• Plasmids pUR4547 and pUR4548 are Saccharomyces cerevisiae episomal expression plasmids, derived from pSYl (Harmsen et al., Gene, 125: 115-123, (1993). From pSYl the Pstl site, located in front of the GAL7 promoter was removed after partial digestion with Pstl, incubation with Klenow fragment and subsequent blunt end ligation. After transformation the desired plasmid could be selected on the basis of restriction pattern analysis.
• the BstEII site in the Leu2 selection marker was removed by replacing the about 410bp Afill/PflMI fragment with a corresponding fragment in which the BstEH site was removed via a three step PCR mutagenesis, using the primers:
• P-flMI BOLI 1 5' -GGGAATTCCAATAGGTGGTTAGCAATCG (SEQ. ID NO. 9)
• AflH BOLI 2 5' -CCCCAAGCTTACATGGTCTTAAGTTGGCGT (SEQ. ID NO. 12)
• PCR-A was performed with primers BOLI 1 and BOLI 4 and resulted in an about 130bp fragment with the PflMI restriction site at the 3 ' -end and the inactivated BstEII site at the 5 '-end.
• PCR-B was performed with primers BOLI 2 and BOLI 3 and resulted in an about 290bp fragment with the Aflll site at the 5 '-end.
• the third PCR was with the fragments obtained from reaction A and B, together with the primers BOLI 1 and BOLI 2.
• the about l. ⁇ kb Sad-Hindi11 fragment was replaced with synthetic fragments, having sequences as presented below, resulting the plasmids pUR4547 and pUR4548, respectively.
• Both plasmids contain the GAL7 promoter and PGK terminator sequences as well as the invertase (SUC2) signal sequence.
• SUC2 invertase
• the DNA sequence encoding the SUC2 signal sequence is followed by the first 5 codons, (encoding Q-V-Q-L-Q) of the HC-V domain (including the Bstll site) , a stuffer sequence, the last six codons (encoding Q-V-T-V-S-S) of the HC-V domain.
• pUR4547 this is followed by two stop codons, an AfHI and HindiII site.
• pUR4548 this sequence is followed by eleven codons encoding the myc-tag, two stop codons, an AfHI and HindiII site.
• Plasmids pUR4547 and pUR4548 were deposited under the Budapest Treaty at the Centraal Bureau voor Schimmelcultures, Baarn on 18th August 1997 with deposition numbers: CBS 100012 and CBS 100013, respectively.
• EPC or a similar arrangement from a state not being a contracting state of the EPC, it is hereby requested that a sample of such deposit, when requested, will be submitted to an expert only.
• the about 6.4kb vector fragment was isolated and ligated with the Pstl-BstEII fragments of about 350bp obtained as described above.
• transformants were selected from minimal medium agar plates (comprising 0.7% yeast nitrogen base, 2% glucose and 2% agar, supplemented with the essential amino acids and bases) .
• R9 pUR4640 (see Figure 1) (SEQ. ID NO. 21-22)
• anti-hCG (alpha unit)
• the about 330bp fragments were purified via agarose gel electrophoresis and isolation from the gel. The fragments were cloned into pUR4421 (see Example 1 in WO 94/25591) which was digested with the same enzymes, resulting in pJS2 (H14) . Subsequently, the about 420bp Eagl -Hindi11 fragment of pJS2 was isolated and ligated in the about 6.6kb Eagl- HindiII vector fragment of the pSYl plasmid of which the Pstl and BstEII sites were removed as described above.
• the resulting plasmid pJS7 was digested with BstEII and Hindlll, after which the purified vector fragment was religated in the presence of a synthetic linker having the following sequence: (SEQ. ID NO. 29-31)
• plasmid pJS9 plasmid pS9.
• the plasmid was digested with Pstl and Hindlll, after which the purified vector fragments of about 7.0kb were ligated with the Pstl -Hindlll fragments of about 350bp of pUR4638 and pUR4640, encoding an anti-RR6 HC-V fragment denoted R9 followed by the myc-tail.
• the resulting S. cerevisiae episomal expression plasmid pUR4619 encodes a anti-hCG-anti-RR6 bispecific bihead preceded by the SUC2 signal sequence and followed by the myc-tail .
• BstEII, XhoI-BstEII fragments of about 0.7kb can be isolated and subsequently cloned into the vector fragment of pUR4547
• biheads can be obtained without the myc tail .
• expression vectors can be constructed in which different promoter systems, e.g. the constitutive GAPDH promoter or different signal sequences, e.g. the mating factor prepro sequence are used.
• transformants were selected from minimal medium agar plates as described in part b(ii) above.
• the transformants were grown overnight in selective minimal medium and subsequently diluted ten times in YPGal medium. After 24 and 48 hours of growth, samples were taken for Western blot analysis.
• monoclonal anti-myc antibodies were used, followed by incubation with poly-clonal rabbit-anti-mouse conjugate with alkaline phosphatase.
• the oligonucleotide linker fragments encode the last amino acid of the N-terminal HC-V fragment (S) and the first amino acid of the C-terminal HC-V fragment, intersected by the connecting linker peptide. This resulted in plasmids pUR5330 to 5334, respectively.
• Plasmid Linker level (mg/1) pUR4619 None 11 pUR5330 S-G-T-S-G-S-Q 36 pUR5331 S-S-S-S-A-S-A-S-S-A-Q 49 pUR5332 S-G-S-P-G-S-P-G-Q 33 pUR5333 S-A-T-T-T-G-S-S-P-G-P-T-Q 56 pUR5334 S-A-N-H-S-G-N-A-S-Q 51
• PINs coated with hCG were incubated with (diluted) medium samples. Subsequently, the PINs were incubated with a RR6- alkaline phosphatase conjugate, in which the azo-dye RR6 was coupled to the alkaline phosphatase via its reactive triazine group. Finally, the alkaline phosphatase enzyme activity was determined after incubation of the PINs with p-nitro-phenyl phosphate and the optical density was measured at 405nm (see Figure 5) .
• a 5 ml column of recombinant Protein A Fast Flow Sepharose (Amersham Pharmacia Biotech) was equilibrated by washing with 10 column volumes of wash buffer (10 mM potassium phosphate, pH 6) , at a flow rate of 2 ml/min.
• wash buffer (10 mM potassium phosphate, pH 6)
• the bi-head fermentation broth was loaded at 2 ml/min in an upwards direction.
• the column was washed with wash buffer until the OD 2 eo reached the baseline.
• Elution was carried out with a linear gradient of 0 - 40 mM citric acid pH 2.5 in the reverse direction, collecting 4 ml fractions into tubes containing 400 ⁇ l of a neutralising agent (1M Tris.Cl, pH 8.5) in order to minimise the effects of the acid.
• a neutralising agent (1M Tris.Cl, pH 8.5
• Peak fractions were checked for purity by running on a 12% SDS-PAGE Ready Gel (Bio-Rad) under standard denaturing conditions. Staining was with GelCode Blue (Pierce & Warriner) . The fractions were concentrated using Macrosep centrifugal concentrators (3 kDa molecular weight cut-off, Pall Filtron Corp.) then buffer exchanged into lOmM potassium phosphate, pH 6 using PD-10 columns (Amersham Pharmacia Biotech) . The final purity of the sample was determined by carrying out a UV scan from 400 - 220 nm and using the value at 280 nm to determine an accurate concentration. The samples were then aliquoted into vials, frozen, freeze dried and stored until required.
• a solution of Reactive Red 6 (RR6) was made up at 10 mg/ml in phosphate buffered saline (PBS) .
• a solution of bovine serum albumin (BSA) was made up at 10 mg/ml in PBS. 200 ⁇ l of the RR6 solution was added to 800 ⁇ l of the BSA solution and the resulting solution was mixed in an end over end rotary mixer for
• RR6 that had conugated to BSA was separated from free RR6 by addition of the reaction mixture (1 ml) to a PD10 column (Pharmacia) previously washed with 10 ml of PBS containing 0.1% sodium azide (PBSA) . The column was then eluted by addition of PBSA (5 ml) and 1 ml aliquots were collected. The RR6-BSA conjugate eluted in fractions 4 and 5.
• a 50 ⁇ l aliqot of Duke blue latex (10 % solids) was added and mixed by inverting.
• the diluted latex was centrifuged at 8000 g for 10 minutes at room temperature, the supernatant removed and the pellet vortexed briefly.
• the pellet was resuspended in 900 ⁇ l of buffer B and to this 100 ⁇ l of the previously prepared RR6-BSA conjugate was added.
• the latex solution was sonicated for 10 s using a sonic probe.
• the solution containing the latex was mixed for 2 h at room temperature and then centrifuged (8000 g, 10 min at room temperature) .
• the latex pellet was washed by resuspending in 1 ml of buffer B and centrifuged once more (8000 g, 10 min at room temperature) .
• the pellet was then resuspended in 1 ml buffer B ready for use.
• the llama bi-heads were tested by self assembling onto RR6-BSA adsorbed latex and detection of hCG in a rapid assay technology (RAT) format. This was performed by mixing the llama bi-head (5 ⁇ l of a 0.1 mg/ml solution) with RR6-BSA adsorbed latex (5 ⁇ l of 0.1 % solids) in 10 ⁇ l of PBSA to which hCG (5 ⁇ l of various concentrations) was added. The resulting solution was added to the bottom of a nitrocellulose strip (6 mm wide x 30 mm long) on which a monoclonal antibody recognising hCG had been adsorbed by plotting in a line (2.5 mg/ml) mid way up the strip.
• RAT rapid assay technology
• the latex- bi-head-hCG solution was allowed to flow up the nitrocellulose strip by capillary action and the strip was then washed by applying PBSA (25 ⁇ l) to the bottom of the strip.
• PBSA 25 ⁇ l
• Figure 6 shows that the llama bi-heads with linkers 3, 4 and 5 gave the highest response in RAT assays. These linkers are structurally more ordered than the comparative examples, flexible linkers 2 and 6 and result in more hCG and more latex captured in the assay. The more ordered linkers promote the correct orientation of the binding domains to achieve more optimal binding than when no linker is used.
• Linker 3 is derived from CWP1 and Linker 5 from CBH1P. Synthetic linkers with some order (linker 4 containing 2 proline residues) can offer increased sensitivity in assays than those with little order (linker 2) .
• Figure 7 shows that the bi-head with linker 4 can detect lower amounts (50 mlU/ml) of hCG than the bi-head with linker 2 and, hence, give a more sensitive assay for hCG.

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