WO2006089678A2 - Antibodies and peptides binding to hiv tat, and uses thereof - Google Patents
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- WO2006089678A2 WO2006089678A2 PCT/EP2006/001404 EP2006001404W WO2006089678A2 WO 2006089678 A2 WO2006089678 A2 WO 2006089678A2 EP 2006001404 W EP2006001404 W EP 2006001404W WO 2006089678 A2 WO2006089678 A2 WO 2006089678A2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1072—Regulatory proteins, e.g. tat, rev, vpt
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates generally to compositions useful for assaying or treating human immunodeficiency virus-1 CHW-I) infection.
- HTV-I human immunodeficiency virus type 1
- RNA High plasma levels of human immunodeficiency virus type 1 (HIV-I) RNA are found during primary infection with HTV-I, the seroconversion illness (C. Bamberger et al, AIDS, 7:(suppl 2):S59 (1993); M. S. Saag et al, Nature Med., 2:625 (1996)), after which they subside as the immune response controls the infection to a variable extent.
- Post seroconversion lower but detectable levels of plasma HIV-I KNA are present and these levels rise with disease progression to again attain high levels at the AIDS stage (M. S. Saag et al, Nature Med., 2:265 (1996)).
- any intervention that inhibits HTV- 1 infectivity during initial infection and/or lowers viral load post .sero-conversion is likely to have a favourable influence on the eventual outcome, delaying or preventing progression to AIDS.
- H-V- 1 human immunodeficiency virus
- vaccines are under development for control of the spread of HTV-I to uninfected humans.
- this effort has largely been directed to proteins of the virus, expressed on the surface of infected cells, which are recognized by cytotoxic T cells with eHrnination of the infected cells, while free virus is blocked and cleared by antibody to surface antigens of the ⁇ rion.
- Limitations of this mode of vaccination are readily apparent for HTV-I. which has demonstrated a great diversity in immunogenic viral epitopes and rapid mutational variations that occur -within and between individuals (B. D. Preston et al. Science, 242:1168(1988): J.
- Tat protein is released extracellularly, making it available to be taken up by other infected cells to enhance transcription of HTV- 1 in the cells and to be taken up by noninfected cells, altering host cell gene activations and rendering the cells susceptible to infection by the virus. Uptake of Tat by cells is very strong, and has been reported as mediated by a short basic sequence of the protein (S. Fawell et al., Proc. Natl. Acad. Sci., USA, 91:664-668 (1994)).
- Tat protein and to the integrin cell surface receptor capable of binding to the Tat protein.
- Two Tat sequences that bind integrin are identified, which are the basic region or domain which is the dominant binding site for the integrin, having a peptide sequence of -Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg- (SEQ ID NO: 18), as well as -Gly-Arg-Gly-Asp-Ser-Pro- (SEQ ID NO: 19).
- compositions and methods for treatment of HIV-I both prophylactically and therapeutically, which are useful to lower the viral levels of HTV-I for the treatment and possible prevention of the subsequent, generally fatal, AIDS disease.
- WO 99/02185 describes the identification of a number of epitopes, i.e. binding regions, recognised by antibodies (antigenic sequences) in the N-terminal linear sequence and the C-terminal linear sequence of exon 1 of Tat.
- Epitope II has been identified as the ten amino acid sequence of amino acids at positions 41 to 50 of exon 1. This has been given the general formula:
- a first aspect of the invention provides an immunoglobulin variable domain comprising all or a fragment or variant of each of peptide sequences SEQ ID No 2, 3 and 4. It is important to note that the first aspect of the invention provides an immunoglobulin variable domain including the peptide sequences (or a fragment Oi variant) of SEQ ID No 2, SEQ ID No 3 and SEQ ID No 4.
- a further aspect of the invention provides an immunoglobulin variable domain having one or more complementarity determining regions (CDRs) encoded by all or a fragment or variant of each of the polynucleotide sequence of SEQ ID No 8, 9 and 10.
- CDRs complementarity determining regions
- a still further aspect of the invention provides an immunoglobulin variable domain comprising all or a fragment or variant of each of peptide sequences SEQ ID No 5, 6 and 7.
- a yet further aspect of the invention provides an immunoglobulin variable domain having CDRs encoded by all or a fragment or variant of each of polynucleotide sequences of SEQ ID No 11 , 12 and 13.
- CAAWDGSLSGHW [SEQ ID NO 7] TTTGATGATTATGGCATGGCCTGGGTCCGCCAAGCTCCAGGG [SEQ ID No 8]
- Antibody molecules consist of two identical heavy-chain and two identical light-chain polypeptides, which are covalently linked by disulphide bonds. Each of the chains is folded into several discrete domains. The N-termiiial domains of all the chains are variable in sequence and therefore called the variable regions (V-regions). The V-regions of one heavy (VH) and one light chain (VL) associate to form the antigen-binding site. The module formed by the combined VH and V L domains is referred to as the Fv (variable fragment) of the antibody. The C-terrninal ends of both heavy and light chains are more conserved in sequence and therefore referred to as the constant regions.
- Heavy chain constant regions are composed of several domains, eg. the heavy chain constant region of the gamma-isotype (IgG) consists of three domains (CHl. CH2, CH3) and a hinge region which connects the CHl and CH2 domains. The hinges of the two heavy chains are covalently linked together b ⁇ disulphide bridges. Light chains have one constant domain which packs against the CHl domain.
- the variable regions themselves each contain 3 clusters of hypervariable residues . , in a framework of more conserved sequences. These hypervariable regions interact with the antigen, and are called the Complementarity Determining Regions (CDRsj. The more conserved sequences are called the Framework Regions (FRs). X-ra3' studies of antibodies have shown that the CDRs form loops which protrude from the top of the molecule, whilst the FRs provide a structural beta-sheet framework.
- immunoglobulin variable domain we include the region of a heavy chain or right chain of an immunoglobulin located at its N-terminal end. which may comprise three complementarity-detennining regions (termed CDRs or CDRl. CDR2 and CDR3) and, optionally, four framework regions (termed FRs or FRl. FR2, FR3 and FR4), as distinct from the constant region located at the C-terminal end of the heavy or light chain.
- the peptides of SEQ ID No 2, 3 and 4 correspond to CDRs of a VH 3 while the peptides of SEQ ID No 5. 6 and 7 correspond to CDRs of a VL.
- fragment includes where one or more of the amino acid residues of the SEQ ID sequence is absent, provided that such a change results in a peptide whose basic properties, for example binding activity, have not significantly been changed.
- hnmunoglobulin variable domains refer to SEQ ID numbers having peptide sequences
- valiant includes where there are one or more amino acid insertions, deletions, or substitutions, either conservative or non-conservative, of the amino acid residues of the SEQ ID sequence, provided that such a change results in a peptide whose basic properties, for example binding activity, have not significantly been changed.
- conservative substitutions ' is intended combinations such as GI3'. Ala; VaI, lie. Leu; Asp. GIu: Asn, GIn: Ser, Thr; Lys ; Arg; and Phe, Tyr.
- valiants may be made using well known methods of protein engineering and site-directed mutagenesis.
- the fragments or variants of the peptide sequences of the immunoglobulin variable domains according to the above aspects of the invention have up to 1, 2, 3. 4 or 5 amino acid residues that are absent and/or variant.
- the immunoglobulin variable domain according to the first aspect of the invention comprises all or a fragment or variant of SEQ ID No 2, 3 and 4.
- the immunoglobulin variable domain may contain 0, 1, 2, 3. 4 or 5 absent and/or variant amino acids in each of the peptide sequences given in SEQ ID No 2. 3 and 4.
- the immunoglobulin variable domain comprises up to 1. 2. 3. 4 or 5 (ie O 5 I 3 2, 3, 4 or 5) absent and/or variant amino acids in total from the peptide sequences given in SEQ H) No 2, 3 and 4; or SEQ E) No 5, 6 and 7.
- the fragments or variants of the peptide sequences of the immunoglobuhn. variable domains according to the above aspects of the invention have only conservative substitutions.
- fragment includes where one or more of the nucleotides is absent, provided that such a change results in a polynucleotide that encodes a peptide whose basic properties, for example binding activity, have not significantly been changed.
- variable domains refer to SEQ ID numbers having a polynucleotide sequences
- variant includes where there are one or more nucleotide insertions, deletions, or substitutions, of the given nucleotide residues of the SEQ ED sequence, provided that such a change results in a pol y nucleotide encoding a peptide whose basic properties, for example binding activity, have not significantly been changed. For example, different codons can be substituted which code for the same amino acidfs) as tlie original codons.
- the substitute codons may code for a different amino acid that will not affect the activity of an encoded peptide or which ma ⁇ ' improve its activity.
- site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In ' Vitro Mutagenesis," Science, 229: 193-1210 (1985), which is incorporated herein by reference. Since such modified polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention.
- the fragments or valiants of the polynucleotide sequences encoding the immuiioglobuhn variable domains according to the above aspects of the invention have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 (ie 0, 1, 2, 3, 4, 5 5 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) nucleotides that are absent and/or variant.
- the immunoglobulin variable domain according to the second aspect of the invention comprises a fragment or variant of SEQ K ) No 8, 9 and 10.
- the irrrmunoglobuhn variable domain may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 (ie 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) absent and/or variant nucleotides in each of the polynucleotide sequences given in SEQ ID No 8, 9 and 10.
- the immunoglobulin variable domain comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 (ie 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) absent and/or variant nucleotides in total from the polynucleotide sequences given in SEQ ID No 8, 9 and 10; or SEQ ID No 11, 2 and 13.
- the fragments or variants of the potynucleotide sequences of the immunoglobulm variable domains according to the above aspects of the invention introduce no or onfy conservative substitutions into the encoded peptide sequence of the immunoglobulin valuable domain.
- binding activity of the peptide(s) is further discussed below in relation to further embodiments of the invention and in the accompanying example.
- an immunoglobulin variable domain of the invention comprises the peptide sequences:
- peptide sequences are encoded by the pol y nucleotide sequences: TTTGATGATTATGGCATGGCCTGGGTCCGCCAAGCTCCAGGG, TCGGGTGTTAGTTGGAATGGCAGTAGGACGCACTATGCAGACTCTGTG AAGGGCCGA 5 GCGAGAGGAGTAGCTGGTCATGACTAC [SEQ ID No 8 . . 9 and 10]
- immunoglobulin variable domain of the invention comprises the peptide sequences:
- peptide sequences are encoded by the polynucleotide sequences:
- the immunoglobulin variable domains of the above aspects of the invention may be s ⁇ ithesised according to methods known in the art.
- the immunoglobulin variable domains may be formed by expression from polynucleotide sequences encoding the immunoglobulin variable domains using techniques of molecular biology, as well known to those skilled in the art. However, chemical peptide synthesis techniques may also be used.
- Peptides may be sj ⁇ ithesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9- fluorei ⁇ dmetlryloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base- labile protecting group is effected using 20% pipei ⁇ dine in N ; N-dimeth3 7 lfo ⁇ namide.
- Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), bul3 7 loxycarbonyl derivative (in the case of lysine and histidine).
- trityl derivative in the case of cysteine
- 4-methoxy-2 ⁇ 6-trimetb3'lbenzenesulphonyl derivative in the case of argi ⁇ ine.
- glutamii ⁇ e o ⁇ asparagine are C-temiinal residues .
- the solid-phase support is based on a polydimetlryl-acrylamide porymer constituted from the three monomers dmiethylacrylamide (backbone- monomer), bisaciyloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent).
- the peptide-to-resin cleavable linked agent used is the acid-labile 44iydiuxymetl ⁇ yl-phenoxyacetic acid derivative. All amino acid derivatives are added as then" preformed S3'rnrnetrica ⁇ anhydride derivatives with the exception of asparagine and glutamine.
- Trichloroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide.
- Arry scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers.
- Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UIC) Ltd. Nottingham NG7 2QJ 5 UIC. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography.
- Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chramatograpl ⁇ y. amino- acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
- FAB fast atom bombardment
- antibody molecules consist of two identical heavy-chain and two identical light-chain polypeptides.
- the N-terminal domains of the chains are variable in sequence and are therefore called the variable regions (V-regions).
- VH variable heavy
- VL light chain
- an embodiment of the above aspects of the invention is where the peptide sequences of SEQ ID No 2, 3 and 4 and the peptide sequences encoded by the polynucleotides of SEQ ID No 8. 9 and 10 are three hypervariable regions/complementarity detenmning regions of an immunoglobulin variable domain.
- the ⁇ nnruno globulin variable domain is a V H domain.
- an alternative embodiment of the above aspects of the invention is where the peptide sequences of SEQ ID No 5, 6 and 7 and me peptide sequences encoded by the pohynucleotides of SEQ ID No 11, 12 and 13 are three hypervariable regions/complementarity detenmning regions of an immunoglobulin variable domain.
- the immunoglobulin variable domain is a VL domain.
- Immunoglobulin valuable domains typically comprise one or more framework regions, for example four framework regions.
- the framework regions may comprise peptide sequences from a number of different animals, for example mice, rats, pigs, sheep, goats or other such mammals routinely used to generate antibodies for research or therapeutic memeposes, preferably the framework regions are human.
- framework regions "" or "FRs" we include regions of the immunoglobulin domain other than the CDRs which form the structural framework within which the CDRs are positioned to form an immunoglobulin binding domain.
- FRl is typically the region at the N-terminus of the variable domain located on the N- terminal side of CDRl; FR2 and FR3 are located between CDRl and CDR2 and between CDR2 and CDR3. respectively; FR4 is located at the C-terminus of the variable domain on the C-terminal side of CDR3.
- Polynucleotide sequences encoding immunoglobulin framework regions (FRs) and immunoglobulin complementarity-determining regions (CDRs) that may be used in the synthesis of immunoglobulin variable domains of the invention ma ⁇ ' be obtained from, pre-existing libraries of sequences that encode antibodies with different binding specificities.
- libraries are well known to those in the art and standard molecular biology techniques (such as polymerase chain reaction (PCR) using oligonucleotide primers specific for variable domain regions, or restriction enzyme digestion) may be used to obtain sequences encoding FRs and CDRs from such libraries.
- libraries containing nucleotide sequences encoding full-length immunoglobulin polypeptides or fragments thereof may be used to obtain sequences for use in the invention.
- the framework regions are derived from a human Ig (immunoglobulin) heavy chain.
- the heavy chain may be derived from the human KOL heavy chain. However, it may also be derived from, for example, the human NEWM or EU heavy chain.
- the hea ⁇ 'y chain may be derived from one of the human germ line heavy chain genes, such as DP47.
- the framework regions may be derived from a human kappa or lambda light chain.
- the light chain is preferably derived from the human REI light chain.
- the light chain ma ⁇ ' be derived from one of the human genu line light chain genes, such as DPL3.
- Sequences encoding framework regions may be sj'nthesised ⁇ e novo using standard technologies.
- the framework sequences ma ⁇ ' overlap with constant parts in the sequences containing the CDRs allowing easy assembly of a full length and complete VH or VL-
- a further aspect of the invention provides an antibody comprising one or more immunoglobulin valuable domains according to the previous aspects of the invention and one or more immunoglobulin constant domains.
- Constant domains may be selected to have desired effector functions appropriate to the intended use of the antibody so constructed.
- human IgG isotypes, IgG 1 and IgGs are effective for complement fixation and cell mediated lysis.
- isotypes such as IgG 2 and IgG 4 .
- classes such as IgM and IgE. may be more suitable.
- Constant domains ma)' comprise peptide sequences from a number of different animals, for example mice, rats, pigs, sheep, goats or other such mammals routinely used to generate antibodies for research or therapeutic purposes.
- Human constant domain DNA sequences preferably in conjunction with their variable domain framework bases can be prepared in accordance with well-known procedures. An example of this is CMdPATH IH available from The Wellcome Foundation Ltd.
- IgGl constant domains which ma)' be used in this aspect of the invention include the "lambda 2' " (light chain), as encoded by the coding regions of the polynucleotide sequence provided in GenBank Accession Number X06875, and the "gamma 1" (heavy chain) as encoded by the coding regions of the poh'iiucleotide sequence provided in GenBank Accession Number Zl 7370 (allotype GIm(Z)J.
- IgG4 constant domains which may be used in this aspect of the invention include the "lambda 2" (light chain), as encoded by the coding regions of the pofynucleotide sequence provided in GenBank Accession Number X06875, and the heavy chain pol y peptide encoded by coding regions of the polynucleotide sequence provided in GenBank Accession Number K01316.
- a further aspect of the invention provides an antibody comprising one or more polypeptides of SEQ ID No 14 or 15 or a fragment or variant thereof.
- the antibody may, for example, be encoded by the polynucleotide sequence of SEQ JD No 16 or 17 or a fragment or variant thereof.
- the pofypepiide sequence of SEQ ID No 14 comprises the peptide sequences of SEQ ID No 2. 3 and 4 as well as framework regions as discussed above and comprises a VH variable domain sequence as well as constant region sequences.
- the pofypeptide sequence of SEQ ID No 15 comprises the peptide sequences of SEQ ID No 5, 6 and 7 as well as framework regions as discussed above and comprises a V L variable domain sequence as well as constant region sequences.
- the pol y nucleotide sequence of SEQ ID No 16 encodes the pol y peptide sequence of SEQ ID No 14 and the pol y nucleotide sequence of SEQ ID No 17 encodes the polypeptide sequence of SEQ ID No 15.
- Amino acids 1 to 116 of SEQ ID No 14 are the VH domain sequence and are encoded by nucleotides 1 to 348 of SEQ ID No.
- Amino acids 1 to 112 of SEQ JD No 15 are the VL domain sequence and are encoded by nucleotides 1 to 336 of SEQ ID No 17.
- SEQ ID No 14 the constant part stalls with AST and in SEQ ID No 15 the constant part with QPK.
- polypeptides of SEQ ID No 14 and 15 or the variable domains thereof are capable of forming an antibody or antibody fragment.
- a "variant" of the polypeptide of SEQ ID No 14 or 15 will have a region which has at least 90% (preferably 95. 96. 97. 98 or 99%) sequence identity with the polypeptide of SEQ ID Nos 14 or 15 as measured by the Bestfit Program of the Wisconsin Sequence Analysis Package, version 8 for Unix.
- the percentage identity may be calculated by reference to a region of at least 50 amino acids (preferably at least 75, 100, 120 or 140) of the candidate variant molecule, and the most similar region of equivalent length in the polypeptide of SEQ ID No 14 or 15, allowing gaps of up to 5%.
- a "vaiiant" of the polypeptide of SEQ ID No 14 or 15 is one hi winch the basic properties of the polypeptide, for example binding activity, have not been significantly changed.
- the percent identity may be determined, for example, by comparing sequence info ⁇ nati on using the GAP computer program, version 6.0 described by Devereux et al. (Nucl Acids res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
- the GAP program utilizes the alignment method of Neddleman and Wunsch (J MoI Biol, 48:443, 1970), as revised by Smith and Waterman (Adv. Appl Math 2.482. 1981).
- the preferred default parameters for the GAP pro grain include : (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non -identities) for nucleotides, and the weighted comparison matrix of Bribskov and Burgess, NucJ. Acids Res.
- a “fragment” of the potypeptide of SEQ ID No 14 comprises at least 100, 200, preferably 300, 350, 400 or 425 amino acids, or 100, preferably, 125, 150, 175 or 200 amino acids of the polypeptide of SEQ ID No 15.
- a “fragment” of the pofypeptide of SEQ ID No 14 or 15 is one in which the basic properties of the polypeptide, for example binding activity, have not been significantly changed.
- fragments or variants of the polypeptide sequences of the SEQ ID Nos 14 and 15 have only conservative substitutions. Preferably, there are no changes to the polypeptide sequences given in SEQ ID No 14 and 15.
- a "variant" of Hie polynucleotide of SEQ ID 16 or 17 is one which is usable to produce a polypeptide or a fragment thereof whose basic properties, for example binding activity, have not been significantly changed.
- different codons can be substituted which code for the same amino acid(s) as the original codons.
- the substitute codons ma ⁇ ' code for a different amino acid that will not affect the activity of the protein or which may improve its activity.
- site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis .
- polynucleotide sequence (or fragments thereof) of SEQ ID No 16 or 17 can be used to obtain other DNA sequences that hybridise with it under conditions of high stringency.
- DNA includes any genomic DNA. Accordingly, this aspect of the invention includes DNA that shows at least 85 per cent preferably 90 per cent, and most preferably 95 per cent homology with the polynucleotide of SEQ H) No 16 or 17.
- DNA-DNA 5 DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0. IXSSC and 6XSSC and at temperatures of between 55°C and 70°C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By “high stringency” we mean 2XSSC and 65 0 C. IXSSC is 0.15M NaCl/0.015M sodium citrate.
- a “fragment" the polynucleotide of SEQ ID No 16 or 17 comprises at least 100O 5 preferably 150O 5 1750, 2000 or 2100 nucleotides of the polynucleotide of SEQ ID No 16, or 1000, preferably, 1500, 1600, 1700 or 1800 nucleotides of the polynucleotide of SEQ H) No 17.
- a “fragment” of the polynucleotide of SEQ ID No 16 or 17 is one in which the basic properties of the encoded polypeptide, for example binding activity, have not been significantly changed.
- a fragment or variant of the pol y peptide or polynucleotide of SEQ ID No 14, 15 5 16 and 17 contains variations of the CDR regions (i.e. SEQ ID No 2, 3, 4. 5. 6. or 1). as discussed above in relation to the iniimmo globulin binding domains of the invention.
- nucleotides in SEQ ID No 16 and 17 do not introduce a frame shift in the open reading frame or a stop codon. as would be appreciated by a person skilled in the art.
- the fragments or variants of the pol y nucleotide sequences of SEQ ID No 16 and 17 introduce only conservative substitutions into the polypeptide sequence of the SEQ ID No 14 and 15.
- antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e.. molecules that contain an antigen-binding site that specifically binds (imniunoreacts with) an antigen.
- Ig immunoglobulin
- Such antibodies include, but are not limited to. polyclonal, monoclonal, chimeric, single chain. F a b, Fab 1 and F (ab' )2 fragments, and an F ab expression library.
- an antibody molecule obtained from humans relates to any of the classes IgG 5 IgM. IgA. IgE and IgD 3 which differ from one another by the nature of the hea ⁇ 7 chain present in the molecule.
- immunoglobulin molecules include Fab-lilce molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single- chain Fv (ScFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird el al (1988) Science 242, 423; Huston et al (1988) Proc. Natl Acad. ScL USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989) Nature 341. 544).
- Fab-lilce molecules Better et al (1988) Science 240, 1041
- Fv molecules Single-chain Fv (ScFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide
- dAbs single domain antibodies
- dAbs single domain antibodies
- ScFv molecules we mean molecules wherein the V H and V L partner domains are linked via a flexible oligopeptide.
- antibody fragments rather than whole antibodies
- the smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue.
- Fab, Fv 5 ScFv and dAb antibod ⁇ ' fragments can all be expressed in and secreted from different cells, such as E. coli, NSO, Chinese Hamster Ovarian (CHO) and human cells thus allowing the facile production of large amounts of the said fragments.
- an immunoglobulin binding domain or antibody of the present invention is distinct from naturally occurring immunoglobulin binding domains and antibodies.
- an embodiment of any of the aspects of the invention is wherein the immunoglobulin binding domain or antibod ⁇ ' is isolated and/or is recombinant.
- an embodiment of tins aspect of the invention is wherein the antibod) 7 further comprises an iininunoglobulin constant domain.
- an embodiment of the previous aspects of the invention is wherein the immunoglobulin variable domain or antibody selectively binds HTV Tat polypeptide.
- the immunoglobulin variable domain or the antibody selectively binds epitope II of HTV Tat polypeptide.
- the Tai polypeptide has a central role in the replication of HIV and contains a number of epitopes, i.e. binding regions, recognised b ⁇ 7 antibodies (antigenic sequences) in the N-terminal linear sequence and the C-terminal linear sequence of exon 1 of Tat.
- Epitope II is a ten amino acid sequence of amino acids at positions 41 to 50 of exon 1.
- TMs has been given the general formula:
- the immunoglobulin variable domain or the antibody recognises and binds to the Tat polypeptide, but may also interact with other proteins through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule.
- Screening assa)'S to determine binding specif ⁇ cit ⁇ ' of an immunoglobulin variable domain or antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et ⁇ l. (Eds). Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N. Y. (198S) 5 Chapter 6.
- binding assays such as, for example, ELISA, affinity determinations using eg Biacore and functional in vitro and in vivo assays, as would be appreciated by a person skilled in the art.
- Further studies include FACS anafysis and ⁇ mnunol ⁇ sto chemistry for assessment of target specificity and cross-reactivity.
- the peptide and polynucleotide sequences of the immunoglobulin binding domains of the invention include frasments and variants of those sequences, provided that such changes result in an immunoglobulin binding domain whose basic properties, for example binding activity, have not significantly been changed. It is possible to determine whether the fragments and variants of the sequences provided have so changed the binding activity of the immunoglobulin binding domain using the methods provided in the examples and discussed herein.
- a further aspect of the invention provides an immunoglobulin variable domain or an antibody capable of binding to epitope II of the Tat pol y peptide and having a binding affinity of at least 10 "s to epitope II of the TAT pofypeptide.
- the value of the binding affinity of the immunoglobulin variable domain or antibody to epitope II of TAT polypeptide is very dependent on the measuring method used.
- the binding affinity 10 ' specified in this aspect of the invention was determined using the Biacore binding affinity method detailed in the examples.
- the immunoglobulin valuable domain or antibody of this aspect of the invention can have a binding affinity of 10 '9 , 10 "30 , 10 " ⁇ , 10 "12 to epitope II of TAT polypeptide when measured using the Biacore binding affxoity method detailed in the examples.
- immunoglobulin variable domain is an immunoglobulin variable domain according to any of the previous aspects of the invention, or the antibody is an antibody according to an ⁇ ' of the previous aspects of the invention.
- Antibodies and immunoglobulin binding domains can be identified using techniques including phage display libraries (Hoogenboom and Winter, J. MoI. Biol, 227:381 (1991); Marks et al, J. MoI. Biol., 222:581 (1991)).
- n-CoDeR ⁇ a technology termed n-CoDeR ⁇ .
- the technology utilises a single human framework in V H and VL respectively, and CDRs are shuffled randomly into their respective positions thereby generating a great variability.
- the library has been used extensively to select high affinity antibody fragments against all types of antigens including haptens, peptides, proteins including human proteins and also carbohydrates. AU parts of the antibodies are full ⁇ ' human and are derived from human donors.
- n-CoDeR ® library is built using a modular approach where some modules (framework sequences) are kept constant while other modules (the CDRs) are allowed to vary.
- the further antibodies or immunoglobulin binding domains of this aspect of the invention are generated using n-CoDeR ® as described in WO 9S/32845.
- a human antibody molecule relates to any of the classes IgG 5 IgM, IgA 3 IgE and IgD 5 which differ from one another by the nature of the heavy chain present hi the molecule. Certain classes have subclasses as well, such as IgG 1 , IgG 2 , and others.
- an embodiment of any of the aspects of the invention is wherein the antibod ⁇ ' is an IgG] antibody, preferably a human IgG] antibody.
- Aj] embodiment of an) 7 of the aspects of the invention is wherein the immunoglobulin variable domain or antibody further comprising a member of a specific binding pair.
- AQ immunoglobulin variable domain or antibody of the invention may be linked with a member of a specific binding pair, e.g. biotin. so that it can be retrieved from solution by use of an anti-tag, e.g. avidin. coupled to a solid support, e.g. magnetic beads.
- a specific binding pair e.g. biotin.
- an anti-tag e.g. avidin. coupled to a solid support, e.g. magnetic beads.
- specific binding pairs are known to those in the art, e.g. maltose and maltose binding protein.
- An embodiment of any of the aspects of the invention is wherein the immunoglobulin variable domain or antibody comprising a further therapeutic entity.
- the further entity may be of use in therapy, e.g. a toxin, an enzyme capable of activating a prodrug, a binding partner or a radioisotope.
- the furfher entity ma)' be a protein such as an enzyme useful in isolating the immunoglobulin variable domain or antibody, e.g. Glutathione S -transferase.
- the further entity may be an oligonucleotide (may be either DNA 5 RNA or PNA).
- the further entity may also be beneficial in imaging, e.g. it ma ⁇ ' be a label.
- a further aspect of the invention provides a polypeptide comprising all or a fragment or variant of each of polypeptide sequences SEQ ID No 2, 3, 4, 5, 6 and 7; or all or a fragment or variant of each of polypeptide sequences amino acids 1 to 116 of SEQ ID No 14 and amino acids 1 to 112 of SEQ ID No 15; or all or a fragment or variant of each of polypeptide sequences SEQ ID No 14 and 15.
- SEQ ID No 2. 3, 4, 5, 6, 7, 14 and 15 and fragments and variants thereof are discussed above. Methods of obtaining such a polypeptide are also discussed above.
- the polypeptide may typically be an antibody (including an antibody fragment, as discussed above).
- a further aspect of the invention provides a polynucleotide or polynucleotides encoding the polypeptide of the preceding aspect of the invention.
- the polynucleotide or polynucleotides ma)' comprise all or a fragment or variant of each of the polynucleotide sequences of SEQ ID No S 5 9. 10. 11, 12 and 13; or all or a fragment or variant of each of the polynucleotide sequences 1 to 348 of SEQ ID No 16 and 1 to 336 of SEQ ID No 17; or all or a fragment or variant of each of the polynucleotide sequences of SEQ ID No 16 and 17.
- Methods of obtaining a polynucleotide of this aspect of the invention include using the well known PCR technique in which a number of overlapping oligonucleotides are used to synthesise a complete polynucleotide of the sequence given.
- Oligonucleotides i.e.. small nucleic acid segments, may be readily prepared b ⁇ ; . for example, directly synthesizing the oRgonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide S3 r nthesizer.
- a further aspect of the invention provides an expression vector comprising a polynucleotide sequence of the previous aspect of the invention.
- the expression vector could express a given polynucleotide in conjunction with one or more further polynucleotide sequences so as to encode a scFv pol y peptide.
- the tenn 'scFv' is discussed above.
- SEQ ID No 16 encodes a heavy chain polypeptide
- SEQ ID No 17 encodes a light chain polypeptide
- Amino acids 1 to 116 of SEQ ID No 14 are the VH domain sequence and are encoded by nucleotides 1 to 348 of SEQ DD No 16.
- Amino acids 1 to 112 of SEQ ID No 15 are the VL domain sequence and are encoded by nucleotides 1 to 336 of SEQ ID No 17.
- the expression vector of this aspect of the invention may comprise the pofyuucleotide sequence of nucleotides 1 to 348 of SEQ ID No 16 (or a variant thereof, as discussed above) and nucleotides 1 to 336 of SEQ ID No 17 (or a variant thereof, as discussed above) separated by a polynucleotide sequence encoding a flexible oligopeptide.
- Such an expression vector would comprise a poh'nucleotide which, when expressed, would produce a scFv pol y peptide, as known to those skilled in the art.
- a further aspect of the invention is a host cell comprising an expression vector according to the invention.
- the sequence ma ⁇ ' be incorporated in a vector having control sequences operably linked to the polynucleotide sequence to control its expression.
- the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted polynucleotide sequence, further polynucleotide sequences so that the protein encoded for by the polynucleotide is produced as a fusion and/or nucleic acid encoding secretion signals so that the protein produced in the host cell is secreted from the cell.
- the protein encoded for by the polynucleotide sequence can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the protein is produced and recovering the protein from the host cells or the surrounding medium.
- Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, yeast, and eukaryotic cells such as COS, NSO or CHO cells.
- the choice of host cell can be used to control the propeities of the protein expressed in those cells. e.g. controlling where the protein is deposited - in the host cells or affecting properties such as its glycos3'lation.
- polynucleotide sequences may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
- the companion DNA will depend upon the nature of the host, the maim& of the introduction of the DNA into the host, and whether episornal maintenance or integration is desired.
- the polynucleotide sequence is inserted into an expression vector, such as a plasrnid, in proper orientation and correct reading frame for expression.
- an expression vector such as a plasrnid
- the polynucleotide sequence ma)' be linked to the appropriate transcriptional and traiislational regulators' control nucleotide sequences recognised b)' the desired host, although such controls are generally available in the expression vector.
- the polynucleotide sequence insert ma ⁇ ' be operatively linked to an appropriate promoter.
- Bacterial promoters include the E.coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the phage ⁇ PR and PL promoters, the phoA promoter and the ⁇ rp promoter.
- Eukaryotic promoters include the CMV immediate early promoter, the HSV ttrymidine kinase promoter, the earl ⁇ ' and late SV40 promoters and the promoters of retroviral LTRs. Other suitable promoters will be known to the skilled artisan.
- the expression constructs will desirably also contain sites for transcription initiation and teimination, and in the transcribed region, a ribosome binding site for translation (Hastings et al,
- Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, potyaden3'lation sequences, enhancer sequences, marker genes and other sequences as appropriate.
- Vectors may be plasmids, viral e.g. phage, or phagernid, as appropriate.
- plasmids viral e.g. phage, or phagernid, as appropriate.
- S3'stems employing: bacteria (e g. E. coll and Bacillus subtilis) transformed with, for example, recombinant bacteriophage, plasmid or cosmid DNA expression vectors; .yeasts [e.g. Saccaromyces cerevisiae) transformed with, for example, yeast expression vectors; insect cell systems transformed with, for example, viral expression vectors (e g baculovirus); plant cell systems transfected with, for example viral or bacterial expression vectors; animal cell systems transfected with, for example, adenovirus expression vectors.
- bacteria e g. E. coll and Bacillus subtilis
- .yeasts e.g. Saccaromyces cerevisiae transformed with, for example, yeast expression vectors
- insect cell systems transformed with, for example, viral expression vectors e g baculovirus
- plant cell systems transfected with, for example viral or bacterial expression vectors e.g baculovirus
- the vectors ma)' include a prolcaryotic replicon, such as the Col El orL for propagation in a prokaryote. even if the vector is to be used for expression in other, non-prokaryotic cell types.
- the vectors may also include an appropriate promoter such as a prolcaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E.coli, transformed therewith.
- a promoter is an expression control element formed b ⁇ a DNA sequence that permits binding of RJSIA polymerase and transcription to occur.
- Promoter sequences compatible with exemplar ⁇ ' bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
- T ⁇ ical prokaryotic vector plasmids are: pUCl ⁇ , pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA 5 USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pPJT5 available from Pharmacia (Piscataway, NJ, USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNHSA, pNH16A ; pNHISA, pNH46A available from Stratagene Cloning S3'stems (La Jolla, CA 92037, USA).
- Useful 3'easl plasmid vectors are pRS403-406 and pRS4l3-416 and are generally available from Stratagene Cloning Systems (La Jolla. CA 92037. USA).
- pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRPl, LEU2 and UJRA3.
- Plasmids pRS4l3-416 are Yeast Centromere plasmids (YCps).
- Methods well known to those skilled in the ail can be used to construct expression vectors containing the coding sequence and, for example appropriate transcriptional or translational controls.
- One such method involves ligation via homopolymer tails.
- Hoinoporymer polydA (or polydC) tails are added to exposed 3 ' OH groups on the DNA fragment to be cloned by terminal deoxynucleotidyl transferases.
- the fragment is then capable of annealing to the polydT (or pofydG) tails added to the ends of a linearised plasmid vector. Gaps left following annealing can be filled by DNA polymerase and the free ends joined 03' DNA ligase.
- Another ' method involves ligation via coheswe ends.
- Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
- a further method uses synthetic molecules called linkers and adaptors.
- DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3' termini and fill in recessed 3' ends.
- Synthetic linkers pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible teniii ⁇ i.
- Adaptors are also chemically sj'nthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end.
- Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnolo -"tg-i 1 es Inc. New Haven. CN. USA.
- a desirable way to modify a polynucleotide is to use the polymerase chain reaction as disclosed by Saila el al (1988) Science 239, 487-491.
- the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA.
- the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
- the vector is then introduced into the host cell through standard techniques. Generally, not all of the hosts will be transformed by the vector and it will therefore be necessary to select for transformed host cells.
- One selection technique involves incorporating into the expression vector a DNA sequence marker, with an ⁇ r necessary control elements, that codes for a selectable trait in the transformed cell. These markers include dihydrofolate reductase. G418 or neomycin resistance for eukaryotic cell culture, and tetracyclin, kanarrrycin or ampiciUin resistance genes for culturina; in E.coli and other bacteria.
- the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
- Host cells that have been transformed by the expression vector are then cultured for a sufficient time and under appropriate conditions known to those skilled hi the. ait to permit the expression of the encoded antibody or immunoglobulin binding domain. which, can then be recovered.
- the antibody or immunoglobulin binding domain can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography. hydrox3dapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (" ⁇ PLC'J is employed for purification.
- ⁇ PLC'J high performance liquid chromatography
- the host cell may be any suitable host cell, for example bacterial, e.g. E. coli, yeast, insect mammalian or plant cell, as would be appreciated by a person skilled in the art.
- Preferred host cells are Chinese Hamster Ovarian (CHO) cells.
- a further aspect of the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an immunoglobulin variable domain and/or an antibody and/or a polypeptide and/or a pol y nucleotide according to the invention and a pharmaceutically acceptable carrier.
- an immunoglobulin variable domain, antibody, polypeptide, polynucleotide or a gene therapy vector of the invention Whilst it is possible for an immunoglobulin variable domain, antibody, polypeptide, polynucleotide or a gene therapy vector of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
- the carrier(s) must be "acceptable” in the sense of being compatible with the invention and not deleterious to the recipients thereof.
- the carriers will be water or saline which will be sterile and pyrogen free.
- the immunoglobulin variable domain, antibody, polypeptide, polynucleotide or gene therapy vector of the invention can also be administered parenteral ⁇ , for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrastemally, intracranial ⁇ , intra-musculaiiy or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
- the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
- Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants. buffers, bacteriostats and solutes which rendei the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
- the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilisedj condition requiring only the addition of the sterile liquid canier, for example water for injections, immediately prior to use.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- An immunoglobulin binding domain or antibody of the invention may be given in monthly injections, facilitating the patient's drug compliance and likely niii ⁇ irnizing adverse events. It may be supplied as a solution for infusion, to be kept at 2-8 0 C, in 10 inM Sodium Phosphate, 150 mM Sodium Chloride at pH 7.4, produced with Water For injection (WFI).
- WFI Water For injection
- a further aspect of the invention provides an immunoglobulin variable domain or antibody or a polypeptide or a polynucleotide or a gene therapy vector or a pharmaceutical composition according to the invention for use in medicine.
- a further aspect of the invention provides the use of an immunoglobulin variable domain and/or an antibody and/or a pofypepytide and/or a polynucleotide and/or a gene therapy vector and/or a pharmaceutical composition according to the invention in the manufacture of a medicament for treating a patient having, or at risk of contracting or developing, HIV/AIDS.
- a further aspect of the invention provides a method of treating HTV/AIDS comprising administering to a patient an immunoglobulin variable domain and/or an antibody and/or a pol y peptide and/or a polynucleotide and/or a gene therap ⁇ ' vector and/or a pharmaceutical composition according to the invention.
- Au embodiment of the use 01 method of treatment aspects of the invention is wherein the patient has a competent immune system and is HIV-I infected.
- An embodiment of the use or method of treatment aspects of the invention is wherein the patient does not have HTV.
- An embodiment of the use or method of treatment aspects of the invention is wherein the patient is incapable of mounting an effective or rapid immune response to infection with HW-I .
- the present invention provides immunoglobulin binding domains and antibodies which can react with the HIV Tat polypeptide to reduce viral levels of HIV, and thus may be useful hi both a therapeutic and proplrylactic context to control the development of ATDS in a large population exposed to, or infected by, HIV which produce upon infection different Tat proteins.
- n-CoDeR ® anti-Tat antibody will probably be given by monthly injections, facilitating the patient's drug compliance and likely niinnnizin X gD a ' dverse events.
- a medicament comprising an immunoglobulin binding domain or antibody of the invention may reduce HIV viral multiplication during any initial acute infection with HIV and minimize chronic viremia leading to ADDS. This may also lower chronic viral multiplication in infected subjects, again minimizing progression to AIDS.
- Such a medicament may be therapeutically administered to a HIV infected human with a competent immune system for treatment or control of viral infection.
- a HIV infected human may be as3'mptoniatic.
- the medicament ma ⁇ ' be administered to an uninfected human for proplxylaxis.
- the medicament may also be useful for treating a patient who is incapable of mounting an effective or rapid immune response to infection with HTV. This may be achieved by chronically administering the medicament.
- suitable for treatment with this medicament are HIV infected patients who are immunocompromised b3 ; disease and unable to mount a strong immune response.
- the medicament of the invention preferably employs the antibody of the invention.
- This medicament is administered as passive immttnotherapy to inhibit viral multiplication and lower the viral load.
- the exogenous antibodies which react with most Tat proteins from HTV provide in the patient an immediate interdiction of the transfer of Tat from virally infected cells to other infected or uninfected cells. Accordingly, the patient may be chronically treated with the antibody composition for a long treatment regimen.
- each dose will comprise between about 5 to 800 mg, preferably 400, 300, 200 or 50 mg. Said dose is preferably given in a concentration of about 5 to 100 mg/ml per injection, preferably 75 mg, 50 mg, or 10 mg.
- the frequency of chronic administration may range from dairy dosages to once or twice a week to once a month, and may depend upon the half-life of the immunoglobulin binding domain/anlibody (e.g. about 7-21 days). However, the duration of chronic treatment for such infected patients is anticipated to be an indefinite, but prolonged period. Other dosage ranges may also be contemplated by one of skill in the art, particularly where administration of the immunoglobulin binding domain/antibody is in conjunction or sequential with other anti-viral treatments.
- anti-viral treatments which may be included in the pharmaceutical composition or medicaments of the invention include anti-retroviral drugs, for example reverse transcriptase inhibitors such as AZT, zidovudine, lamivudine, nevirapine or 3TC. or protease inhibitor drugs, for example indinavir orritonavir; or entry inhibitors, such as enfurvitide.
- anti-retroviral drugs for example reverse transcriptase inhibitors such as AZT, zidovudine, lamivudine, nevirapine or 3TC.
- protease inhibitor drugs for example indinavir orritonavir
- entry inhibitors such as enfurvitide.
- the genotype of the HTV is PW-I .
- Figure 2 Inhibition experiment using 1 % initially HLV-I IIIB-infected Jurlcat cells with increasing concentrations of n-CoDeR ' anti-Tat antibodj'. Virus levels were determined through a p24 detecting ELISA at day 7 post infection.
- Example 1 Characterisation of an antibody that binds to Epitope II of HIV Tat polypeptide
- the antibody is directed against epitope II (SEQ 1 ) of the excreted regulatory protein Tat encoded by HIV-I .
- n ⁇ CoDeR Sl anti-Tat antibody is a fully human recombinant IgGj antibody expressed in Chinese Hamster Ovarian (CHO) cells. It ma ⁇ ' e.g. be supplied as a solution for infusion, to be kept at 2-8°C, in 10 mM Sodium Phosphate, 150 roM Sodium Chloride at pH 7.4, produced with Water For Injection ( WFl).
- the peptide sequence of the Gamma 1 chain of the antibody is provided above as SEQ ID No 14; the corresponding polynucleotide sequence of the Gamma 1 chain of the antibody is provided above as SEQ ID No 16.
- the peptide sequence of the Lambda chain of the antibody is provided above as SEQ ID No 15; the corresponding polynucleotide sequence of the Gamma 1 chain of the antibody is provided above as SEQ ID No 17.
- AU antigens are biotinylated peptides.
- ELISA analysis confirms that n-CoDeR ® anti-Tat antibody binds specifically to the target epitope in a concentration dependent mariner. Twelve two fold serial dilutions of n-CoDeR ® anti-Tat antibody ranging from l ⁇ g/ml were incubated on peptide coated plates (peptide was conjugated to BSA). The plate was washed and then probed with HRP labelled rabbit anti-human IgG and developed with PIERCE super signal. Luminesence was measured at 700 nm. Half maximal binding was achieved at 0.7 nM of n-CoDeR ® anti-Tat antibody. The data is presented in Figure 1.
- n-CoDeR ⁇ anti-Tat antibody to inhibit HTV-I replication has been tested in a set of experiments performed at Karolinslca Institute. The experiments have been based on HTV-I IHB infected Jurkat cells (human T-cell line) in which viral replication has been monitored through an ELISA directed against the surface protein p24 (Re el aL 1995 ).
- n-CoDeR (fi) anti-Tal antibody has been calculated from inhibition experiments performed with 1 % and 3 % initially infected cells and resulted in 0.05 ⁇ g/ml and 0.07 ⁇ g/rnl, respectively (Table 3).
- n-CoDeR ® anti-Tat antibody to inhibit viral replication in Jurkat cells expressing Tat with arnino acid variations near the target epitope (same variations that are present in patient isolates) has been tested in a new set of experiments, recentfy performed. The result suggests that these amino acid variations do not influence the ability of the antibody to bind to and neutralise Tat.
- Biacore sj'stems utilize the phenomenon of surface plasmon resonance (SPR) to perform antibody antigen interaction anafysis.
- SPR surface plasmon resonance
- the refractive index at the interface between the surface and a solution flowing over the surface changes.
- the sensorgram shows an increasing response as the antibodies binds (antibody binding peptide). The response remains constant if the interaction reaches equilibrium.
- the response decreases as the immuncomplex dissociate.
- the kinetics of the complete interaction i.e.
- the rates of complex formation (lc a ) and dissociation (lt d ), can be determined from the information in a sensorgram.
- the BIAevaluation software (Biacore soft ware) used generates the values of k a and k ⁇ by fitting the data to interaction models. The software evaluation of the graph is done by numerical integration methods and global fitting methods.
- There are two general ways to calculate the total affinity of an interaction either by using the level of binding at equilibrium (seen as a constant signal) as a function of sample (antibody) concentration or it can also be determined from ' the kinetic estimations hi the sensogram.
- the equilibrium constant ICD is the ratio of the kinetic rate constants, k d /k a .
- Biotinylated peptides were immobilized on avidin coupled Biacore sensor cliip SA and BSA conjugated peptide was coupled direct])' to dextran matrix of a CMS chip.
- Steps were taken to ensure comparable molar iinmobilisation concentrations of the respective antigens.
- the immobilisation level was kept low in order to minimize the influence of avidity.
- Five different concentrations of each antibod ⁇ ' were injected consecutively on the chip and the recorded affinity was the summery of the data from all of the injections. Resulting binding curves were analysed with BIAevahiation. No background binding to the chip matrix or to non relevant peptides were detected.
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Abstract
The present invention relates to an immunoglobulin variable domain comprising all or a fragment or variant of each of peptide sequences SEQ ID No 2,3 and 4. Generally the invention relates to compositions useful for assaying or treating human immunodeficiency virus- 1(HIV-I) infection. There is also provided antibodies and polypeptides comprising immunoglobulin variable domains, polynucleotides, vectors, host cells encoding immunoglobulin variable domains, pharmaceutical compositions comprising and uses of immunoglobulin variable domains.
Description
PEPTIDES AND USES THEREOF.
The present invention relates generally to compositions useful for assaying or treating human immunodeficiency virus-1 CHW-I) infection.
High plasma levels of human immunodeficiency virus type 1 (HIV-I) RNA are found during primary infection with HTV-I, the seroconversion illness (C. Bamberger et al, AIDS, 7:(suppl 2):S59 (1993); M. S. Saag et al, Nature Med., 2:625 (1996)), after which they subside as the immune response controls the infection to a variable extent. Post seroconversion, lower but detectable levels of plasma HIV-I KNA are present and these levels rise with disease progression to again attain high levels at the AIDS stage (M. S. Saag et al, Nature Med., 2:265 (1996)). Approximately 50% of subjects have a symptomatic illness at seroconversion (B. Tindall and D. A. Cooper, AIDS. 5:1 (1991)) and symptomatic seroconversion is associated with an increased risk for the development of AIDS, probably because a severe primary illness is likely related to an early and extensive spread of MTV.
Inhibition of viral multiplication during the initial infection will likely reduce the subsequent development of chronic viremia leading to ATDS. Current medical practice, with administration of antiviral drugs for defined "at risk" situations, such as needle sticks with contaminated blood or pregnancy in HTV infected mothers, supports this concept. ' .
Post sero-conversion levels of HIV-I RNA in plasma have proven to be the most powerful prognosticator of the likelihood of progression to AIDS (J. W. Mellors et al, Science, 272:1167 (1996); M. S. Saag et al, Nature Med., 2:265 (1996); R. W. Coombs et al, J. Inf. Dis., 174:704 (1996); S. L. Welles et al, J. M. Dis., 174:696 (1990)). Other measures of viral load, such as cellular RNA (K. Saksela et al, Proc. Natl Acad. Sci. USA, 91:1104 (1994)) and cellular HIV proviral DNA (T- H. Lee et al, J. Acq. hnrn. Def. Syndromes, 7:381 (1994)) similarly establish the
CONFIRMATION fcOPY
importance of the initial infection in establishing viral loads that determine future disease progression.
Thus, any intervention that inhibits HTV- 1 infectivity during initial infection and/or lowers viral load post .sero-conversion is likely to have a favourable influence on the eventual outcome, delaying or preventing progression to AIDS.
A variety of methods are now employed to treat patients infected with human immunodeficiency virus (H-V- 1). including treatment with certain combinations of protease inhibitor drugs. Unfortunately, however, this type of treatment is associated with serious side effects in some patients.
Alternatively, vaccines are under development for control of the spread of HTV-I to uninfected humans. 'However, this effort has largely been directed to proteins of the virus, expressed on the surface of infected cells, which are recognized by cytotoxic T cells with eHrnination of the infected cells, while free virus is blocked and cleared by antibody to surface antigens of the λάrion. Limitations of this mode of vaccination are readily apparent for HTV-I. which has demonstrated a great diversity in immunogenic viral epitopes and rapid mutational variations that occur -within and between individuals (B. D. Preston et al. Science, 242:1168(1988): J.
D. Roberts et aL Science, 242:1171 (1988); A. R. Meyerhans et al., Cell, 58:901
(1989); K. Kusnwi et al., J. Virol, 66:875 (1992); B. A. Larder et aL, Science,
243:1731 (1989); M. S. Sang et al, N. Engl. J. Med., 329:1065 (1993); M. A.
' Sande/et al., JAMA, 270:2583 (1993); M. Seligmann et al. Lancet, 343:871 (1994): G. Meyers et al., Human retroviruses and AIDS 1993, 1- V, A compilation and analysis of nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los Alamos, N.Mex.)
Variation in strains of HIV-I and frequent mutations of virion proteins have prevented successful application of conventional vaccine approaches (W. E. Paul,
Cell 82:177 (1995); J. E. Osborn, J. Acq. Imm. Def. Syndr. Hum. Retroviral,
9:26 (1995)). Mutation and selection of resistant valiants is the central problem -in
developing a successful HIV-] vaccine (M. D. Daniel et al.. Science, 258: 1938 (1992); N. L. Letvin. N. Engl. J. Med., 329:1400 (1993); M. Clerici et al., AIDS, 8:1391 (1994;; S. M. Wolinsky et al. Science, 272:537 (1996;).
Other approaches to HIV-I treatment have focused on the transact! vating (tat) gene of HIV-I5 which produces a protein (TatJ essential for transcription of the virus. The tat gene and its protein have been sequenced and examined for involvement in proposed treatments of HR7 (see, e.g., U.S. Pat. No. 5,158,877;
U.S. Pat. No. 5,238,882; U.S. Pat. No. 5,110,802; International Patent Application No. WO92/0787L published May 14, 1992; International Patent Application No.
WO91/10453, published JuL 25, 1991: International Patent Application No.
WO91/09958, published JuI. H3 1991; International Patent Application No.
WO87/02989, published May 2I5 1987). Tat protein is released extracellularly, making it available to be taken up by other infected cells to enhance transcription of HTV- 1 in the cells and to be taken up by noninfected cells, altering host cell gene activations and rendering the cells susceptible to infection by the virus. Uptake of Tat by cells is very strong, and has been reported as mediated by a short basic sequence of the protein (S. Fawell et al., Proc. Natl. Acad. Sci., USA, 91:664-668 (1994)).
International Patent Application No. WO92/14755, published Sep. 3, 1992, relates to the Tat protein and to the integrin cell surface receptor capable of binding to the Tat protein. Two Tat sequences that bind integrin are identified, which are the basic region or domain which is the dominant binding site for the integrin, having a peptide sequence of -Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg- (SEQ ID NO: 18), as well as -Gly-Arg-Gly-Asp-Ser-Pro- (SEQ ID NO: 19). This specification demonstrates that a number of peptides corresponding to these Tat sequences and the corresponding integilns block in vitro cell binding to Tat coated plates, as do antibodies to the appropriate integrins. However, the -specification also shows that these reagents do not block uptake of functional Tat by cells (see Example 9 in WO92/14755). thus nullifying the proposed mechanism of action for therapeutic benefit in HIV infection.
Both monoclonal and polyclonal antibodies to Tat protein have been produced in animals and shown to block uptake of Tat protein in vitro (see. e.g.. D. Brake et al,
J. Virol, 64:962 (1990); D. Mann et al EMBO J., 10:1733 (1991); J. Abraham et al. cited aboλ'e; P. Auron et al, cited above; M. Jaye et.al, cited above; G. Zauli et al. cited above). More recent reports showed that monoclonal or polyclonal antibodies to Tat protein added to tissue culture medium attenuated HW-I infection in vitro (L. Steinaa et al, Arch. Virol., 139:263 (1994); M. Re et al, J.
Acq. Imm. Def. Syndr. Hum. Retroviral., 10:408 (1995); and G. Zauli et al, J. Acq. Imm. Def. Syndr. Hum. Retroviral., 10:306 (1995)).
Despite the growing knowledge about HTV-I disease progression, there remains a need in the art for the development of compositions and methods for treatment of HIV-I, both prophylactically and therapeutically, which are useful to lower the viral levels of HTV-I for the treatment and possible prevention of the subsequent, generally fatal, AIDS disease.
As discussed above the Tat polypeptide has a central role in the replication of HTV. WO 99/02185 describes the identification of a number of epitopes, i.e. binding regions, recognised by antibodies (antigenic sequences) in the N-terminal linear sequence and the C-terminal linear sequence of exon 1 of Tat. Epitope II has been identified as the ten amino acid sequence of amino acids at positions 41 to 50 of exon 1. This has been given the general formula:
KXLGISYGRK [SEQ ID No 1], where X is Ala or GIy "
A first aspect of the invention provides an immunoglobulin variable domain comprising all or a fragment or variant of each of peptide sequences SEQ ID No 2, 3 and 4.
It is important to note that the first aspect of the invention provides an immunoglobulin variable domain including the peptide sequences (or a fragment Oi variant) of SEQ ID No 2, SEQ ID No 3 and SEQ ID No 4.
A further aspect of the invention .provides an immunoglobulin variable domain having one or more complementarity determining regions (CDRs) encoded by all or a fragment or variant of each of the polynucleotide sequence of SEQ ID No 8, 9 and 10.
A still further aspect of the invention provides an immunoglobulin variable domain comprising all or a fragment or variant of each of peptide sequences SEQ ID No 5, 6 and 7.
A yet further aspect of the invention provides an immunoglobulin variable domain having CDRs encoded by all or a fragment or variant of each of polynucleotide sequences of SEQ ID No 11 , 12 and 13.
The following amino acid and polynucleotide sequences are referred to in the above aspects of the invention. .
FDDYGMAWVRQAPG [SEQ ID NO 2]
SGVSWNGSRTEIYADSVKGR [SEQ ID NO 3]
ARGVAGHDY [SEQ ID No 4]
CSGSRSNIGSHWF [SEQ ID NO 5]
DNNIORPS [SEQ ID NO O] .
CAAWDGSLSGHW [SEQ ID NO 7]
TTTGATGATTATGGCATGGCCTGGGTCCGCCAAGCTCCAGGG [SEQ ID No 8]
TCGGGTGTTAGTTGGAATGGCAGTAGGACGCACTATGCAGACTCTGTG AAGGGCCGA [SEQ ID No 9]
GCGAGAGGAGTAGCTGGTCATGACTAC [SEQ ID NO 10]
TGTTCTGGAAGCAGGTCCAACATCGGAAGTCATTATGTATTC [SEQ ID No l l]
GACAATAATAAGCGACCCTCA [SEQ TD NO 12]
TGTGCAGCATGGGATGGCAGCCTGAGTGGCCATGTGGTA [SEQ ID NO 13]
Antibody molecules consist of two identical heavy-chain and two identical light-chain polypeptides, which are covalently linked by disulphide bonds. Each of the chains is folded into several discrete domains. The N-termiiial domains of all the chains are variable in sequence and therefore called the variable regions (V-regions). The V-regions of one heavy (VH) and one light chain (VL) associate to form the antigen-binding site. The module formed by the combined VH and VL domains is referred to as the Fv (variable fragment) of the antibody. The C-terrninal ends of both heavy and light chains are more conserved in sequence and therefore referred to as the constant regions.
Heavy chain constant regions are composed of several domains, eg. the heavy chain constant region of the gamma-isotype (IgG) consists of three domains (CHl. CH2, CH3) and a hinge region which connects the CHl and CH2 domains. The hinges of the two heavy chains are covalently linked together b}^ disulphide bridges. Light chains have one constant domain which packs against the CHl domain.
The variable regions themselves each contain 3 clusters of hypervariable residues., in a framework of more conserved sequences. These hypervariable regions interact with the antigen, and are called the Complementarity Determining Regions (CDRsj. The more conserved sequences are called the Framework Regions (FRs). X-ra3' studies of antibodies have shown that the CDRs form loops which protrude from the top of the molecule, whilst the FRs provide a structural beta-sheet framework.
By "immunoglobulin variable domain" we include the region of a heavy chain or right chain of an immunoglobulin located at its N-terminal end. which may comprise three complementarity-detennining regions (termed CDRs or CDRl. CDR2 and CDR3) and, optionally, four framework regions (termed FRs or FRl. FR2, FR3 and FR4), as distinct from the constant region located at the C-terminal end of the heavy or light chain.
The peptides of SEQ ID No 2, 3 and 4 correspond to CDRs of a VH3 while the peptides of SEQ ID No 5. 6 and 7 correspond to CDRs of a VL.
Where the immunoglobulin variable domains according to the above aspects of the invention refer to SEQ ID numbers having peptide sequences, then "fragment" includes where one or more of the amino acid residues of the SEQ ID sequence is absent, provided that such a change results in a peptide whose basic properties, for example binding activity, have not significantly been changed.
Similarly, where the hnmunoglobulin variable domains according to the above aspects of the invention refer to SEQ ID numbers having peptide sequences, then "valiant" includes where there are one or more amino acid insertions, deletions, or substitutions, either conservative or non-conservative, of the amino acid residues of the SEQ ID sequence, provided that such a change results in a peptide whose basic properties, for example binding activity, have not significantly been changed.
By "conservative substitutions"' is intended combinations such as GI3'. Ala; VaI, lie. Leu; Asp. GIu: Asn, GIn: Ser, Thr; Lys; Arg; and Phe, Tyr.
Such valiants may be made using well known methods of protein engineering and site-directed mutagenesis.
Preferably, the fragments or variants of the peptide sequences of the immunoglobulin variable domains according to the above aspects of the invention have up to 1, 2, 3. 4 or 5 amino acid residues that are absent and/or variant. For example, the immunoglobulin variable domain according to the first aspect of the invention comprises all or a fragment or variant of SEQ ID No 2, 3 and 4. The immunoglobulin variable domain may contain 0, 1, 2, 3. 4 or 5 absent and/or variant amino acids in each of the peptide sequences given in SEQ ID No 2. 3 and 4.
However, it is preferred that the immunoglobulin variable domain comprises up to 1. 2. 3. 4 or 5 (ie O5 I3 2, 3, 4 or 5) absent and/or variant amino acids in total from the peptide sequences given in SEQ H) No 2, 3 and 4; or SEQ E) No 5, 6 and 7.
Preferably, the fragments or variants of the peptide sequences of the immunoglobuhn. variable domains according to the above aspects of the invention have only conservative substitutions. Preferably, there are no absent and/or variant amino acids to the peptide sequences given in SEQ ID No 2. 3 and 4; or SEQ ID No 5, 6 and 7.
"Where the immunoglobulin variable domains according to the above aspects of the invention refer to SEQ ID numbers having polynucleotide sequences, then "fragment" includes where one or more of the nucleotides is absent, provided that such a change results in a polynucleotide that encodes a peptide whose basic properties, for example binding activity, have not significantly been changed.
Similarly, where the immunoglobulin variable domains according to the above aspects of the invention refer to SEQ ID numbers having a polynucleotide sequences, then "variant" includes where there are one or more nucleotide insertions, deletions,
or substitutions, of the given nucleotide residues of the SEQ ED sequence, provided that such a change results in a polynucleotide encoding a peptide whose basic properties, for example binding activity, have not significantly been changed. For example, different codons can be substituted which code for the same amino acidfs) as tlie original codons. Alternatively, the substitute codons may code for a different amino acid that will not affect the activity of an encoded peptide or which ma}' improve its activity. For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In 'Vitro Mutagenesis," Science, 229: 193-1210 (1985), which is incorporated herein by reference. Since such modified polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention.
Preferably, the fragments or valiants of the polynucleotide sequences encoding the immuiioglobuhn variable domains according to the above aspects of the invention have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 (ie 0, 1, 2, 3, 4, 55 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) nucleotides that are absent and/or variant. For example, the immunoglobulin variable domain according to the second aspect of the invention comprises a fragment or variant of SEQ K) No 8, 9 and 10. Hence the irrrmunoglobuhn variable domain may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 (ie 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) absent and/or variant nucleotides in each of the polynucleotide sequences given in SEQ ID No 8, 9 and 10.
However, it is preferred that the immunoglobulin variable domain comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 (ie 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) absent and/or variant nucleotides in total from the polynucleotide sequences given in SEQ ID No 8, 9 and 10; or SEQ ID No 11, 2 and 13. Preferably, there are either no absent and/or variant nucleotides, or only variant nucleotides that do not lead to a change in the encoded amino acid sequence, in
the polynucleotide sequences given in SEQ ID No 8, 9 and 10: or SEQ ID No 113 2 and 13.
It is also preferred that the absence and/or \.7ariance of nucleotides in SEQ ID No 8, 9 and 10. or SEQ ID No 11, 12 and 13 do not introduce a frame shift in the open reading frame or a stop codon. as would be appreciated by a person skilled in the art.
Preferabfy, the fragments or variants of the potynucleotide sequences of the immunoglobulm variable domains according to the above aspects of the invention introduce no or onfy conservative substitutions into the encoded peptide sequence of the immunoglobulin valuable domain. Preferabhy, there are no absent and/or variant nucleotides to the polynucleotide sequences given in SEQ ID No 8, 9 and 10, or SEQ ID No 11, 12 and 13.
"Significantly" in the above context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.
The mention above of the "binding activity" of the peptide(s) is further discussed below in relation to further embodiments of the invention and in the accompanying example.
Preferably, an immunoglobulin variable domain of the invention comprises the peptide sequences:
FDDYGMAWVRQAPG, SGVSWNGSRTHYADSVKGR, ARGVAGHDY [SEQ ID No 2, 3 and 4].
These peptide sequences are encoded by the polynucleotide sequences:
TTTGATGATTATGGCATGGCCTGGGTCCGCCAAGCTCCAGGG, TCGGGTGTTAGTTGGAATGGCAGTAGGACGCACTATGCAGACTCTGTG AAGGGCCGA5 GCGAGAGGAGTAGCTGGTCATGACTAC [SEQ ID No 8.. 9 and 10]
Alternatively the immunoglobulin variable domain of the invention comprises the peptide sequences:
CSGSRSNIGSHYVF, DNNICRPS, CAAWDGSLSGHW [SEQ ID NO 5, 6 and 7]
These peptide sequences are encoded by the polynucleotide sequences:
TGTTCTGGAAGCAGGTCCAACATCGGAAGTCATTATGTATTC, GACAATAATAAGCGACCCTCA,
TGTGCAGCATGGGATGGCAGCCTGAGTGGCCATGTGGTA [SEQTD NO H312 and 13]
The immunoglobulin variable domains of the above aspects of the invention may be s}αithesised according to methods known in the art. Typically, the immunoglobulin variable domains may be formed by expression from polynucleotide sequences encoding the immunoglobulin variable domains using techniques of molecular biology, as well known to those skilled in the art. However, chemical peptide synthesis techniques may also be used.
Peptides may be sjαithesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9- fluoreiηdmetlryloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base- labile protecting group is effected using 20% pipeiϊdine in N;N-dimeth37lfoπnamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid),
bul37loxycarbonyl derivative (in the case of lysine and histidine). trityl derivative (in the case of cysteine) and 4-methoxy-2Λ6-trimetb3'lbenzenesulphonyl derivative (in the case of argiαine). Where glutamiiαe oτ asparagine are C-temiinal residues., use is made of the 4.4'-dhiethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimetlryl-acrylamide porymer constituted from the three monomers dmiethylacrylamide (backbone- monomer), bisaciyloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent). The peptide-to-resin cleavable linked agent used is the acid-labile 44iydiuxymetlτyl-phenoxyacetic acid derivative. All amino acid derivatives are added as then" preformed S3'rnrnetricaϊ anhydride derivatives with the exception of asparagine and glutamine. which are added using a reversed N5N- dicyclohexyl-carbodiimide/l-hydiOxybenzotriazole mediated coupling procedure. AU coupling and deprotection reactions are monitored using niiihrydrin, triαitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoro acetic acid containing a 50% scavenger mix. Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesised. Trichloroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Arry scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UIC) Ltd. Nottingham NG7 2QJ5 UIC. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chramatograplτy. amino- acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
Techniques for the preparation of polynucleotides are elaborated in Sambrook. J. et al, 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, NY; or Ausubel. F. M. e1 al.f 1989. Current Protocols in Molecular Biology. John Wiley & Sons. New York N. Y.. both of which are incorporated herein by reference in their entirety. Further techniques, for example techniques using chemical or erizyrnatic S3'nthesis. will also be well known to those skilled in the art.
As discussed above, antibody molecules consist of two identical heavy-chain and two identical light-chain polypeptides. The N-terminal domains of the chains are variable in sequence and are therefore called the variable regions (V-regions). The V-regions of one heavy (VH) and one light chain (VL) associate to form the antigen-binding site.
An embodiment of the above aspects of the invention is where the peptide sequences of SEQ ID No 2, 3 and 4 and the peptide sequences encoded by the polynucleotides of SEQ ID No 8. 9 and 10 are three hypervariable regions/complementarity detenmning regions of an immunoglobulin variable domain. Preferably, the ήnnruno globulin variable domain is a VH domain.
An alternative embodiment of the above aspects of the invention is where the peptide sequences of SEQ ID No 5, 6 and 7 and me peptide sequences encoded by the pohynucleotides of SEQ ID No 11, 12 and 13 are three hypervariable regions/complementarity detenmning regions of an immunoglobulin variable domain. Preferably, the immunoglobulin variable domain is a VL domain.
Immunoglobulin valuable domains typically comprise one or more framework regions, for example four framework regions. "Vi7InIe the framework regions may comprise peptide sequences from a number of different animals, for example mice, rats, pigs, sheep, goats or other such mammals routinely used to generate antibodies for research or therapeutic puiposes, preferably the framework regions are human.
By "".framework regions"" or "FRs" we include regions of the immunoglobulin domain other than the CDRs which form the structural framework within which the CDRs are positioned to form an immunoglobulin binding domain. FRl is typically the region at the N-terminus of the variable domain located on the N- terminal side of CDRl; FR2 and FR3 are located between CDRl and CDR2 and between CDR2 and CDR3. respectively; FR4 is located at the C-terminus of the variable domain on the C-terminal side of CDR3.
Polynucleotide sequences encoding immunoglobulin framework regions (FRs) and immunoglobulin complementarity-determining regions (CDRs) that may be used in the synthesis of immunoglobulin variable domains of the invention ma}' be obtained from, pre-existing libraries of sequences that encode antibodies with different binding specificities. Such libraries are well known to those in the art and standard molecular biology techniques (such as polymerase chain reaction (PCR) using oligonucleotide primers specific for variable domain regions, or restriction enzyme digestion) may be used to obtain sequences encoding FRs and CDRs from such libraries. It will be understood that libraries containing nucleotide sequences encoding full-length immunoglobulin polypeptides or fragments thereof (for example, encoding immunoglobulin variable domains or regions thereof) may be used to obtain sequences for use in the invention.
Advantageously, the framework regions are derived from a human Ig (immunoglobulin) heavy chain. The heavy chain may be derived from the human KOL heavy chain. However, it may also be derived from, for example, the human NEWM or EU heavy chain. Moreover, the heaΛ'y chain may be derived from one of the human germ line heavy chain genes, such as DP47.
Alternatively, the framework regions may be derived from a human kappa or lambda light chain. The light chain is preferably derived from the human REI light chain.
However, it may also be derived from, for example, the human EU light chain.
Moreover, the light chain ma}' be derived from one of the human genu line light chain genes, such as DPL3.
Sequences encoding framework regions may be sj'nthesised άe novo using standard technologies. For convenience the framework sequences ma}' overlap with constant parts in the sequences containing the CDRs allowing easy assembly of a full length and complete VH or VL-
A further aspect of the invention provides an antibody comprising one or more immunoglobulin valuable domains according to the previous aspects of the invention and one or more immunoglobulin constant domains.
Constant domains may be selected to have desired effector functions appropriate to the intended use of the antibody so constructed. For example, human IgG isotypes, IgG1 and IgGs are effective for complement fixation and cell mediated lysis. For other purposes other isotypes, such as IgG2 and IgG4. or other classes, such as IgM and IgE. may be more suitable.
Constant domains ma)' comprise peptide sequences from a number of different animals, for example mice, rats, pigs, sheep, goats or other such mammals routinely used to generate antibodies for research or therapeutic purposes.
For human therap3', it is particularly desirable to use constant domains from human isotypes to minimise antiglobulin responses during therapy. Human constant domain DNA sequences, preferably in conjunction with their variable domain framework bases can be prepared in accordance with well-known procedures. An example of this is CMdPATH IH available from The Wellcome Foundation Ltd.
Examples of IgGl constant domains which ma)' be used in this aspect of the invention include the "lambda 2'" (light chain), as encoded by the coding regions of the polynucleotide sequence provided in GenBank Accession Number X06875, and the "gamma 1" (heavy chain) as encoded by the coding regions of the
poh'iiucleotide sequence provided in GenBank Accession Number Zl 7370 (allotype GIm(Z)J.
Examples of IgG4 constant domains which may be used in this aspect of the invention include the "lambda 2" (light chain), as encoded by the coding regions of the pofynucleotide sequence provided in GenBank Accession Number X06875, and the heavy chain polypeptide encoded by coding regions of the polynucleotide sequence provided in GenBank Accession Number K01316.
A further aspect of the invention provides an antibody comprising one or more polypeptides of SEQ ID No 14 or 15 or a fragment or variant thereof.
The antibody may, for example, be encoded by the polynucleotide sequence of SEQ JD No 16 or 17 or a fragment or variant thereof.
The following amino acid and polynucleotide sequences axe included in this aspect of the invention.
EVQ]XESGGGLVQPGGSIJILSCAASGFTFDDYGMAWVRQAPGKGLEWV SGVSWNGSRTHYADSVKGRFΉSRDNSKNTLYLQMNSLRAEDTAVΎYCA RGVAGHDYWGQGTLVWSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVWSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICM^OHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEΛ^ΖFNWYΛΦGVEVHNAICTKPREEQYNS T^RVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQV ΎTLPPSRDELTICNQVSLTCLΛ/-KGFYPSDIAΛ^E^/ESNGQPENNYKTTPPVL DSDGSFPLYSKLTVDKSRWQQGNWSCSVMHEAIJINEIYTQKSLSLSPGK
[SEQ ID No 14]
QSVLTQPPSASGTPGQRVπSCSGSRSNIGSHYWWYQQLPGTAPKLLIYD NNKRPSGWDRFSGSKSGTSASIAISGLRSEDEAD YYCAAWDGSLSGHVV FGGGTKLWLGQPKAAPSVTLITPSSEELQANKATLVCUSDFYPGAVTVA
MCADSSPVICAGVETTTPSICQSI^NKY^ASSIXSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS [SEQ ID NO 15]
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGG TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATG GCATGGCCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGTGGGTAT CGGGTGTTAGTTGGAATGGCAGTAGGACGCACTATGCAGACTCTGTGA AGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATC TGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTG CGAGAGGAGTAGCTGGTCATGACTACTGGGGCCAGGGTACACTGGTCA CCGTGAGCTCAGGTGAGTCGTACGCTAGCAAGCTTTCTGGGGCAGGCC AGGCCTGACCTTGGCTTTGGGGCAGGGAGGGGGCTAAGGTGAGGCAG GTGGCGCCAGCCAGGTGCACACCCAATGCCCATGAGCCCAGACACTGG ACGCTGAACCTCGCGGACAGTTAAGAACCCAGGGGCCTCTGCGCCCTG GGCCCAGCTCTGTCCCACACCGCGGTCACATGGCACCACCTCTCTTGC AGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAG CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAAAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAA GCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCA GTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCT CTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCTGGCTTTTTCCCC AGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCAC ACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGG GAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCA CTCCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCA ATCTTCTCTCTGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCC ACCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCA1AGGCG GGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCG
GGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGG GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA TGATCTCCCGGACCCCTGAGGTCACATGCCTTGGTGGTGGACGTGAGCC ACGAAGACCCTGAGGTCAAGTTCA-ACTGGTACGTGGACGGCGTGGAG GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA TGGCAAGGAGTACAAGTGC-AAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGC GAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGA GTGACCGCTGTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC GTGATGCATGAGGCTCTGCACAA-CCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAA [SEQ ID No 16]
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAG AGGGTCACCATCTCTTGTTCTGGAAGCAGGTCCAACATCGGAAGTCAT TATGTATTCTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTC ATCTATGACAATAATAAGCGACCCTCAGGGGTCCCTGACCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGG TCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGGCAGCCTG AGTGGCCATGTGGTATTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT GAGTAGAACGTACGCTAGCAAGCTTGGATCCACGATCCTGAGCAAGGA CCTCTGCCCTCCCTGTTCAGACCCTTGCTTGCCTCAGCAGGTCATTACA ACCACTTCACCTCTGACCGCAGGGGCAGGGGACTAGATAGAATGACCT ACTGAGCCTCGTCTGTCTGTCTGTCTGTCTCTCTGTTTGTCTGTCTGTCT CTCTGTTTGTCTCTCTGTCTGTCTGACAGGCGCAGGCTGGGTCTCTAAG CCTTGTTCTGTTCTGGCCTCCTCAGTCTGGGTTCTTGTCGGAACAGCTTT GCCCTTGGGTTACCTGGGTTCCATCTCCTGGGGAATTGGGAACAAGGG
GTCTGAGGGAGGCACCTCCTGGGAGACTTTAGAAGGACCCAGTGCCCT CGGGGCTGATGCTCGGGAATCACAGAGCTGCTGACCCAGAGCCAGGAT CCAGACCCAGAATGAGGTAGGAGGTGGAGGGGCTGCCCTGGCTCGTCT GGGGGCTGCCAGGGACTGAGCCCTGAGCCAGCCTGAGACTCAGGAAA CCCCGTCAGGAGGGAGAAGGGAGAAGCAGACTCTGGACACCAGAAAG CCAGGGGAAGGGTCACAAAAGGAGTGGATGTGACGGAAGGGCGGGCT CCTGGGTCTCTTCAGAACATATCCCCTGTGCCCAGGGGGATCAGAGCTG GCAGAGTCCACTGCGTGAAAGCCCCACTGCTATGACCAGGTAGCCGGG ACGTGGGGTGGATGCCAGAAAAGACTCCATGGAATAAGAGAGAGCCC AGGACAGCAGGCAGGCTCTCCGATCCCCCCAGGCCCTTGCCCCATACA CGGGCTCCAGAACACACATTTGGCTGGAACAGCCTGAGGGACCAAAA GGCCCCAGTATCCCACAGAGCTGAGGAGCCAGGCCAGAAAAGTAACC CCAGAGTTCGCTGTGCAGGAGAGACACAGAGCTCTCTTTATCTGTCAG GATGGCAGGAGGGGACAGGGTCAGGGCGCTGAGGGTCAGATGTCGGT GTTGGGGGCCAAGGCCCCGAGAGATCTCAGGACAGGTGGTCAGGTGTC TAAGGTAAAACAGCTCCCCGTGCAGATCAGGACATAGTGGAAAACAC CCTGACCCCTCTGCCTGGCATAGACCTTCAGACACAGAGCCCCTGAAC AAGGGCACCCCAACACCTCATCATATACTGAGGTCAGGGGCTCCCCAG GTGGACACCAGGACTCTGACCCCCTGCCCCTCATCCACCCCGCAGGTC AGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGA GCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTA CCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCA AGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAG TACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCC CACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGA GAAGACAGTGGCCCCTACAGAATGTTCA [SEQ ID No 17]
The pofypepiide sequence of SEQ ID No 14 comprises the peptide sequences of SEQ ID No 2. 3 and 4 as well as framework regions as discussed above and comprises a VH variable domain sequence as well as constant region sequences. Similarly, the pofypeptide sequence of SEQ ID No 15 comprises the peptide sequences of SEQ ID No 5, 6 and 7 as well as framework regions as discussed
above and comprises a VL variable domain sequence as well as constant region sequences.
The polynucleotide sequence of SEQ ID No 16 encodes the polypeptide sequence of SEQ ID No 14 and the polynucleotide sequence of SEQ ID No 17 encodes the polypeptide sequence of SEQ ID No 15. Amino acids 1 to 116 of SEQ ID No 14 are the VH domain sequence and are encoded by nucleotides 1 to 348 of SEQ ID No
16. Amino acids 1 to 112 of SEQ JD No 15 are the VL domain sequence and are encoded by nucleotides 1 to 336 of SEQ ID No 17. Thus, in SEQ ID No 14 the constant part stalls with AST and in SEQ ID No 15 the constant part with QPK.
The polypeptides of SEQ ID No 14 and 15 or the variable domains thereof are capable of forming an antibody or antibody fragment.
A "variant" of the polypeptide of SEQ ID No 14 or 15 will have a region which has at least 90% (preferably 95. 96. 97. 98 or 99%) sequence identity with the polypeptide of SEQ ID Nos 14 or 15 as measured by the Bestfit Program of the Wisconsin Sequence Analysis Package, version 8 for Unix. The percentage identity may be calculated by reference to a region of at least 50 amino acids (preferably at least 75, 100, 120 or 140) of the candidate variant molecule, and the most similar region of equivalent length in the polypeptide of SEQ ID No 14 or 15, allowing gaps of up to 5%.
A "vaiiant" of the polypeptide of SEQ ID No 14 or 15 is one hi winch the basic properties of the polypeptide, for example binding activity, have not been significantly changed.
The percent identity may be determined, for example, by comparing sequence infoπnati on using the GAP computer program, version 6.0 described by Devereux et al. (Nucl Acids res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Neddleman and Wunsch (J MoI Biol, 48:443, 1970), as
revised by Smith and Waterman (Adv. Appl Math 2.482. 1981). The preferred default parameters for the GAP pro grain include : (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non -identities) for nucleotides, and the weighted comparison matrix of Bribskov and Burgess, NucJ. Acids Res. 14:6745, 1986 as described by Schwaits and Da3'hoff, eds, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each S3'mbol in each gap; and (3) no penalty for end gaps.
A "fragment" of the potypeptide of SEQ ID No 14 comprises at least 100, 200, preferably 300, 350, 400 or 425 amino acids, or 100, preferably, 125, 150, 175 or 200 amino acids of the polypeptide of SEQ ID No 15. A "fragment" of the pofypeptide of SEQ ID No 14 or 15 is one in which the basic properties of the polypeptide, for example binding activity, have not been significantly changed.
Preferably, fragments or variants of the polypeptide sequences of the SEQ ID Nos 14 and 15 have only conservative substitutions. Preferably, there are no changes to the polypeptide sequences given in SEQ ID No 14 and 15.
It is possible to determine whether the fragments and variants of the sequences provided have so changed the binding activity of the polypeptide of SEQ ID No 14 or 15 using methods provided in the example and outline further below.
A "variant" of Hie polynucleotide of SEQ ID 16 or 17 is one which is usable to produce a polypeptide or a fragment thereof whose basic properties, for example binding activity, have not been significantly changed. For example, different codons can be substituted which code for the same amino acid(s) as the original codons. Alternatively, the substitute codons ma}' code for a different amino acid that will not affect the activity of the protein or which may improve its activity. For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro
Mutagenesis.," Science, 229: 193-1210 (1985). which is incorporated herein by reference. Since such modified polynucleotides can be obtained b)7 the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention.
Moreover, it will be recognised by those skilled in the art that the polynucleotide sequence (or fragments thereof) of SEQ ID No 16 or 17 can be used to obtain other DNA sequences that hybridise with it under conditions of high stringency. Such DNA includes any genomic DNA. Accordingly, this aspect of the invention includes DNA that shows at least 85 per cent preferably 90 per cent, and most preferably 95 per cent homology with the polynucleotide of SEQ H) No 16 or 17.
DNA-DNA5 DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0. IXSSC and 6XSSC and at temperatures of between 55°C and 70°C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By "high stringency" we mean 2XSSC and 650C. IXSSC is 0.15M NaCl/0.015M sodium citrate.
A "fragment" the polynucleotide of SEQ ID No 16 or 17 comprises at least 100O5 preferably 150O5 1750, 2000 or 2100 nucleotides of the polynucleotide of SEQ ID No 16, or 1000, preferably, 1500, 1600, 1700 or 1800 nucleotides of the polynucleotide of SEQ H) No 17. A "fragment" of the polynucleotide of SEQ ID No 16 or 17 is one in which the basic properties of the encoded polypeptide, for example binding activity, have not been significantly changed.
It is possible to determine whether the fragments and variants of the sequences provided have so changed the binding activity of the polypeptide encoded by SEQ ID No 16 or 17 using methods provided in the example and outline further below.
Preferably, a fragment or variant of the polypeptide or polynucleotide of SEQ ID No 14, 155 16 and 17 contains variations of the CDR regions (i.e. SEQ ID No 2, 3,
4. 5. 6. or 1). as discussed above in relation to the iniimmo globulin binding domains of the invention.
It is also preferred that the absence and/or variance of nucleotides in SEQ ID No 16 and 17 do not introduce a frame shift in the open reading frame or a stop codon. as would be appreciated by a person skilled in the art.
Preferabfy. the fragments or variants of the polynucleotide sequences of SEQ ID No 16 and 17 introduce only conservative substitutions into the polypeptide sequence of the SEQ ID No 14 and 15. Preferably, there are no changes to the polynucleotide sequences given in SEQ ID No 16 and 17.
The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e.. molecules that contain an antigen-binding site that specifically binds (imniunoreacts with) an antigen. Such antibodies include, but are not limited to. polyclonal, monoclonal, chimeric, single chain. Fab, Fab1 and F(ab')2 fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any of the classes IgG5 IgM. IgA. IgE and IgD3 which differ from one another by the nature of the hea^7 chain present in the molecule. Certain classes have subclasses as well, such as IgGi- IgGi, IgG3. IgG4 and others. Furthermore;, hi humans, the light chain ma)' be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
These immunoglobulin molecules include Fab-lilce molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single- chain Fv (ScFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird el al (1988) Science 242, 423; Huston et al (1988) Proc. Natl Acad. ScL USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989) Nature 341. 544). A general review of the
techniques involved in the S3'iτthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nanire 349., 293-299.
By "ScFv molecules" we mean molecules wherein the VH and VL partner domains are linked via a flexible oligopeptide.
The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv5 ScFv and dAb antibod}' fragments can all be expressed in and secreted from different cells, such as E. coli, NSO, Chinese Hamster Ovarian (CHO) and human cells thus allowing the facile production of large amounts of the said fragments.
Whole antibodies, and F(ab')o fragments are ''bivalent". By "bivalent" we mean that the said antibodies and F(ab')2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent having only one antigen combining sites.
We consider that an immunoglobulin binding domain or antibody of the present invention is distinct from naturally occurring immunoglobulin binding domains and antibodies. However, an embodiment of any of the aspects of the invention is wherein the immunoglobulin binding domain or antibod}' is isolated and/or is recombinant.
An embodiment of tins aspect of the invention is wherein the antibod)7 further comprises an iininunoglobulin constant domain.
An embodiment of the previous aspects of the invention is wherein the immunoglobulin variable domain or antibody selectively binds HTV Tat polypeptide. Preferabfy, the immunoglobulin variable domain or the antibody selectively binds epitope II of HTV Tat polypeptide.
As discussed above the Tai polypeptide has a central role in the replication of HIV and contains a number of epitopes, i.e. binding regions, recognised b}7 antibodies (antigenic sequences) in the N-terminal linear sequence and the C-terminal linear sequence of exon 1 of Tat. Epitope II is a ten amino acid sequence of amino acids at positions 41 to 50 of exon 1. TMs has been given the general formula:
KXLGISYGRX [SEQ ID NO I]5 where X is Ala or GIy.
By "selectively binds" we include that the immunoglobulin variable domain or the antibody recognises and binds to the Tat polypeptide, but may also interact with other proteins through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assa)'S to determine binding specifϊcit}' of an immunoglobulin variable domain or antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et άl. (Eds). Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N. Y. (198S)5 Chapter 6.
Further methods that may be used in determining the binding characteristics of an immunoglobulin variable domain antibody or antibody include binding assays such as, for example, ELISA, affinity determinations using eg Biacore and functional in vitro and in vivo assays, as would be appreciated by a person skilled in the art. Further studies include FACS anafysis and ήmnunolήsto chemistry for assessment of target specificity and cross-reactivity.
Still further examples of methods of detemiining the binding specificity of an immunoglobulin binding domain or antibody of the invention are provided in the accompanying examples.
As mentioned above, the peptide and polynucleotide sequences of the immunoglobulin binding domains of the invention include frasments and variants
of those sequences, provided that such changes result in an immunoglobulin binding domain whose basic properties, for example binding activity, have not significantly been changed. It is possible to determine whether the fragments and variants of the sequences provided have so changed the binding activity of the immunoglobulin binding domain using the methods provided in the examples and discussed herein.
A further aspect of the invention provides an immunoglobulin variable domain or an antibody capable of binding to epitope II of the Tat polypeptide and having a binding affinity of at least 10"s to epitope II of the TAT pofypeptide.
The value of the binding affinity of the immunoglobulin variable domain or antibody to epitope II of TAT polypeptide is very dependent on the measuring method used. The binding affinity 10' specified in this aspect of the invention was determined using the Biacore binding affinity method detailed in the examples.
Preferabfy, the immunoglobulin valuable domain or antibody of this aspect of the invention can have a binding affinity of 10'9, 10"30, 10"π, 10"12 to epitope II of TAT polypeptide when measured using the Biacore binding affxoity method detailed in the examples.
An embodiment of this aspect of the invention wherein the immunoglobulin variable domain is an immunoglobulin variable domain according to any of the previous aspects of the invention, or the antibody is an antibody according to an}' of the previous aspects of the invention.
Antibodies and immunoglobulin binding domains can be identified using techniques including phage display libraries (Hoogenboom and Winter, J. MoI. Biol, 227:381 (1991); Marks et al, J. MoI. Biol., 222:581 (1991)).
Recently a novel type of technology was developed for generation of variability in human antibody libraries. Soderlind et al, Nat Biotechnology (2000) 18(8): 852-85 and WO 98/32845. both of which are incorporated herein by reference, disclose a
technology termed n-CoDeRφ. The technology utilises a single human framework in VH and VL respectively, and CDRs are shuffled randomly into their respective positions thereby generating a great variability. The library has been used extensively to select high affinity antibody fragments against all types of antigens including haptens, peptides, proteins including human proteins and also carbohydrates. AU parts of the antibodies are full}' human and are derived from human donors. Thus, even though the librae is restricted to one type of framework it is possible to find solutions to all types of antigenic topographies with variations in charge and hydrophobicity within the library. In fact, it has been demonstrated that the library can reach into paits of sequence space where normal human antibodies cannot (Borrebaeck CA, Ohlin M. Nat Biotechnol. (2002) Dec;20(12): 1189-90). The n-CoDeR® library is built using a modular approach where some modules (framework sequences) are kept constant while other modules (the CDRs) are allowed to vary.
Hence, it is preferred that the further antibodies or immunoglobulin binding domains of this aspect of the invention are generated using n-CoDeR® as described in WO 9S/32845.
Methods by which to determine whether an immunoglobulin binding domain or anfibod}' has a suitable binding affinity to epitope II of the TAT polypeptide are provided in the example below.
In general, a human antibody molecule relates to any of the classes IgG5 IgM, IgA3 IgE and IgD5 which differ from one another by the nature of the heavy chain present hi the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, and others.
An embodiment of any of the aspects of the invention is wherein the antibod}' is an IgG] antibody, preferably a human IgG] antibody.
Aj] embodiment of an)7 of the aspects of the invention is wherein the immunoglobulin variable domain or antibody further comprising a member of a specific binding pair.
AQ immunoglobulin variable domain or antibody of the invention may be linked with a member of a specific binding pair, e.g. biotin. so that it can be retrieved from solution by use of an anti-tag, e.g. avidin. coupled to a solid support, e.g. magnetic beads. Further examples of specific binding pairs are known to those in the art, e.g. maltose and maltose binding protein.
An embodiment of any of the aspects of the invention is wherein the immunoglobulin variable domain or antibody comprising a further therapeutic entity.
For example, the further entity may be of use in therapy, e.g. a toxin, an enzyme capable of activating a prodrug, a binding partner or a radioisotope. The furfher entity ma)' be a protein such as an enzyme useful in isolating the immunoglobulin variable domain or antibody, e.g. Glutathione S -transferase. The further entity may be an oligonucleotide (may be either DNA5 RNA or PNA). The further entity may also be beneficial in imaging, e.g. it ma}' be a label.
A further aspect of the invention provides a polypeptide comprising all or a fragment or variant of each of polypeptide sequences SEQ ID No 2, 3, 4, 5, 6 and 7; or all or a fragment or variant of each of polypeptide sequences amino acids 1 to 116 of SEQ ID No 14 and amino acids 1 to 112 of SEQ ID No 15; or all or a fragment or variant of each of polypeptide sequences SEQ ID No 14 and 15. SEQ ID No 2. 3, 4, 5, 6, 7, 14 and 15 and fragments and variants thereof are discussed above. Methods of obtaining such a polypeptide are also discussed above. The polypeptide may typically be an antibody (including an antibody fragment, as discussed above).
A further aspect of the invention provides a polynucleotide or polynucleotides encoding the polypeptide of the preceding aspect of the invention. The polynucleotide or polynucleotides ma)' comprise all or a fragment or variant of each of the polynucleotide sequences of SEQ ID No S5 9. 10. 11, 12 and 13; or all or a fragment or variant of each of the polynucleotide sequences 1 to 348 of SEQ ID No 16 and 1 to 336 of SEQ ID No 17; or all or a fragment or variant of each of the polynucleotide sequences of SEQ ID No 16 and 17.
SEQ ID No 8, 9, 1O5 11, 12, 13, 16 and 17 and fragments and variants thereof are discussed above.
Methods of obtaining a polynucleotide of this aspect of the invention include using the well known PCR technique in which a number of overlapping oligonucleotides are used to synthesise a complete polynucleotide of the sequence given. Oligonucleotides, i.e.. small nucleic acid segments, may be readily prepared b};. for example, directly synthesizing the oRgonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide S3rnthesizer.
A further aspect of the invention provides an expression vector comprising a polynucleotide sequence of the previous aspect of the invention.
The expression vector could express a given polynucleotide in conjunction with one or more further polynucleotide sequences so as to encode a scFv polypeptide. The tenn 'scFv' is discussed above.
For example, SEQ ID No 16 encodes a heavy chain polypeptide, while SEQ ID No 17 encodes a light chain polypeptide. Amino acids 1 to 116 of SEQ ID No 14 are the VH domain sequence and are encoded by nucleotides 1 to 348 of SEQ DD No 16. Amino acids 1 to 112 of SEQ ID No 15 are the VL domain sequence and are encoded by nucleotides 1 to 336 of SEQ ID No 17. Therefore the expression vector of this aspect of the invention may comprise the pofyuucleotide sequence of nucleotides 1 to 348 of SEQ ID No 16 (or a variant thereof, as discussed above) and nucleotides 1
to 336 of SEQ ID No 17 (or a variant thereof, as discussed above) separated by a polynucleotide sequence encoding a flexible oligopeptide. Such an expression vector would comprise a poh'nucleotide which, when expressed, would produce a scFv polypeptide, as known to those skilled in the art.
A further aspect of the invention is a host cell comprising an expression vector according to the invention.
In order to obtain expression of a poh'nucleotide sequence, the sequence ma}' be incorporated in a vector having control sequences operably linked to the polynucleotide sequence to control its expression. The vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted polynucleotide sequence, further polynucleotide sequences so that the protein encoded for by the polynucleotide is produced as a fusion and/or nucleic acid encoding secretion signals so that the protein produced in the host cell is secreted from the cell. The protein encoded for by the polynucleotide sequence can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the protein is produced and recovering the protein from the host cells or the surrounding medium. Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, yeast, and eukaryotic cells such as COS, NSO or CHO cells. The choice of host cell can be used to control the propeities of the protein expressed in those cells. e.g. controlling where the protein is deposited - in the host cells or affecting properties such as its glycos3'lation.
The polynucleotide sequences ma)' be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA "will depend upon the nature of the host, the maim& of the introduction of the DNA into the host, and whether episornal maintenance or integration is desired.
Generally, the polynucleotide sequence is inserted into an expression vector, such as a plasrnid, in proper orientation and correct reading frame for expression. If
necessary, the polynucleotide sequence ma)' be linked to the appropriate transcriptional and traiislational regulators' control nucleotide sequences recognised b)' the desired host, although such controls are generally available in the expression vector. Thus, the polynucleotide sequence insert ma}' be operatively linked to an appropriate promoter. Bacterial promoters include the E.coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the phage λ PR and PL promoters, the phoA promoter and the τrp promoter. Eukaryotic promoters include the CMV immediate early promoter, the HSV ttrymidine kinase promoter, the earl}' and late SV40 promoters and the promoters of retroviral LTRs. Other suitable promoters will be known to the skilled artisan. The expression constructs will desirably also contain sites for transcription initiation and teimination, and in the transcribed region, a ribosome binding site for translation (Hastings et al,
International Patent No. WO 98/16643, published 23 April 1998).
Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, potyaden3'lation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagernid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambroolc et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of polynucleotide sequences, for example in preparation of polynucleotide constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds., JoIm Wiley & Sons, 1992 and Sambroolc & Russell (2001) Molecular Cloning, a laboratory manual, Cold Spring Harbor Press, Cold Spring Harbor. NY, LTSA.
Many expression s)rstems are known, including S3'stems employing: bacteria (e g. E. coll and Bacillus subtilis) transformed with, for example, recombinant bacteriophage, plasmid or cosmid DNA expression vectors; .yeasts [e.g. Saccaromyces cerevisiae) transformed with, for example, yeast expression vectors; insect cell systems transformed with, for example, viral expression vectors
(e g baculovirus); plant cell systems transfected with, for example viral or bacterial expression vectors; animal cell systems transfected with, for example, adenovirus expression vectors.
The vectors ma)' include a prolcaryotic replicon, such as the Col El orL for propagation in a prokaryote. even if the vector is to be used for expression in other, non-prokaryotic cell types. The vectors may also include an appropriate promoter such as a prolcaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E.coli, transformed therewith.
A promoter is an expression control element formed bγ a DNA sequence that permits binding of RJSIA polymerase and transcription to occur. Promoter sequences compatible with exemplar}' bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
T}φical prokaryotic vector plasmids are: pUClδ, pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA5 USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pPJT5 available from Pharmacia (Piscataway, NJ, USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNHSA, pNH16A; pNHISA, pNH46A available from Stratagene Cloning S3'stems (La Jolla, CA 92037, USA).
A typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, NJ, USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-I cells. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscatawa)', NJ, USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
Useful 3'easl plasmid vectors are pRS403-406 and pRS4l3-416 and are generally available from Stratagene Cloning Systems (La Jolla. CA 92037. USA). Plasmids pRS403. pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRPl, LEU2 and UJRA3. Plasmids pRS4l3-416 are Yeast Centromere plasmids (YCps).
Methods well known to those skilled in the ail can be used to construct expression vectors containing the coding sequence and, for example appropriate transcriptional or translational controls. One such method involves ligation via homopolymer tails. Hoinoporymer polydA (or polydC) tails are added to exposed 3 ' OH groups on the DNA fragment to be cloned by terminal deoxynucleotidyl transferases. The fragment is then capable of annealing to the polydT (or pofydG) tails added to the ends of a linearised plasmid vector. Gaps left following annealing can be filled by DNA polymerase and the free ends joined 03' DNA ligase.
Another' method involves ligation via coheswe ends. Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
A further method uses synthetic molecules called linkers and adaptors. DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3' termini and fill in recessed 3' ends. Synthetic linkers, pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible teniiiαi. Adaptors are also chemically sj'nthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnolo -"tg-i1es Inc. New Haven. CN. USA.
A desirable way to modify a polynucleotide is to use the polymerase chain reaction as disclosed by Saila el al (1988) Science 239, 487-491. In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
The vector is then introduced into the host cell through standard techniques. Generally, not all of the hosts will be transformed by the vector and it will therefore be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence marker, with an}r necessary control elements, that codes for a selectable trait in the transformed cell. These markers include dihydrofolate reductase. G418 or neomycin resistance for eukaryotic cell culture, and tetracyclin, kanarrrycin or ampiciUin resistance genes for culturina; in E.coli and other bacteria. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
Host cells that have been transformed by the expression vector are then cultured for a sufficient time and under appropriate conditions known to those skilled hi the. ait to permit the expression of the encoded antibody or immunoglobulin binding domain. which, can then be recovered.
The antibody or immunoglobulin binding domain can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography. hydrox3dapatite chromatography and
lectin chromatography. Most preferably, high performance liquid chromatography ("ΗPLC'J is employed for purification.
The host cell may be any suitable host cell, for example bacterial, e.g. E. coli, yeast, insect mammalian or plant cell, as would be appreciated by a person skilled in the art. Preferred host cells are Chinese Hamster Ovarian (CHO) cells.
A further aspect of the invention provides a pharmaceutical composition comprising an immunoglobulin variable domain and/or an antibody and/or a polypeptide and/or a polynucleotide according to the invention and a pharmaceutically acceptable carrier.
Whilst it is possible for an immunoglobulin variable domain, antibody, polypeptide, polynucleotide or a gene therapy vector of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.
The immunoglobulin variable domain, antibody, polypeptide, polynucleotide or gene therapy vector of the invention can also be administered parenteral^, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrastemally, intracranial^, intra-musculaiiy or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants. buffers, bacteriostats and solutes which rendei the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilisedj condition requiring only the addition of the sterile liquid canier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
An immunoglobulin binding domain or antibody of the invention may be given in monthly injections, facilitating the patient's drug compliance and likely niiiϊirnizing adverse events. It may be supplied as a solution for infusion, to be kept at 2-80C, in 10 inM Sodium Phosphate, 150 mM Sodium Chloride at pH 7.4, produced with Water For injection (WFI).
A further aspect of the invention provides an immunoglobulin variable domain or antibody or a polypeptide or a polynucleotide or a gene therapy vector or a pharmaceutical composition according to the invention for use in medicine.
A further aspect of the invention provides the use of an immunoglobulin variable domain and/or an antibody and/or a pofypepytide and/or a polynucleotide and/or a gene therapy vector and/or a pharmaceutical composition according to the invention in the manufacture of a medicament for treating a patient having, or at risk of contracting or developing, HIV/AIDS.
A further aspect of the invention provides a method of treating HTV/AIDS comprising administering to a patient an immunoglobulin variable domain and/or an antibody and/or a polypeptide and/or a polynucleotide and/or a gene therap}' vector and/or a pharmaceutical composition according to the invention.
Au embodiment of the use 01 method of treatment aspects of the invention is wherein the patient has a competent immune system and is HIV-I infected.
An embodiment of the use or method of treatment aspects of the invention is wherein the patient does not have HTV.
An embodiment of the use or method of treatment aspects of the invention is wherein the patient is incapable of mounting an effective or rapid immune response to infection with HW-I .
The present invention provides immunoglobulin binding domains and antibodies which can react with the HIV Tat polypeptide to reduce viral levels of HIV, and thus may be useful hi both a therapeutic and proplrylactic context to control the development of ATDS in a large population exposed to, or infected by, HIV which produce upon infection different Tat proteins.
Compared to other HTV therapies. n-CoDeR® anti-Tat antibody will probably be given by monthly injections, facilitating the patient's drug compliance and likely niinnnizin XgD a ' dverse events.
A medicament comprising an immunoglobulin binding domain or antibody of the invention may reduce HIV viral multiplication during any initial acute infection with HIV and minimize chronic viremia leading to ADDS. This may also lower chronic viral multiplication in infected subjects, again minimizing progression to AIDS.
Such a medicament may be therapeutically administered to a HIV infected human with a competent immune system for treatment or control of viral infection. Such an infected human may be as3'mptoniatic. Similarly, the medicament ma}' be administered to an uninfected human for proplxylaxis.
The medicament may also be useful for treating a patient who is incapable of mounting an effective or rapid immune response to infection with HTV. This may be achieved by chronically administering the medicament. Among such patients suitable for treatment with this medicament are HIV infected patients who are immunocompromised b3; disease and unable to mount a strong immune response.
In later stages of HIV infection, the likelihood of generating effective titers of antibodies is less, due to the immune impairment associated with the disease. Also among such patients are HIV infected pregnant women, neonates of infected mothers, and unimmunized patients with putative exposure (e.g.. a human who has been inadvertently "stuck" with a needle used by an HIV infected human). For such patients, the medicament of the invention preferably employs the antibody of the invention. This medicament is administered as passive immttnotherapy to inhibit viral multiplication and lower the viral load. The exogenous antibodies which react with most Tat proteins from HTV provide in the patient an immediate interdiction of the transfer of Tat from virally infected cells to other infected or uninfected cells. Accordingly, the patient may be chronically treated with the antibody composition for a long treatment regimen.
The amount of immunoglobulin binding domain/antibody required to produce an exogenous effect in the patient without significant adverse side effects varies depending upon the pharmaceutical composition emplθ3'ed. In infected patients, generally, each dose will comprise between about 5 to 800 mg, preferably 400, 300, 200 or 50 mg. Said dose is preferably given in a concentration of about 5 to 100 mg/ml per injection, preferably 75 mg, 50 mg, or 10 mg.
The frequency of chronic administration may range from dairy dosages to once or twice a week to once a month, and may depend upon the half-life of the immunoglobulin binding domain/anlibody (e.g. about 7-21 days). However, the duration of chronic treatment for such infected patients is anticipated to be an indefinite, but prolonged period. Other dosage ranges may also be contemplated by one of skill in the art, particularly where administration of the immunoglobulin
binding domain/antibody is in conjunction or sequential with other anti-viral treatments.
Further anti-viral treatments which may be included in the pharmaceutical composition or medicaments of the invention include anti-retroviral drugs, for example reverse transcriptase inhibitors such as AZT, zidovudine, lamivudine, nevirapine or 3TC. or protease inhibitor drugs, for example indinavir orritonavir; or entry inhibitors, such as enfurvitide.
Preferably, the genotype of the HTV is PW-I .
Methods by which the presence and/or amount of HW Tat polypeptide bound to a immunoglobulin binding domain and/or an antibody of the invention as well known to those skilled in the art and include, for example, western blotting or ELISA.
All documents referred to herein are, for the avoidance of doubt hereby incorporated by reference.
The invention is now described by reference to the following, non-limiting, figures and examples.
Figure 1. Dose response ELISA of n-CoDeR® anti-Tat antibody against target epitope BSA conjugated peptide.
Figure 2. Inhibition experiment using 1 % initially HLV-I IIIB-infected Jurlcat cells with increasing concentrations of n-CoDeR ' anti-Tat antibodj'. Virus levels were determined through a p24 detecting ELISA at day 7 post infection.
Example 1: Characterisation of an antibody that binds to Epitope II of HIV Tat polypeptide
We have generated a fully human recombinant IgGj antibody from the n-CoDeR* phage display library. The antibody is directed against epitope II (SEQ 1 ) of the excreted regulatory protein Tat encoded by HIV-I .
Physical, chemical, and pharmaceutical properties and formulation
n~CoDeRSl anti-Tat antibody is a fully human recombinant IgGj antibody expressed in Chinese Hamster Ovarian (CHO) cells. It ma}' e.g. be supplied as a solution for infusion, to be kept at 2-8°C, in 10 mM Sodium Phosphate, 150 roM Sodium Chloride at pH 7.4, produced with Water For Injection ( WFl).
The peptide sequence of the Gamma 1 chain of the antibody is provided above as SEQ ID No 14; the corresponding polynucleotide sequence of the Gamma 1 chain of the antibody is provided above as SEQ ID No 16.
The peptide sequence of the Lambda chain of the antibody is provided above as SEQ ID No 15; the corresponding polynucleotide sequence of the Gamma 1 chain of the antibody is provided above as SEQ ID No 17.
Table 1. CDR sequences. The CDRs are also indicated in the specification above.
In vitro studies have been performed to characterise the affinity, specificity and biological activity of an n-CoDeR Φ'"' anti-Tat antibody.
Affinity
To test the binding kinetics of n-CoDeR~' anti-Tat antibody, an assay was conducted using Biacore and data were analysed using BIAevaruation software. The ka, kd and KD were determined and are shown in Table 2 below.
Table 2. Affinity measurements of D-CoDeR* anti-Tat antibody against target peptides The peptides are named GG284 and GG285 and exhibit an amino acid variations (boldedj immediate]}' upstream of the target epitope (underlined).
AU antigens are biotinylated peptides.
Binding Characteristics by ELISA
ELISA analysis confirms that n-CoDeR® anti-Tat antibody binds specifically to the target epitope in a concentration dependent mariner. Twelve two fold serial dilutions of n-CoDeR® anti-Tat antibody ranging from lμg/ml were incubated on peptide coated plates (peptide was conjugated to BSA). The plate was washed and then probed with HRP labelled rabbit anti-human IgG and developed with PIERCE super signal. Luminesence was measured at 700 nm. Half maximal binding was achieved at 0.7 nM of n-CoDeR® anti-Tat antibody. The data is presented in Figure 1.
Functional in vitro studies
The ability of n-CoDeRφ anti-Tat antibody to inhibit HTV-I replication has been tested in a set of experiments performed at Karolinslca Institute. The experiments
have been based on HTV-I IHB infected Jurkat cells (human T-cell line) in which viral replication has been monitored through an ELISA directed against the surface protein p24 (Re el aL 1995 ).
Several experiments have been performed with similar results. An example is shown in Figure 2.
The IC50 value for n-CoDeR(fi) anti-Tal antibody has been calculated from inhibition experiments performed with 1 % and 3 % initially infected cells and resulted in 0.05 μg/ml and 0.07 μg/rnl, respectively (Table 3).
Table 3. IC50 values calculated for antibody n-CoDeR® anti-Tat antibody in 1 % and 3 % HIV 1 TUB infected Jurkat cells, respectively.
The ability of n-CoDeR® anti-Tat antibody to inhibit viral replication in Jurkat cells expressing Tat with arnino acid variations near the target epitope (same variations that are present in patient isolates) has been tested in a new set of experiments, recentfy performed. The result suggests that these amino acid variations do not influence the ability of the antibody to bind to and neutralise Tat.
4. References
Brake DA5 Goudsmit J, Krone WJ3 Schammel P5 Appleby N, Meloen RH5 Debouck C. Characterization of murine monoclonal antibodies to the tat protein from human immunodeficiency virus type 1. J Virol. 1990;64(2):962-965.
Re5 M. C, et.al. Effect of antibody to HIV-I Tat protein on viral replication in vitro and progression of HIV-I disease in vivo. J. Acq. Irmn. Def. Syndx. And Hum. Retroviral. 1995;10:408-415.
Steinaa, L., Sorensen. A. M., Nielsen, J. O.. Hansen. J. E. Antibody to HIV-I Tat protein inhibits the repication of virus in culture. Arch. Virol. 1994; 139(3-4) :263- 271.
Example 2: Antibody affinity evaluation with Biacore
Biacore sj'stems utilize the phenomenon of surface plasmon resonance (SPR) to perform antibody antigen interaction anafysis. As molecules adhere to a sensor surface (chip), the refractive index at the interface between the surface and a solution flowing over the surface changes. When a sample of antibodies is passed over the immobilized antigen on the sensor surface the changes in index is recorded as a sensogram. The sensorgram shows an increasing response as the antibodies binds (antibody binding peptide). The response remains constant if the interaction reaches equilibrium. When sample is changed to only buffer, the response decreases as the immuncomplex dissociate. The kinetics of the complete interaction, i.e. the rates of complex formation (lca) and dissociation (ltd), can be determined from the information in a sensorgram. The BIAevaluation software (Biacore soft ware) used generates the values of ka and kά by fitting the data to interaction models. The software evaluation of the graph is done by numerical integration methods and global fitting methods. There are two general ways to calculate the total affinity of an interaction either by using the level of binding at equilibrium (seen as a constant signal) as a function of sample (antibody) concentration or it can also be determined from' the kinetic estimations hi the sensogram. For a simple 1 :1 interactions the equilibrium constant ICD is the ratio of the kinetic rate constants, kd/ka.
Biotinylated peptides were immobilized on avidin coupled Biacore sensor cliip SA and BSA conjugated peptide was coupled direct])' to dextran matrix of a CMS chip.
Steps were taken to ensure comparable molar iinmobilisation concentrations of the respective antigens. The immobilisation level was kept low in order to minimize the influence of avidity. Five different concentrations of each antibod}' were injected consecutively on the chip and the recorded affinity was the summery of the data from all of the injections. Resulting binding curves were analysed with BIAevahiation. No background binding to the chip matrix or to non relevant peptides were detected.
Claims
1. An immunoglobulin variable domain comprising all or a fragment or variant of each of peptide sequences SEQ ID No 2, 3 and 4.
2. An immunoglobulin variable domain having complementarity determining regions (CDRs) encoded by all or a fragment or variant of each of the polynucleotide sequences of SEQ ID No S5 9 and 10.
3. An immunoglobulin variable domain comprising all or a fragment or variant of each of peptide sequences SEQ ID No 5, 6 and 7.
4. An immunoglobulin variable domain having CDRs encoded by all or a fragment or variant of each of the polynucleotide sequences of SEQ ID No 11. 12 and 13.
5. The inxmunoglobulin variable domain according to claims 1 and 2 wherein the immunoglobulin variable domain is a VH domain.
6. The immunoglobulin variable domain according to claims 3 and 4 wherein the immunoglobulin variable domain is a VL domain
7. An immunoglobulin variable domain according to any one of the previous claims comprising one or more framework regions.
S. An immunoglobulin variable domain according to claim 7 wherein the framework regions are human.
9. An antibody comprising one or more immunoglobulin variable domains according to any one of the previous claims and one or more immunoglobulin constant domains.
10. An antibody comprising one or more polypeptides of SEQ ID No 14 or 15 or a fragment or variant thereof; or comprising one or more polypeptides of amino acids 1 to 116 of SEQ ID No 34 or amino acids 1 to 112 of SEQ ID No IS5 or a fragment or variant thereof.
3 1. An immunoglobulin variable domain according to any one of claims 1 to 8 or an antibody according to claim 9 or 10 wherein the immunoglobulin variable domain or the antibody selectively binds HIV Tat potypeptide.
12. The immunoglobulin variable domain or an antibody according to claim 11 wherein the immunoglobulin variable domain or the antibody selectively binds epitope II of HTV Tat polypeptide.
13. An immunoglobulin variable domain or an antibody capable of binding to epitope II of the Tat polypeptide and having a binding affinity of at least 10~8 to epitope II of the TAT polypeptide.
14. The immunoglobulin variable domain or antibody according to claim 13 comprising an immunoglobulin variable domain according to an}' one of claims 1 to 9 or an antibody according to claims 10 or 11.
15. An antibody according to any of the previous claims wherein the antibodj' is an IgGj antibod}'.
16. An immunoglobulin variable domain or antibody according to any one of the previous claims further comprising a member of a specific binding pair.
17. An immunoglobulin variable domain or antibody according to an}' one of the previous claims comprising a further therapeutic entity.
18. A polypeptide comprising all or a fragment or variant of each of pofypeptide sequences SEQ ID No 2, 3, 4. 5. 6 and 7; or all or a fragment or variant of each of polypeptide sequences amino acids 1 to 116 of SEQ ID No 14 and amino acids 1 to 1 12 of SEQ ID No 15; or all or a fragment or variant of each, of polypeptide sequences SEQ ID No 14 and 15
19. A polynucleotide or pofynucleotides encoding the polypeptide of claim 18.
20. The polynucleotide or polynucleotides according to claim 18 comprising all or a fragment or variant of each of the polynucleotide sequences of SEQ ID No 8, 9, 10. 11. 12 and 13; or all or a fragment or variant of each of the polynucleotide sequences 1 to 348 of SEQ ID No 16 and 1 to 336 of SEQ ID No 17; or all or a fragment or variant of each of the polynucleotide sequences of SEQ ID No 16 and 17.
21. Ail expression vector comprising a polynucleotide sequence of claim 19 or 20.
22. A host cell comprising an expression vector according to claim 21.
23. A pharmaceutical composition comprising an immunoglobulin variable domain and/or antibody according to any one of the previous claims and/or a polypeptide according to claim 18 and/or a polynucleotide according to claim 19 or 20 and a pharmaceutically acceptable carrier.
24. An immunoglobulin variable domain or antibody according to. any one of the previous claims or a polypeptide according to claim 18 or a polynucleotide according to claim 19 oi' 20 or a pharmaceutical composition according to claim 23 for use in medicine.
25. Use of an immunoglobulin variable domain and/or an antibody according to any one of the previous claims and/or a pol)'peptide according to claim 18 and/or a polynucleotide according to claim 19 and/or a pharmaceutical composition according claim 23 in the manufacture of a medicament for treating a patient having, or at risk of contracting or developing. HTV/ AIDS.
26. A method of preventing or treating HIV/ AIDS comprising administering to a patient an immunoglobulin variable domain and/or an antibody according to any one of the previous claims and/or a polypeptide according to claim 18 and/or a polynucleotide according to claim 19 or 20 or a pharmaceutical composition according claim 23.
27. The use of claim 25 oi the method of claim 26 wherein the patient has a competent immune system and is HTV-I infected.
28. The use of 25 or the method of claim 26 wherein the patient does not have
HΓV.
29. The use of claim 25 or the method of claim 26 wherein the patient is incapable of mounting an effective or rapid immune response to infection with HTV-I.
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WO2009083225A3 (en) * | 2007-12-28 | 2010-09-16 | Bioinvent International Ab | Antibody-containing formulation for the use for treating cardiovascular diseases associated with atherosclerosis |
US20120315276A1 (en) * | 2009-12-10 | 2012-12-13 | Bayer Intellectual Property Gmbh | Neutralizing prolactin receptor antibodies and their therapeutic use |
US10550198B2 (en) | 2015-09-24 | 2020-02-04 | Daiichi Sankyo Company, Limited | Anti-garp antibody |
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Cited By (5)
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WO2009083225A3 (en) * | 2007-12-28 | 2010-09-16 | Bioinvent International Ab | Antibody-containing formulation for the use for treating cardiovascular diseases associated with atherosclerosis |
US20120315276A1 (en) * | 2009-12-10 | 2012-12-13 | Bayer Intellectual Property Gmbh | Neutralizing prolactin receptor antibodies and their therapeutic use |
US10550198B2 (en) | 2015-09-24 | 2020-02-04 | Daiichi Sankyo Company, Limited | Anti-garp antibody |
US11046780B2 (en) | 2015-09-24 | 2021-06-29 | Daiichi Sankyo Company, Limited | Anti-garp antibody |
US12006372B2 (en) | 2015-09-24 | 2024-06-11 | Daiichi Sankyo Company, Limited | Anti-GARP antibody |
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