WO2000069914A2 - Anticorps - Google Patents

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Publication number
WO2000069914A2
WO2000069914A2 PCT/GB2000/001910 GB0001910W WO0069914A2 WO 2000069914 A2 WO2000069914 A2 WO 2000069914A2 GB 0001910 W GB0001910 W GB 0001910W WO 0069914 A2 WO0069914 A2 WO 0069914A2
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Prior art keywords
hab
egp
nucleotide sequence
fragment
seq
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PCT/GB2000/001910
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English (en)
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WO2000069914A3 (fr
Inventor
Hendricus Renerus Jacobus Mattheus Hoogenboom
Anneke Reurs
Sigrid Herma Wilma Beiboer Beiboer
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Oxford Biomedica (Uk) Limited
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Priority to AU49350/00A priority Critical patent/AU4935000A/en
Publication of WO2000069914A2 publication Critical patent/WO2000069914A2/fr
Publication of WO2000069914A3 publication Critical patent/WO2000069914A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to antibodies.
  • the present invention relates to human antibodies that recognise an EGP-2 antigen.
  • the present invention relates to human antibodies obtainable by selection screening of immunoglobulin genes from a phage display library. It also relates to processes for the production of these antibodies and in vitro and in vivo applications of these antibodies in diagnosis and immunotherapy.
  • TAAs tumour associated antigens
  • CEA carcinoembryonic antigen
  • TAG72 TAG72
  • c-erB2 underglycosylated MUC-1
  • p53 TAA
  • EGP40 epithelial glycoprotein-2 antigen
  • EGP-2 is a type I 38 kDa transmembrane glycoprotein which is present on almost all cell types derived from the ectoderm and endoderm germlineages (Gottlinger et al 1986) and has been reported to function as an intercellular adhesion molecule (Litvinov et al 1994). Recently, EGP-2 was shown to modulate cadherin-based intercellular junctions, causing a redistribution of E-cadherin and catenins and a downregulation of ⁇ -catenin in transfected L-cells and epithelial cells (Litvinov et al 1997).
  • tumour metastasis may play a role in tumour metastasis by decreasing the amount of cadherin-based junctions, thus destabilizing intercellular contacts.
  • malignant cells may be distinguished from their non- malignant counterparts by the expression of tumour-associated antigens such as (EGP-2).
  • murine antibodies to EGP-2 have been used in radioimmune detection trials (Balaban et al 1991; Kosterink et al 1995) as well as in phase I and II clinical trials (Frodin et al 1988; Riethmuller et al 1994; Elias et al 1990).
  • MOC- 31 whole antibody and ScFv fragments thereof having the same specificity (see Roovers et al 1998).
  • EGP-2 could not be detected by MOC-31 (De Jonge et al 1993).
  • Mabs with the specificity of MOC-31 may be useful in treatment of colorectal cancer, since the EGP-2 antigen does not appear to be shed into the circulation.
  • the half life of murine Mabs in the circulation is relatively short compared to human immunoglobulin.
  • the Fc portion of murine Mabs may not elicit Antibody Dependent Cellular Cytoxicity (ADCC) or Cellular Dependent Cytoxicity (CDC) as effectively as the Fc portion of a human antibody.
  • ADCC Antibody Dependent Cellular Cytoxicity
  • CDC Cellular Dependent Cytoxicity
  • classic methods for producing monoclonal antibodies are slow, laborious and expensive. By way of example, animals must be immunised repeatedly and the entire process, including cloning and screening and recloning of hybridomas may require a year or more.
  • the present invention seeks to provide human antibodies and derivatives, variants and fragments thereof that recognise that EGP-2 antigen by selection screening of immunoglobulin genes from a phage display library.
  • the present invention provides a recombinant human antibody (Hab) that recognises the EGP-2 antigen.
  • human antibody means an antibody that recognises the EGP-2 antigen comprising human light chain variable regions (NL) and human heavy chain variable (VH) regions which are obtainable by selection screening of immunoglobulin genes from a phage display library.
  • variable regions refers to the variable regions, or domains, of the light chains (VL) and heavy chains (VH) which contain the determinants for binding recognition specificity and for the overall affinity of the Hab or Hab fragment for EGP-2.
  • the variable domains of each pair of light (NL) and heavy chains (NH) are involved in antigen recognition and form the antigen binding site.
  • the domains of the light and heavy chains have the same general structure and each domain has four framework (FR) regions, whose sequences are relatively conserved, connected by three complementarity determining regions (CDRs).
  • the FR regions maintain the structural integrity of the variable domain.
  • the CDRs are the polypeptide segments within the variable domain that mediate binding of the antigen.
  • constant region refers to the domains of the light (CL) and heavy (CH) chain of the Hab or Hab fragment which provide structural stability and other biological functions such as antibody chain association, secretion, transplacental mobility, and complement binding, but which are not involved with binding EGP-2.
  • CL light
  • CH heavy chain of the Hab or Hab fragment
  • the amino acid sequence and corresponding exon sequences in the genes of the constant region will be dependent upon the species from which it is derived. However, variations in the amino acid sequence leading to allotypes are relatively limited for particular constant regions within a species.
  • allelic genes are an antigenic determinant (or epitope) that distinguishes allelic genes.
  • variable region of each chain is joined to the constant region by a linking polypeptide sequence.
  • the linkage sequence is coded by a "J" sequence in the light chain gene, and a combination of a "D” sequence and a "J” sequence in the heavy chain gene.
  • guided selection means a sequential shuffling of human and murine V-genes.
  • Chimeric antibody for purposes of this invention refers to an antibody having either a heavy and light chain encoded by a nucleotide sequence derived from a murine immunoglobulin gene and either a heavy and light chain encoded by a nucleotide sequence derived from a human immunoglobulin gene.
  • antibody or “immunoglobulin” includes fragments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with or recognising EGP-2.
  • proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab', Fv fragments, and single chain antibodies (scFv) containing a VL and VH domain joined by a peptide linker.
  • the scFvs may be covalently or non-covalently linked to form antibodies having two or more binding sites.
  • the Hab or the Hab fragment of the present invention is a high affinity Hab or Hab fragment with an off-rate from 10 "2 s " 1 to 10 "4 s "1 .
  • the Hab or the Hab fragment of the present invention is a high affinity Hab or Hab fragment with an off-rate of about 2.4 x 10 "4 s "1 .
  • off-rate means the dissociation rate (k ot ⁇ ) of a Hab or Hab fragment from an antigen. In the context of the present invention, it is measured using BIAevaluation software (Pharmacia). A low off rate is desirable as it reflects the affinity of an Fab fragment for an antigen.
  • affinity is defined in terms of the dissociation rate or off-rate (k off ) of a Hab or Hab fragment from an EGP-2 antigen. The lower the off-rate the higher the affinity that a Hab or Hab fragment has for an antigen such as EGP-2.
  • the "HAb” can be the peptide per se also as well as being part of a fusion protein.
  • the present invention includes any HAb derivatives that have EGP-2 binding activity.
  • the present invention includes the entire HAb, HAb derivatives and biologically-active HAb fragments. These include a Hab or Hab fragment with EGP-2 binding activity that has amino acid substitutions or have sugars or other molecules attached to amino acid functional groups.
  • the HAb is an isolated HAb and/or purified HAb.
  • the Hab or Hab fragment can be obtainable from or produced by any suitable source, whether natural or not, or it may be a synthetic HAb, a semi-synthetic HAb, a derivatised HAb, or a recombinant HAb.
  • the non-native HAb includes a HAb at least a portion of which has been prepared by recombinant DNA techniques or a HAb produced by chemical synthesis techniques or combinations thereof.
  • the non-native Hab or Hab fragment has been prepared by use of recombinant techniques.
  • the sequence of the Hab or Hab fragment of the present invention may be the same as that of the naturally occuring form or it may be a variant, homologue, fragment or derivative thereof.
  • variants in relation to the nucleotide sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the expression product of the resultant nucleotide sequence has EGP-2 binding affinity, preferably having at least the same EGP-2 binding affinity as the expression product of a sequence identified as SEQ ID No 1 or SEQ ID No 2 or SEQ ID No 9 or SEQ ID No 10.
  • sequence identity preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
  • sequence identity preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
  • sequence identity preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
  • Relative sequence identity can also be determined by commercially available computer programs that can calculate % identity between two or more sequences using any suitable algorithm for determining identity, using for example default parameters.
  • a typical example of such a computer program is CLUSTAL.
  • the BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html. which is incorporated herein by reference.
  • the search parameters are defined as follows, can be advantageously set to the defined default parameters.
  • substantially identical when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more.
  • the default threshold for EXPECT in BLAST searching is usually 10.
  • BLAST Basic Local Alignment Search Tool
  • blastp, blastn, blastx, tblastn, and tblastx these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (see http://www.ncbi.nih.gov/BLAST blast_help.html) with a few enhancements.
  • the BLAST programs were tailored for sequence similarity searching, for example to identify D homologues to a query sequence. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al (1994) Nature Genetics 6:119-129.
  • J 0 blastp - compares an amino acid query sequence against a protein sequence database.
  • blastn compares a nucleotide query sequence against a nucleotide sequence database.
  • blastx compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.
  • tblastn compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands).
  • tblastx compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • BLAST uses the following search parameters:
  • HISTOGRAM - Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual).
  • DESCRIPTIONS Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page).
  • EXPECT The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).
  • CUTOFF - Cutoff score for reporting high-scoring segment pairs.
  • the default value is calculated from the EXPECT value (see above).
  • HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual).
  • significance thresholds can be more intuitively managed using EXPECT.
  • ALIGNMENTS Restricts database sequences to the number specified for which high- scoring segment pairs (HSPs) are reported: the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).
  • MATRIX - Specify an alternate scoring matrix for BLASTP.
  • BLASTX, TBLASTN and TBLASTX The default matrix is BLOSUM62 (Henikoff & Henikoff. 1992).
  • the valid alternative choices include: PAM40, PAM120, PAM250 and IDENTITY.
  • No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response.
  • FILTER - Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17:149-163, or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States (1993) Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
  • Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs. It is not unusual for nothing at all to be masked by SEG, XNU, or both, when applied to sequences in SWISS-PROT, so filtering should not be expected to always yield an effect. Furthermore, in some cases, sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect.
  • NCBI-gi causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.
  • sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.
  • no gap penalties are used when determining sequence identity.
  • the present invention also encompasses nucleotide sequences that are complementary to the sequences presented herein, or any variant, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar promoter sequences in other organisms.
  • the present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences presented herein, or any fragment or derivative thereof.
  • Hybridization means a "process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY) as well as the process of amplification as carried out in polymerase chain reaction technologies as described in Dieffenbach CW and GS Dveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY). Also included within the scope of the present invention are nucleotide sequences that are capable of hybridizing to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987.
  • the present invention covers nucleotide sequences that can hybridise to the nucleotide sequences of the present invention under stringent conditions (e.g. 65°C and O.lxSSC).
  • stringent conditions e.g. 65°C and O.lxSSC.
  • the present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any fragment or derivative thereof. Likewise, the present invention encompasses nucleotide sequences that are complementary to sequences that are capable of hybridising to the sequence of the present invention. These types of nucleotide sequences are examples of variant nucleotide sequences.
  • the term “variant” encompasses sequences that are complementary to sequences that are capable of hydridising to the nucleotide sequences presented herein.
  • amino acid sequence refers to peptide, polypeptide or protein sequences or portions thereof.
  • the present invention provides a polypeptide consisting essentially of a Hab or a Hab fragment.
  • the present invention also covers nucleotide sequence(s) coding for the Hab or Hab fragment of the present invention and variant, homologue, fragment or derivative thereof.
  • the Hab or Hab fragment encoding sequence is an isolated HAb or Hab fragment encoding sequence and/or a purified Hab or Hab fragment coding sequence.
  • the Hab or Hab fragment coding sequence can be obtainable from or produced by any suitable source, whether natural or not, or it may be synthetic, semi-synthetic or recombinant.
  • nucleotide sequence refers to an oligonucleotide sequence or polynucleotide sequence, and variants, homologues, fragments and derivatives thereof (such as portions thereof).
  • the nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin which may be double-stranded or single-stranded whether representing the sense or antisense strand.
  • the DNA sequence can be, for example, a synthetic DNA sequence, a recombinant DNA sequence (i.e. prepared by use of recombinant DNA techniques), a cDNA sequence or a partial genomic DNA sequence, including combinations thereof.
  • the DNA is or comprises cDNA.
  • nucleotide sequence can include more than one nucleotide sequence.
  • nucleotide sequence means DNA. More preferably, the term “nucleotide sequence” means DNA prepared by use of recombinant DNA techniques (i.e. recombinant DNA).
  • the present invention relates to a DNA sequence (preferably a cDNA sequence) encoding a Hab or Hab fragment.
  • cDNA sequences encoding a Hab or Hab fragment.
  • the present invention provides a nucleotide sequence consisting essentially of a Hab or Hab fragment coding sequence.
  • recombinant means a Hab or Hab fragment prepared by use of recombinant DNA techniques.
  • Altered Hab or Hab fragment encoding polynucleotide sequences which may be used in accordance with the invention include different nucleotide residues resulting in a polynucleotide that encodes the same or a functionally equivalent Hab or Hab fragment which Hab or Hab fragment may have deletions, insertions or substitutions therein.
  • deletion is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
  • an "insertion” or “addition” is a change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring HAb or HAb encoding nucleotide sequence.
  • substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
  • alleles of the Hab or Hab fragment are included within the scope of the present invention.
  • an "allele” or “allelic sequence” is an alternative form of the Hab or Hab fragment. Alleles result from a mutation, i.e., a change in the nucleotide sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • the present invention also relates to DNA fragments comprising the DNA sequences of the present invention or allelic variations of such sequences.
  • a highly preferred aspect of the present invention relates to a Hab or Hab fragment comprising the amino acid sequence SEQ ID No 9 or SEQ ID No 10 of the present invention.
  • the present invention relates to an isolated Hab or Hab fragment comprising the amino acid sequence of SEQ ID No. 9 or SEQ ID No 10.
  • Another highly preferred aspect of the present invention relates to a Hab or Hab fragment comprising the amino acid sequence SEQ ID No 1 or SEQ ID No 2 of the present invention.
  • the present invention relates to an isolated Hab or Hab fragment comprising the amino acid sequence of SEQ ID No. 1 or SEQ ID No 2.
  • the present invention also relates to DNA comprising the DNA sequence of SEQ ID No. 17 or an allelic variation thereof.
  • the present invention also relates to DNA comprising the DNA sequence of SEQ ID No. 18 or an allelic variation thereof.
  • a highly preferred aspect of the present invention relates to recombinant DNA comprising the DNA sequence of SEQ ID No. 17 and SEQ ID No 18 or an allelic variation thereof.
  • the present invention also relates to DNA comprising the DNA sequence of SEQ ID No.
  • the present invention also relates to DNA comprising the DNA sequence of SEQ ID No.
  • a highly preferred aspect of the present invention relates to recombinant DNA comprising the DNA sequence of SEQ ID No. 25 and SEQ ID No 26 or an allelic variation thereof.
  • immunological activity is defined as the capability of the natural. recombinant or synthetic HAb or any fragment thereof, to induce a specific immune response in appropriate animals or cells on binding with a EGP-2 antigen.
  • the nucleotide sequence and/or the Hab or Hab fragment thereof are in an isolated and/or purified form. Protein purification methods are also well known in the art.
  • the Hab or Hab fragment of the present invention can be produced by recombinant DNA methods or synthetic peptide chemical methods that are well known to those of ordinary- skill in the art.
  • the Hab or Hab fragment can be synthesized by techniques well known in the art, as exemplified by "Solid Phase Peptide Synthesis: A Practical Approach” E. Atherton and R. C. Sheppard, IRL Press, Oxford England. Similarly, multiple fragments can be synthesized which are subsequently linked together to form larger fragments. These synthetic peptide fragments can also be made with amino acid substitutions at specific locations in order to test for agonistic and antagonistic activity in vitro and in vivo. Peptide fragments that possess high affinity binding to tissues can be used to isolate the EGP-2 antigen on affinity columns.
  • the Hab or HAb fragment can be synthesized in a standard microchemical facility and purity checked with HPLC and mass spectrophotometry. Methods of peptide synthesis, HPLC purification and mass spectrophotometry are commonly known to those skilled in these arts.
  • the present invention also provides a Hab or Hab fragment of the present invention which can be expressed under in vitro and in vivo conditions in a transformed host cell into which has been incorporated the foregoing DNA sequences or allelic variations thereof and which can be used in the prevention and/or treatment of cancer related diseases.
  • vector includes expression vectors and transformation vectors.
  • expression vector means a construct capable of in vivo or in vitro expression.
  • transformation vector means a construct capable of being transferred from one species to another.
  • Preferred vectors for use in accordance with the present invention are recombinant viral vectors, in particular recombinant retroviral vectors (RRV) such as lentiviral vectors, adenoviral vectors including a combination of retroviral vectors.
  • RRV recombinant retroviral vector
  • the term "recombinant retroviral vector” (RRV) refers to a vector with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell includes reverse transcription and integration into the target cell genome.
  • the RRV carries non-viral coding sequences which are to be delivered by the vector to the target cell.
  • An RRV is incapable of independent replication to produce infectious retroviral particles within the final target cell.
  • the RRV lacks a functional gag- pol and/or env gene and/or other genes essential for replication.
  • Preferred vectors for use in accordance with the present invention are recombinant pox viral vectors such as fowl pox virus (FPV), entomopox virus, vaccinia virus such as NYVAC, canarypox virus, MVA or other non-replicating viral vector systems such as those described for example in WO 9530018.
  • FMV fowl pox virus
  • entomopox virus vaccinia virus
  • vaccinia virus such as NYVAC
  • canarypox virus canarypox virus
  • MVA non-replicating viral vector systems
  • Pox viruses may be engineered for recombinant gene expression and for the use as recombinant live vaccines in a dual immunotherapeutic approach.
  • live attenuated viruses such as viruses, as delivery vehicles and/or vector based vaccine candidates, stems from their ability to elicit cell mediated immune responses.
  • the viral vectors as outlined above, are capable of being employed as delivery vehicles and as vector based vaccine candidates because of the immunogenicity of their constitutive proteins, which act as adjuvants to enhance the immune response, thus rendering a nucleotide sequence of interest (NOI) such as a nuelcotide sequence encoding an anti EGP-2 Hab of the present invention more immunogenic.
  • NOI nucleotide sequence of interest
  • the pox virus vaccination strategies have used recombinant techniques to introduce NOIs into the genome of the pox virus. If the NOI is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant pox virus to be infectious, that is to say to infect foreign cells and thus to express the integrated NOI.
  • the recombinant pox virus prepared in this way can be used as live vaccines for the prophylaxis and/or treatment of pathologic and infectious disease and/or cancer.
  • Other requirements for pox viral vector delivery systems include good immunogenicity and safety.
  • MVA is a replication-impaired vaccinia strain with a good safety record. In most cell types and normal human tissue, MVA does not replicate.
  • MVA Limited replication of MVA is observed in a few transformed cell types such as BHK21 cells. Carroll et al (1997 Vaccine 15: 387-394) have shown that the recombinant MVA is equally as good as conventional recombinant vaccinia vectors at generating a protective CD8+T cell response and is an efficacious alternative to the more commonly used replication competent vaccinia virus.
  • the vaccinia virus strains derived from MVA. or independently developed strains having the features of MVA which make MVA particularly suitable for use in a vaccine, are also suitable for use as a delivery vehicle in the present invention.
  • the present invention provides a hybrid viral vector system for in vivo gene delivery, which system comprises one or more primary viral vectors which encode a secondary viral vector, the primary vector or vectors capable of infecting a first target cell and of expressing therein the secondary viral vector, which secondary vector is capable of transducing a secondary target cell.
  • the primary vector is obtainable from or is based on an adenoviral vector and/or the secondary viral vector is obtainable from or is based on a retroviral vector preferably a lentiviral vector.
  • target vector refers to a vector whose ability to infect/transfect transduce a cell or to be expressed in a target cell is restricted to certain cell types within the host organism, usually cells having a common or similar phenotype.
  • nucleotide sequence of the present invention is operably linked to a transcription unit.
  • each transcription unit generally comprises at least a promoter, an optional enhancer and a polyadenylation signal.
  • promoter is used in the normal sense of the art, e.g. an RNA polymerase binding site.
  • the promoter may contain an enhancer element.
  • the term “enhancer” includes a DNA sequence which binds to other protein components of the transcription initiation complex and thus facilitates the initiation of transcription directed by its associated promoter.
  • cell includes any suitable organism.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • transformed cell means a cell having a modified genetic structure.
  • a cell has a modified genetic structure when a vector according to the present invention has been introduced into the cell.
  • organism includes any suitable organism.
  • the organism is a mammal.
  • the organism is a human.
  • transgenic organism means an organism comprising a modified genetic structure.
  • the organism has a modified genetic structure since a vector according to the present invention has been introduced into the organism.
  • the present invention provides a Hab or Hab fragment that recognises EGP-2. This is advantageous because:
  • the present invention is also advantageous for the following reasons: (iv) it provides a Hab or Hab fragment that recognises EGP-2 and which has a greater specificity than conventional human antibodies raised against EGP-2;
  • the present invention also provides a method comprising transforming a host cell with a or the nucleotide sequence(s) of the present invention.
  • the present invention also provides a method comprising culturing a transformed host cell - which cell has been transformed with a or the nucleotide sequence(s) according to the present invention under conditions suitable for the expression of the Hab or Hab fragment encoded by said nucleotide sequence(s).
  • the present invention also provides a method comprising culturing a transformed host cell - which cell has been transformed with a or the nucleotide sequence(s) according to the present invention or a derivative, homologue. variant or fragment thereof - under conditions suitable for the expression of the Hab or Hab fragment encoded by said nucleotide sequence(s): and then recovering said Hab or Hab fragment from the transformed host cell culture.
  • Hab or Hab fragment encoding nucleotide sequences, fusion proteins or functional equivalents thereof may be used to generate recombinant DNA molecules that direct the expression thereof in appropriate host cells.
  • HAbs and HAb fragments may be produced in recombinant E. coli. or yeast expression systems, and purified with column chromatography.
  • Fab, Fv, ScFv antibody fragments can all be expressed in and secreted from E. Coli, thus allowing the production of large amounts of the such fragments.
  • a or the nucleotide sequence(s) encoding the Hab or Hab fragment of the present invention is operably linked to a promoter sequence capable of directing expression of the Hab or Hab fragment encoding nucleotide sequence(s) in a suitable host cell.
  • the transformed host cell When inserted into the host cell, the transformed host cell may be cultured under suitable conditions until sufficient levels of the Hab or Hab fragment are achieved after which the cells may be lysed and the Hab or Hab fragment is isolated.
  • Host cells transformed with the Hab or Hab fragment encoding nucleotide sequence(s) may be cultured under conditions suitable for the expression and recovery of the Hab or Hab fragment from cell culture.
  • the protein produced by a recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing the Hab or Hab fragment encoding nucleotide sequences can be designed with signal sequences which direct secretion of the Hab or Hab fragment encoding nucleotide sequences through a particular prokaryotic or eukaryotic cell membrane.
  • the Hab or Hab fragment may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3 - 26328 1), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, WA).
  • a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the Hab or Hab fragment is useful to facilitate purification.
  • nucleotide sequences of the present invention may be engineered in order to alter a the Hab or Hab fragment encoding sequence(s) for a variety of reasons, including but not limited to. alterations which modify the cloning, processing and/or expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns or to change codon preference.
  • a or the HAb natural, modified or recombinant encoding nucleotide sequence(s) may be ligated to a heterologous sequence to encode a fusion protein.
  • the present invention encompasses the use of fusion proteins comprising the HAb or Hab fragment or an enzymatically active fragment or derivative thereof linked to an affinity tag such as glutathione-S-transferase (GST), biotin, His6, a c-myc tag (see Emrich et al 1993 Biocem Biophys Res Commun 197(1): 214- 220).
  • HA hemagglutinin
  • the fused recombinant protein comprises an antigenic co- protein such as GST, beta-galactosidase or the lipoprotein D from Haemophilus influenzae which are relatively large co-proteins, which solubilise and facilitate production and purification thereof.
  • the fused protein may comprise a carrier protein such as bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • the marker sequence is a hexa- histidine peptide, as provided in the pQE vector (Qiagen Inc) and described in Gentz et al (1989 PNAS 86: 821-824).
  • fusion proteins are readily expressable in yeast culture (as described in Mitchell et al 1993 Yeast 5: 715-723) and are easily purified by affinity chromatography.
  • a fusion protein may also be engineered to contain a cleavage site located between the nucleotide sequence encoding the Hab or Hab fragment and the heterologous protein sequence, so that the Hab or Hab fragment may be cleaved and purified away from the heterologous moiety.
  • an assay for the target protein may be conducted using the entire, bound fusion protein.
  • the co-protein may act as an adjuvant in the sense of providing a generalised stimulation of the immune system.
  • the co-protein may be attached to either the amino or carboxy terminus of the first protein.
  • the presence/absence of marker gene expression suggests that the nucleotide sequence and/or its Hab or Hab fragment is also present, its presence and expression should be confirmed.
  • the Hab or Hab fragment encoding nucleotide sequence is inserted within a marker gene sequence, recombinant cells containing the Hab or Hab fragment coding regions may be identified by the absence of the marker gene function.
  • a marker gene may be placed in tandem with a HAb or Hab fragment encoding nucleotide sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the Hab or Hab fragment as well.
  • Additional methods to quantitate the expression of a particular molecule include radiolabeling (Melby PC et al 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated. Quantitation of multiple samples may be speeded up by running the assay in an ELISA format where the Hab or Hab fragment of interest is presented in various dilutions and a spectrophotometric or calorimetric response gives rapid quantitation.
  • Altered Hab or Hab fragment nucleotide sequences which may be used in accordance with the present invention include deletions, insertions or substitutions of different nucleotide residues resulting in a nucleotide sequence that encodes the same or a functionally equivalent Hab.
  • the expressed Hab or Hab fragment may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent Hab or Hab fragment.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the binding affinity of the Hab or Hab fragment is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid: positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the present invention also encompasses gene therapy whereby the Hab or Hab fragment encoding nucleotide sequence(s) of the present invention is regulated in vivo.
  • expression regulation may be accomplished by administering compounds that bind to the Hab or Hab fragment encoding nucleotide sequence(s) of the present invention, or control regions associated with the Hab or Hab fragment encoding nucleotide sequence of the present invention, or its corresponding RNA transcript to modify the rate of transcription or translation.
  • the Hab or Hab fragment encoding nucleotide sequence(s) of the present invention may be under the expression control of an expression regulatory element, usually a promoter or a promoter and enhancer.
  • the enhancer and/or promoter may be preferentially active in a hypoxic or ischaemic or low glucose environment, such that the Hab or Hab fragment encoding nucleotide sequence(s) is preferentially expressed in the particular tissues of interest, such as in the environment of a tumour, arthritic joint or other sites of ischaemia.
  • any significant biological effect or deleterious effect of the Hab or Hab fragment encoding nucleotide sequence(s) on the individual being treated may be reduced or eliminated.
  • the enhancer element or other elements conferring regulated expression may be present in multiple copies.
  • the enhancer and/or promoter may be preferentially active in one or more specific cell types - such as any one or more of macrophages, endothelial cells or combinations thereof.
  • Further examples include respiratory airway epithelial cells, hepatocytes. muscle cells, cardiac myocytes, synoviocytes, primary mammary epithelial cess and post- mitotically terminally differentiated non-replicating cells such as macrophages and/or neurons.
  • the promoter and/or enhancer may be constitutively efficient, or may be tissue or temporally restricted in their activity.
  • tissue restricted promoters/enhancers are those which are highly active in tumour cells such as a promoter/enhancer from a MUC ⁇ gene, a CEA gene or a 5T4 antigen gene.
  • temporally restricted promoters/enhancers are those which are responsive to ischaemia and/or hypoxia, such as hypoxia response elements or the promoter/enhancer of a grp7% or a grp94 gene.
  • the alpha fetoprotein (AFP) promoter is also a tumour-specific promoter.
  • One preferred promoter-enhancer combination is a human cytomegalovirus (hCMV) major immediate early (MIE) promoter/enhancer combination.
  • the promoters of the present invention are tissue specific. That is, they are capable of driving transcription of a Hab or Hab fragment encoding nucleotide sequence(s) in one tissue while remaining largely “silent" in other tissue types.
  • tissue specific means a promoter which is not restricted in activity to a single tissue type but which nevertheless shows selectivity in that they may be active in one group of tissues and less active or silent in another group.
  • a desirable characteristic of the promoters of the present invention is that they posess a relatively low activity in the absence of activated hypoxia-regulated enhancer elements, even in the target tissue.
  • One means of achieving this is to use "silencer" elements which suppress the activity of a selected promoter in the absence of hypoxia.
  • hypooxia means a condition under which a particular organ or tissue receives an inadequate supply of oxygen.
  • the level of expression of a or the Hab or Hab fragment encoding nucleotide sequence(s) under the control of a particular promoter may be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (that is, deletion analysis). This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity or the smallest region conferring hypoxia sensitivity.
  • tissue specific promoters may be particularly advantageous in practising the present invention.
  • these promoters may be isolated as convenient restriction digestion fragments suitable for cloning in a selected vector.
  • promoter fragments may be isolated using the polymerase chain reaction. Cloning of the amplified fragments may be facilitated by incorporating restriction sites at the 5' end of the primers.
  • the Hab or HAb fragments of the present invention may be used in combination with other compositions and procedures for the treatment of diseases.
  • the HAb or HAb fragments may also be used in combination with conventional treatments of diseases such as cancer.
  • a tumor may be treated conventionally with surgery, radiation or chemotherapy combined with a HAb or HAb fragment or a HAb or HAb fragment may be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize any residual primary tumor.
  • the Hab or Hab fragment can be delivered with a therapeutically effective agent at the same moment in time and at the same site.
  • the Hab or Hab fragment and the therapeutically effective agent may be delivered at a different time and to a different site.
  • the Hab or Hab fragment and the therapeutically effective agent may even be delivered in the same delivery vehicle for the prevention and/or treatment of cancer.
  • Therapeutic strategies based on the use of the Hab or Hab fragment include the recruitment and activation of T cells by using a fusion of an EGP-2 reactive antibody fragment with the bacterial superantigen staphylococcal enterotoxin (Dohlsten et al 1994) or by using bispecific antibodies, directed to both EGP-2 and the T-cell CD3 antigen (Kroesen et al 1994).
  • Anti-EGP-2 antibodies may also be conjugated to different bacterial toxins to yield potent immunotoxins (LeMaistre et al 1987; Zimmermann et al 1997).
  • Hab or HAb fragments may be used in combination with cytotoxic agents for the prevention and/or treatment of angiogenesis and/or cancer.
  • Cytotoxic agents such as ricin. linked to HAb, high affinity HAb fragments, HAb antisera, HAb receptor agonists and antagonists provide a tool for the destruction of cells that bind HAb. These cells may be found in many locations, including but not limited to, micrometastases and primary tumours.
  • Hab or HAb fragments may be used in combination with a pro-drug activating enzyme in gene therapy.
  • the HAb or HAb fragments may be used in combination with another nucleotide sequences of interest (NOI) or NOIs which encode a pro-drug activation enzyme or enzymes which have no significant effect or no deleterious effect until the individual is treated with one or more pro-drugs upon which the enzyme or enzymes act.
  • NOI nucleotide sequences of interest
  • NOIs which encode a pro-drug activation enzyme or enzymes which have no significant effect or no deleterious effect until the individual is treated with one or more pro-drugs upon which the enzyme or enzymes act.
  • NOI nucleotide sequences of interest
  • a pro-drug activating enzyme may be delivered to a tumour site for the treatment of a cancer.
  • a suitable pro-drug is used in the treatment of the patient in combination with the appropriate pro-drug activating enzyme.
  • An appropriate pro-drug is administered in conjunction with the vector.
  • pro-drugs include: etoposide phosphate (with alkaline phosphatase, Senter et al 1988 Proc Nafl Acad Sci 85: 4842- 4846); 5-fluorocytosine (with cytosine deaminase.
  • pro-drug activation enzymes for use in the invention include a 5 thymidine phosphorylase which activates the 5-fluoro-uracil pro-drugs capc ⁇ tabine and furtulon; thymidine kinase from Herpes Simplex Virus which activates ganciclovir; a cytochrome P450 which activates a pro-drug such as cyclophosphamide to a DNA damaging agent; and cytosine deaminase which activates 5-fluorocytosine.
  • an enzyme of human origin is used. 10
  • suitable promoter which may be a promoter driving expression of a ribozyme(s), or a different promoter or promoters, such as in one or more specific cell types.
  • the expression products encoded by the NOIs may be proteins which are secreted from the cell. Alternatively the NOI expression products are not secreted and are active within 25 the cell. In either event, it is preferred for the NOI expression product to demonstrate a bystander effector or a distant bystander effect; that is the production of the expression product in one cell leading to the killing of additional, related cells, either neighbouring or distant (e.g. metastatic), which possess a common phenotype.
  • Suitable NOIs for use in the invention in the treatment or prophylaxis of cancer include NOIs encoding proteins which: destroy the target cell (for example a ribosomal toxin), act as: tumour suppressors (such as wild-type p53); activators of anti-tumour immune mechanisms (such as cytokines. co-stimulatory molecules and immunoglobulins); inhibitors of angiogenesis; or which provide enhanced drug sensitivity (such as pro-drug activation enzymes); indirectly stimulate destruction of target cell by natural effector cells (for example, strong antigen to stimulate the immune system or convert a precursor substance to a toxic substance which destroys the target cell (for example a prodrug 5 activating enzyme).
  • NOIs encoding proteins which: destroy the target cell (for example a ribosomal toxin), act as: tumour suppressors (such as wild-type p53); activators of anti-tumour immune mechanisms (such as cytokines. co-stimulatory molecules and immunoglobulins); inhibitor
  • Encoded proteins could also destroy bystander tumour cells (for example with secreted antitumour antibody-ribosomal toxin fusion protein), indirectly stimulate destruction of bystander tumour cells (for example cytokines to stimulate the immune system or procoagulant proteins causing local vascular occlusion) or convert a precursor substance to a toxic substance which destroys bystander tumour cells (eg an 10 enzyme which activates a prodrug to a diffusible drug).
  • bystander tumour cells for example with secreted antitumour antibody-ribosomal toxin fusion protein
  • indirectly stimulate destruction of bystander tumour cells for example cytokines to stimulate the immune system or procoagulant proteins causing local vascular occlusion
  • convert a precursor substance to a toxic substance which destroys bystander tumour cells eg an 10 enzyme which activates a prodrug to a diffusible drug.
  • NOI(s) encoding antisense transcripts or ribozymes which interfere with expression of cellular genes for tumour persistence (for example against aberrant myc transcripts in Burkitts lymphoma or against bcr-abl transcripts in chronic myeloid 15 leukemia.
  • tumour persistence for example against aberrant myc transcripts in Burkitts lymphoma or against bcr-abl transcripts in chronic myeloid 15 leukemia.
  • combinations of such NOIs is also envisaged.
  • hypoxia regulatable therapeutic NOIs can be found in PCT/GB95/00322 (WO-A-9521927).
  • a vector is a tool that allows or faciliates the transfer of an entity from one environment to another.
  • some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell.
  • entities such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment)
  • the vector may then serve to maintain the heterologous DNA within the cell or may act as a unit of DNA replication.
  • vectors used in recombinant DNA techniques include plasmids, chromosomes, artificial chromosomes or viruses.
  • the vector can be delivered by viral or non- viral techniques.
  • Non-viral delivery systems include but are not limited to DNA transfection methods.
  • transfection includes a process using a non-viral vector to deliver a gene to a target mammalian cell.
  • Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection. compacted DNA-mediated transfection, liposomes. immunoliposomes. lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), multivalent cations such as spermine.
  • cationic lipids or polylysine 1. 2. -bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.
  • DOTAP -bis (oleoyloxy)-3-(trimethylammonio) propane
  • DOTAP -cholesterol complexes
  • Viral delivery systems include but are not limited to adenovirus vector, an adeno- associated viral (AAV) vector, a herpes viral vector, a retroviral vector, a lentiviral vector a baculoviral vector or a pox viral vector.
  • AAV adeno-associated viral
  • retro viruses include but are not limited to: murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
  • MMV murine leukemia virus
  • HCV human immunodeficiency virus
  • EIAV equine infectious anaemia virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • FuSV Fujinami sarcoma virus
  • retroviruses A detailed list of retroviruses may be found in Coffin et al ("Retro viruses "" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).
  • Lentiviruses can be divided into primate and non-primate groups.
  • primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIN).
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • lentivirus family and other types of retroviruses are that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et al 1992 EMBO. J 11 : 3053-3058; Lewis and Emerman 1994 J. Virol. 68: 510-516).
  • retroviruses - such as MLV - are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
  • vectors include ex vivo delivery systems, which include but are not limited to DNA transfection methods such as electroporation. DNA biolistics. lipid- mediated transfection. compacted DNA-mediated transfection.
  • the vector may be a plasmid DNA vector.
  • the vector may be a recombinant viral vectors. Suitable recombinant viral vectors include adenovirus vectors, adeno-associated viral (AAV) vectors. Herpes-virus vectors, or retroviral vectors which are preferred. In the case of viral vectors, gene delivery is mediated by viral infection of a target cell.
  • AAV adeno-associated viral
  • retroviral vectors which are preferred.
  • gene delivery is mediated by viral infection of a target cell.
  • the vector of the present invention may be configured as a split-intron vector.
  • a split intron vector is described in PCT patent application GB98/02885 and GB/98/02867.
  • adenoviruses can be used to transduce target cells to become transient retroviral producer cells that could stably infect neighbouring cells.
  • retroviral producer cells engineered to express a Hab or Hab fragment and/or an NOI(s) of the present invention can be implanted in organisms such as animals or humans for use in the treatment of cancer.
  • the targeting of cells expressing EGP-2 antigen with a HAb or Hab fragment of the present invention facilitates the development of drugs to modulate the activity of cells expressing EGP-2.
  • Different Habs or HAb fragments can be synthesized for use in several applications including but not limited to the linkage of a Hab or Hab fragment to cytotoxic agents for targeted killing of cells that bind the Hab or Hab fragment.
  • the Hab or HAb fragment of the present invention can be coupled to other molecules using standard methods.
  • the amino and carboxyl termini of Hab or HAb fragment may be isotopically and nonisotopically labeled with many techniques, for example radiolabeling using conventional techniques (tyrosine residues- chloramine T. iodogen. lactoperoxidase; lysine residues- Bolton-Hunter reagent).
  • These coupling techniques are well known to those skilled in the art.
  • the coupling technique is chosen on the basis of the functional groups available on the amino acids including, but not limited to amino. sulfhydral, carboxyl. amide, phenol, and imidazole.
  • Various reagents used to effect these couplings include among others, glutaraldehyde, diazotized benzidine. carbodiimide, and p-benzoquinone.
  • the Hab and HAb fragments of the present invention may be chemically coupled to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules and other compounds for a variety of applications.
  • the efficiency of the coupling reaction is determined using different techniques appropriate for the specific reaction. For example, radiolabeling of an HAb peptide with 12 J is accomplished using chloramine T and Na 12 J of high specific activity. The reaction is terminated with sodium metabisulfite and the mixture is desalted on disposable columns. The labeled peptide is eluted from the column and fractions are collected. Aliquots are removed from each fraction and radioactivity measured in a gamma counter. In this manner, the unreacted Na 1_:> I is separated from the labeled Hab or Hab fragment. The peptide fractions with the highest specific radioactivity are stored for subsequent use such as analysis of the ability to bind to a Hab or Hab fragment.
  • the Hab or Hab fragment of the invention is coupled to a scintigraphic radiolabel, a cytotoxic compound or radioisotope, an enzyme for converting a non- toxic prodrug into a cytotoxic drug, a compound for activating the immune system in order to target the resulting conjugate to a colon tumour, or a cell-stimulating compound.
  • a scintigraphic radiolabel a cytotoxic compound or radioisotope
  • an enzyme for converting a non- toxic prodrug into a cytotoxic drug a compound for activating the immune system in order to target the resulting conjugate to a colon tumour, or a cell-stimulating compound.
  • Such conjugates have a "binding portion", which consists of the antibody of the invention, and a “functional portion " , which consists of the radiolabel. toxin or enzyme.
  • the antibody may alternatively be used alone in order simply to block the activity of the EGP-2 antigen, particularly by physically interfering with its binding of another compound.
  • the binding portion and the functional portion of the conjugate may be linked together by any of the conventional ways of cross linking polypeptides, such as those generally described in O'Sullivan et al (Anal. Biochem 1979: 100, 100-108).
  • one portion may be enriched with thiol groups and the other portion reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3- (2-pyridyldithio)propionate (SPDP).
  • NHS iodoacetic acid
  • SPDP N-succinimidyl-3- (2-pyridyldithio)propionate
  • Amide and thioether bonds for example achieved with m-maleimidobenzoyl-N -hydro xysuccinimide ester, are generally more stable in vivo than disulphide bonds.
  • the functional portion may be linked via the carbohydrate portion using the linking technology in EP 0 088 695.
  • the functional portion of the conjugate may be an enzyme for converting a non-toxic prodrug into a toxic drug, for example the conjugates of Bagshawe and his colleagues (Bagshawe (1987) Br. J. Cancer 56, 531; Bagshawe et al (Br. J. Cancer 1988: 58, 700); WO 88/07378) or cyanide-releasing systems (WO 91/11201).
  • the whole enzyme may not be necessary for the whole enzyme to be present in the conjugate but, of course, the catalytic portion must be present.
  • So-called “abzymes” may be used, where a Hab or Hab fragment is raised to a compound involved in the reaction one wishes to catalyse, usually the reactive intermediate state. The resulting antibody can then function as an enzyme for the reaction.
  • the conjugate may be purified by size exclusion or affinity chromatography. and tested for dual biological activities.
  • the antigen immunoreactivity may be measured using an enzyme-linked immunosorbent assay (ELISA) with immobilised antigen and in a live cell radio-immunoassay.
  • ELISA enzyme-linked immunosorbent assay
  • An enzyme assay may be used for ⁇ -glucosidase using a substrate which changes in absorbance when the glucose residues are hydrolysed.
  • a substrate which changes in absorbance when the glucose residues are hydrolysed.
  • oNPG o-nitrophenyl- ⁇ -D-glucopyranoside
  • 2-nitrophenol which is measured spectrophotometrically at 405 nm.
  • the stability of the conjugate may be tested in vitro initially by incubating at 37°C in serum, followed by size exclusion FPLC analysis. Stability in vivo can be tested in the same way in mice by analysing the serum at various times after injection of the conjugate. In addition, it is possible to radiolabel the Hab or Hab fragment with " J, and the enzyme with l I before conjugation, and to determine the biodistribution of the conjugate, free Hab or Hab fragment and free enzyme in mice.
  • the conjugate may be produced as a fusion compound by recombinant DNA techniques whereby a length of DNA comprises respective regions encoding the two portions of the conjugate either adjacent to one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
  • two of the functional portions of the compound may overlap wholly or partly.
  • the DNA is then expressed in a suitable host in known ways.
  • the present invention also includes diagnostic methods and kits for detection and measurement of EGP-2 in biological fluids and tissues, and for localization of EGP-2 in tissues.
  • the Hab or Hab fragments can also be used in a diagnostic method and kit to detect and quantify antibodies capable of binding EGP-2. These kits may permit detection of circulating EGP-2 which, in certain situations, may indicate the spread of micrometastases by primary tumours in situ. Patients that have such circulating anti-EGP-2 antibodies may be more likely to develop tumours and cancers, and may be more likely to have recurrences of cancer after treatments or periods of remission. Kits for measurement of EGP-2 are also contemplated as part of the present invention.
  • the Hab and Hab fragments of the present invention that possess high titer and specificity can be used to establish easy to use kits for rapid, reliable, sensitive, and specific measurement and localization of EGP-2 in extracts of plasma, urine, tissues, and in cell culture media.
  • assay kits include but are not limited to the following techniques; competitive and non-competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry.
  • competitive and non-competitive assays radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry.
  • competitive and non-competitive assays radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assay
  • a radioimmunoassay (RIA) kit One example of an assay kit commonly used in research and in the clinic is a radioimmunoassay (RIA) kit. After successful radioiodination and purification of a HAb or a HAb fragment, the antiserum possessing the highest titer is added at several dilutions to tubes containing a relatively constant amount of radioactivity, such as 10,000 cpm. in a suitable buffer system. Other tubes contain buffer or preimmune serum to determine the non-specific binding.
  • a radioimmunoassay (RIA) kit After successful radioiodination and purification of a HAb or a HAb fragment, the antiserum possessing the highest titer is added at several dilutions to tubes containing a relatively constant amount of radioactivity, such as 10,000 cpm. in a suitable buffer system. Other tubes contain buffer or preimmune serum to determine the non-specific binding.
  • An immunohistochemistry kit may also be used for localization of EGP-2 in tissues and cells.
  • This immunohistochemistry kit provides instructions, a Hab or Hab fragment, and possibly blocking serum and secondary antiserum linked to a fluorescent molecule such as fluorescein isothiocyanate, or to some other reagent used to visualize the primary antiserum.
  • Immunohistochemistry techniques are well known to those skilled in the art.
  • This immunohistochemistry kit permits localization of EGP-2 in tissue sections and cultured cells using both light and electron microscopy. It is used for both research and clinical purposes. For example, tumours are biopsied or collected and tissue sections cut with a microtome to examine sites of EGP-2 production. Such information is useful for diagnostic and possibly therapeutic purposes in the detection and treatment of cancer.
  • the dosage of the Hab or Hab fragment of the present invention will depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. Depending upon the half-life of the Hab or Hab fragment in the particular animal or human, the Hab or Hab fragment can be administered between several times per day to once a week. It is to be understood that the present invention has application for both human and veterinary use. The methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render 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 ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, 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.
  • the Hab or Hab fragments of the present invention may be effective in treating cancer related diseases.
  • the present invention includes the method of treating cancer related disease with an effective amount of a Hab or Hab fragment of the present invention.
  • the Hab and HAb fragments of the present invention can be provided as isolated and substantially purified proteins and protein fragments in pharmaceutically acceptable compositions using formulation methods known to those of ordinary skill in the art. These compositions can be administered by standard routes. These include but are not limited to: oral, rectal, ophthalmic (including intravitreal or intracameral), nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal. intracranial, intratracheal, and epidural) transdermal. intraperitoneal. intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine. or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) routes.
  • the Hab or Hab fragment formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the Hab or Hab fragments of the present invention may be incorporated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the Hab or Hab fragment is slowly released systemically.
  • biodegradable polymers and their use are described, for example, in detail in Brem et al (J. Neurosurg 1991 74:441-446).
  • Osmotic minipumps may also be used to provide controlled delivery of high concentrations of Habs or Hab fragments through cannulae to the site of interest, such as directly into a metastatic growth or into the vascular supply to that tumor.
  • the Hab or Hab fragments of the present invention may be linked to cytotoxic agents which are infused in a manner designed to maximize delivery to the desired location.
  • cytotoxic agents which are infused in a manner designed to maximize delivery to the desired location.
  • ricin-linked high affinity Hab or HAb fragments are delivered through a cannula into vessels supplying the target site or directly into the target.
  • agents are also delivered in a controlled manner through osmotic pumps coupled to infusion cannulae.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question.
  • the Hab or Hab fragment conjugates may be administered in any suitable way, usually parenterally, for example intravenously or intraperitoneally. in standard sterile, non- pyrogenic formulations of diluents and carriers, for example isotonic saline (when administered intravenously).
  • the pro-drug is administered, usually as a single infused dose, or the tumour is imaged.
  • the Hab or Hab fragment conjugate may be immunogenic. cyclosporin or some other immunosuppressant can be administered to provide a longer period for treatment but usually this will not be necessary.
  • the timing between administrations of the Hab or Hab fragment conjugate and pro-drug may be optimised in a non-inventive way since tumour/normal tissue ratios of conjugate (at least following intravenous delivery) are highest after about 4-6 days, whereas at this time the absolute amount of conjugate bound to the tumour, in terms of percent of injected dose per gram, is lower than at earlier times.
  • the optimum interval between administration of the Hab or Hab fragment conjugate and the pro-drug will be a compromise between peak tumour concentration of enzyme and the best distribution ratio between tumour and normal tissues.
  • the dosage of the Hab or Hab fragment conjugate will be chosen by the physician according to the usual criteria. At least in the case of methods employing a targeted enzyme such as ⁇ - glucosidase and intravenous amygdalin as the toxic pro-drug, 1 to 50 daily doses of 0J to 10.0 grams per square metre of body surface area, preferably 1.0-5.0 g/m" are likely to be appropriate. For oral therapy, three doses per day of 0.05 to 10. Og, preferably 1.0-S.Og. for one to fifty days may be appropriate.
  • the dosage of the Hab or Hab fragment conjugate will similarly be chosen according to normal criteria, particularly with reference to the type, stage and location of the tumour and the weight of the patient.
  • the duration of treatment will depend in part upon the rapidity and extent of any immune reaction to the Hab or Hab fragment conjugate.
  • the functional portion of the Hab or Hab fragment conjugate, when the the Hab or Hab fragment conjugate is used for diagnosis usually comprises and consist of a radioactive atom for scintigraphic studies, for example technetium 99m ( 9m Tc) or iodine - 123 ( 12J I), or a spin label for nuclear magnetic resonance (nmr) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-313.
  • a radioactive atom for scintigraphic studies for example technetium 99m ( 9m Tc) or iodine - 123 ( 12J I), or a spin label for nuclear magnetic resonance (nmr) imaging (also known as magnetic resonance imaging
  • the functional portion of the Hab or Hab fragment may comprise a highly radioactive atom, such as ⁇ odine-131. rhenium-186. rhenium-188, yttrium-90 or lead-212, which emits enough to destroy neighbouring cells, or a cytotoxic chemical compound such as methotrexate. adriamicin, vinca alkaliods (vincristine. ⁇ inblastine. etoposidej, daunorubicin or other intercalating agents.
  • a highly radioactive atom such as ⁇ odine-131. rhenium-186. rhenium-188, yttrium-90 or lead-212, which emits enough to destroy neighbouring cells, or a cytotoxic chemical compound such as methotrexate. adriamicin, vinca alkaliods (vincristine. ⁇ inblastine. etoposidej, daunorubicin or other intercalating agents.
  • radio- or other labels may be incorporated in the Hab or Hab fragment conjugate in known ways.
  • the peptide may be biosynthesised or may be synthesised by chemical amino acid synthesis using suitable amino acid precursors involving, for
  • OQm 1 ' 1 R ⁇ 1 RR example, fluorine-19 in place of hydrogen.
  • Labels such as Tc, ⁇ I, Rh. Rh and 11 'in can be attached via a cysteine residue in the peptide.
  • Yttrium-90 can be attached via a lysine residue.
  • the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscinigraphy" (Chatal, CRC Press 1989) describes other methods in detail.
  • compositions comprising an effective amount of a Hab or Hab fragment expression product or a Hab or Hab fragment encoding nucleotide sequence of the present invention can be used in the treatment of cancer related disorders.
  • Such disorders include but not limited to: solid tumours; blood born tumours such as leukemias; tumor metastasis; benign tumours, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; wound granulation; cor
  • Figure 1 which is a graphical representation which shows the off-rate screening of half human (chimeric) Fab clones
  • Figure 2a shows a DNA sequence alignment of selected human variable light chains obtained by guided selection, and MOC-31 VL;
  • Figure 2b shows an amino acid sequence alignment of selected human variable chains obtained via guided selections, and the MOC-31 heavy chain
  • Figure 3 which shows the nucleotide sequence alignment of the selected human light chain from clone C3;
  • Figure 4 shows a comparison of amino acid sequences from the selected human light chain clone (C3), the selected human heavy chain clone (9E) and the murine MOC-31 heavy and light chains;
  • Figure 5 shows a graphical representation of results from a specificity ELISA of clones selected after the second shuffle
  • Figure 6 which shows the nucleotide sequence alignment of the selected human heavy chain from clone 9E
  • Figure 7 shows a graphical representation of results from a specificity ELISA using human Fab LD9 and control Fab binding to different recombinant antigens
  • Figure 8 shows the staining of tissue cryosections of A: colorectal carcinoma and B: normal colonic epithelium with human Fab LD9;
  • Figure 9 shows the complete nucleotide sequence and deduced aminoacid sequence of the VH and VL genes of human Fab LD9;
  • Figure 10 shows the staining of tissues with a MOC-31 derived scFv antibody fragment
  • Figure 11 shows the FACS analysis of human Fab LD9 and control Fab binding to the EGP-2 positive colon cancer cell line CaCo2;
  • Figure 12 shows the binding of soluble scFv MOC-31 and Fab's C3 and 9E to CaC02 cells in flow cytometry.
  • Figure 1 which shows the off-rate screening of half human (chimeric) Fab clones.
  • Figure 2a shows a DNA sequence alignment of selected human variable light chains obtained by guided selection, and MOC-31 VL.
  • the DNA sequences are aligned to Vg/38K, the human germline gene with closest DNA sequence to the selected human light chains as deduced using V-base.
  • Capital letters indicate nucleotide changes that induce amino acid modifications; small characters are slient mutations.
  • Figure 2b shows an amino acid sequence alignment of selected human variable chains obtained via guided selections, and the MOC-31 heavy chain.
  • the amino acid sequences of the selected human chains are aligned to the human germline gene with the closest DNA sequence as deduced using V-base.
  • A alignment of the light chains
  • B alignment of the heavy chains.
  • Capital letters indicate nucleotide changes that induce amino acid modifications; small characters are slient mutations.
  • Figure 3 which shows the nucleotide sequence alignment of the selected human light chain from clone C3. In the top line, the nucleotide sequence of clone C3 is shown.
  • Figure 4 shows a comparison of amino acid sequences from the selected human light chain clone (C3), the selected human heavy chain clone (9E) and the murine MOC-31 heavy and light chains.
  • the corresponding framework regions (FR) 1 to 4 and complementarity determining regions (CDR) 1 to 3 are grouped together.
  • FR framework regions
  • CDR complementarity determining regions
  • Figure 5 shows the specificity ELISA of clones selected after the second shuffle.
  • EGP-2, HEL, TT, BSA represent Epithelial Glycoprotein-2, Hen Egg White Lysozyme, Tetanus Toxoid and Bovine Serum Albumin respectively;
  • Figure 6 which shows the nucleotide sequence alignment of the selected human heavy chain from clone 9E.
  • the nucleotide sequence of clone 9E is shown.
  • the sequences are shown using the convention that a dashed line indicates identity with clone 9E and a letter signifies the location of an nucleic acid different from that in clone 9E.
  • the DNA sequence of the selected human heavy chain from clone 9E was aligned using the Kabat Vbase;
  • Figure 7 shows results from a specificity ELISA using human Fab LD9 and control Fab binding to different recombinant antigens.
  • Figure 8 shows the staining of tissue cryosections of A: colorectal carcinoma and B: normal colonic epithelium with human Fab LD9.
  • Bound soluble Fab was detected with the 9E10 antibody recognising a C-terminal c-myc derived epitope tag present in the antibody construct and peroxydase-conjugated rabbit anti mouse immunoglobulines. Staining was performed with di-aminobenzidin (DAB) / H 2 0 2 as substrate.
  • DAB di-aminobenzidin
  • Figure 9 shows the complete nucleotide sequence and deduced aminoacid sequence of the VH and VL genes of human Fab LD9.
  • Figure 10 shows the staining of tissues with a MOC-31 derived scFv antibod ⁇ fragment.
  • Bound antibody fragments were detected with the 9E10 antibody, recognising a C-terminal c-myc derived epitope tag present in the recombinant antibodies and peroxydase-conjugated rabbit anti mouse immunoglobulines. Di-aminobenzidin / H 2 0 2 was used as substrate for the staining reaction.
  • Figure 11 shows the FACS analysis of human Fab LD9 and control Fab binding to the EGP-2 positive colon cancer cell line CaCo2.
  • Human Fab LD9, cloned as gene III fusion in the phagemid vector pCESl (de Haard et al, 1999) and an anti-tetanus toxoid (control) Fab were rescued with helper phage M13K07 as described (Marks et al, 1991).
  • Recombinant phages expressing either Fab LD9 (thick line), or the anti tetanus toxoid Fab (thin line) were then used to stain colorectal cancer cell line CaCo2.
  • Bound phages were detected with a polyclonal sheep antiserum to fd phage (Pharmacia, Uppsala, Sweden) and FITC-labeled rabbit anti goat immunoglobulines (Dako, Glostrup, Denmark). Cells were analysed by flow cytometry using a FACS Calibur (Becton & Dickinson, Heidelberg, Germany).
  • Figure 12 shows the binding of soluble scFv MOC-31 and Fab ' s C3 and 9E to CaC02 cells in flow cytometry.
  • Thin line is negative control (9E10); thick line is binding of scFv MOC-31; covered half human Fab C3 (nearly completely masked by the MOC-31 line); ( — ) human Fab 9E.
  • a naive human VLCL library (9x10 6 clones), which was prepared from total R ⁇ A isolated from the spleen of a pseudomixoma peritonei patient.
  • V-genes of MOC-31 were cloned in pCESl Fab format vector (De Haard et al, 1999) as indicated (Roovers et al, 1998), and the VH-CH1 of MOC-31 was combined with the human VLCL library.
  • the ApaLl-Ascl fragment of the cloned mouse VLCLs was replaced by the VLCLs from the non-immunized human VLCL library.
  • the obtained library was subjected to four rounds of selection on biotinylated antigen using a decreasing amount of antigen with each round. A small panel of human light chains with related sequences was identified.
  • the selected human VL was combined with a human VHCH1 library which retained the heavy chain CDR3 (HCDR3) of MOC-31.
  • the VHCHl library was prepared by PCR amplification of the VH genes from human peripheral blood lymphocyte (PBL) cDNA and spleen cDNA.
  • PBL peripheral blood lymphocyte
  • spleen cDNA human peripheral blood lymphocyte
  • 207 5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG (AG)TC ACC TTG AAG GAG TCT GG-3'; 206:5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC (GC)AG GTC CAG CTG GT(AG) CAG TCT GG-3';
  • Biotinylated EGP-2 was prepared by expressing and purifying recombinant EGP-2 in a baculovirus system as described by Strassburg et al 1992 and biotinylating with NHS-SS- biotin (Pierce) according the manufacturers descriptions. The biotinylated EGP-2 was tested with MOC-31 on a streptavidin-chip in BIAcore2000.
  • phage were incubated on a rotator wheel for one hour in 2% M-PBST (PBS supplied with 2% Marvel -skimmed milk and 0.1% Tween-20);
  • the infected TGI cells were plated on 2xTY (16 g Bacto-trypton. 10 g yeast- extract and 5 g NaCl per litre) agar plates containing 2% glucose and 100 ⁇ g/ml ampicillin and were incubated overnight at 30°C;
  • Soluble Fab's were produced as described by Roovers et al (1998).
  • the cultures were inoculated with E. coli TGI (K12, O(lac-pro), supE, thi, hsdD5/F' traD36, proA r B + , lacP, / ⁇ cZDM15) harbouring the Fab in pCESl.
  • Individual bacterial clones were picked and production of soluble Fab was induced by activation of the upstream Lac Z promoter with isopropyl- ⁇ -1-thiogalactopyranoside (IPTG) as described by Marks et al (1991).
  • ELISA using soluble Fabs was performed on purified, recombinant EGP-2 in order to identify binding Fabs from the individual clones selected.
  • ELISA plates (Costar, Cambridge, MA, USA) were coated overnight with 1 ⁇ g ml "1 EGP-2 in phosphate- buffered saline (PBS), washed three times with PBS-T [PBS, 0.5% (v/v) Tween 20]. three times with PBS and blocked for 1 h at room temperature (RT) with 2% MPBS [2% (w/v) Marvel - skimmed milk powder - in PBS].
  • induced bacterial supernatants were added [50% (v/v) in 2% MBS] and incubated for 1.5 h at RT. Bound antibody fragments were detected with the 9E10 antibody [50% (v/v) hybridoma supernatant in 2% MPBS], peroxidase-conjugated rabbit anti-mouse immunoglobins [Dako. Glustrup. Denmark: 0.1% (v/v) in 2% MPBS] and stained with trimethylbenzidine (TMB) and hydrogen peroxide. Optical density was measured at 450 nm. This assay is described in detail by Roovers et al (1998 ibid).
  • the assay was carried out as described above but the wells were coated with antigens such as 10 ⁇ g/ml Bovine Serum Albumin (Sigma) in PBS, 3 mg/ml Hen Egg White Lysozyme (Boehringer Mannheim) in 0J M NaHC0 3 (pH 9.6), 10 ⁇ g/ml Tetanus Toxoid in 0.1 M NaHC0 3 (pH 9.6) and 0.5 ⁇ g/ml EGP-2 in PBS.
  • antigens such as 10 ⁇ g/ml Bovine Serum Albumin (Sigma) in PBS, 3 mg/ml Hen Egg White Lysozyme (Boehringer Mannheim) in 0J M NaHC0 3 (pH 9.6), 10 ⁇ g/ml Tetanus Toxoid in 0.1 M NaHC0 3 (pH 9.6) and 0.5 ⁇ g/ml EGP-2 in PBS.
  • the nucleotide sequences of the selected Fabs were determined using the dideoxy sequencing method of Sanger. Products of the sequencing reaction were analysed on a semiautomated sequencer (Alf Express; Pharmacia). The oligonucleotides used were:
  • EGP-2 (Strassburg et al 1992) was covalently coupled to a SA/CM5 sensor chip (Pharmacia, Uppsala, Sweden) via amine coupling, resulting in an antigen surface of 530/1667 resonance units (RU). IMAC-purified monovalent Fab antibody fragments were then run over the surface at high flow rate to ensure saturation of binding. From the obtained sensorgrams, dissociation rates (off-rates) were determined by curve-fitting on the first 20 sec of the dissociation phase using the BIAevaluation software (Pharmacia. Uppsala. Sweden). All kinetic measurements were performed on this antigen surface.
  • cryosections of different tissues were cut and mounted on 3-aminopropyl-2-ethoxysilane (APTS-) coated slides. Sections were air dried, fixed for lOmin at RT with 1% (w/v) paraformaldehyde and air-dried again. Slides were blocked with 10% (v/v) foetal calf serum (FCS) in PBS for 30min at RT and then incubated with recombinant antibody fragments. 9E10 antibody and peroxydase-coupled rabbit anti mouse immunoglobulins (Dako. Glostrup. Denmark). DAB/H 0 2 staining was finally used to visualise bound antibody fragments using standard procedures.
  • FCS foetal calf serum
  • the cloned scFv antibody fragments of MOC-31 showed a variable degree of dimerisation.
  • the mouse heavy chain V gene was cloned into the pCESl Fab format vector (de Haard et al ibid).
  • the obtained chimeric VHCH was combined with both the kappa (K) and lambda ( ⁇ ) human VLVL libraries which were separately generated yielding two libraries of 10 clones (K) and 2 x 10 7 clones ( ⁇ ) respectively.
  • Figure 3 shows the DNA sequence from the light chain of clone C3 Fab which had the lowest off-rate ((k 0 ff 3.0 x 10 "4 s "1 ).
  • a DNA sequence alignment (also shown in Figure 3) revealed that the light chain of clone C3 Fab is fully human and originated from the Kabat Vklll family.
  • a comparison with the germline sequence (EMBL Accession No. X01668) indicated that 10 nucleotide and 5 amino acid substitutions were found.
  • Fig 4 shows that the amino acid sequence of the obtained human light chain from clone C3 differs moderately from the original MOC-31. In particular, no homology was found between the complementarity determining regions (CDRs) of the variable human light chain (VL) from clone C3 and those from MOC-31 VL.
  • CDRs complementarity determining regions
  • the selected human light chain was first cloned into pCESl absent in VH chains to avoid contamination of the MOC-31 VH.
  • cDNA of either PBL or spleen origin was amplified using primer H3 EGP-2 primer, to introduce the MOC-31 CDR3 sequence.
  • the PCR product was then cloned into pCESl vector containing the selected human light chain from clone C3.
  • the resulting libraries from PBL (2 x 10 7 clones) and spleen (1.2 x 10 7 clones) were subjected to three rounds of selection.
  • Fabs were blotted on nitrocellulose and detected with 9E10 and peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAKO, Glostrop, Sweden). This dot blot analysis showed that the clones did produce Fabs, but only in small amounts.
  • Phage display was also used to directly select fully human, high affinity antibody fragments specific for the EGP-2 antigen.
  • the libraries that were used in this study were prepared by PCR amplification of the V-genes from human PBL cDNA and spleen cDNA. Random priming was used for the light chain and IgM-specific priming for the heavy chain which were cloned in two steps into a phagemid vector (pCESl : Hoogenboom et al 1998). Individually cloned heavy and light chain variable region libraries were then combined in an efficient 2-step cloning procedure, permitting the cloning of in total 3.7 x 10 10 independent Fab clones. The design of the library permitted the monitoring of selections with polyclonal phage preparations and the large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore.
  • the performance of the library was tested by isolating on average 13 Fabs specific for protein antigens and even more Fab's for antigen fragments (haptens). After three rounds of panning, two different antibodies C2 and LD9 in a Fab format (on the basis of DNA fingerprint analysis) were found that recognised the EGP-2 antigen in ELISA.
  • Clone C2 (Table V) had a relatively fast off-rate (2 x 10 v “ 2 ⁇ s - " K ) but clone LD9 had an off- rate (2.4 x 10 '4 s "1 ) that was slightly better than that of the high-affinity murine scFv fragments MOC31 (Roovers et al 1998 ibid).
  • the fully human Fab was also tested in immunohistochemistry. As shown in Figure 10 the Fab specifically bound EGP-2 positive tissues. The Fab reacted with normal and malignant non-squamous epithelia and did not stain malignant tissue reported negative for the antigen (melanoma; Herlyn et al 1979).
  • the murine Mab MOC-31 has the best binding characteristic in terms of the lowest dissociation rate ("off-rate") of a series of anti-EGP-2 antibodies tested.
  • the V-genes of the MOC-31 Mab were cloned as single chain variable fragment (scFv) which competed with the original hybridoma antibodies for binding to EGP-2 antigen.
  • the off-rate of the MOC-31 scFv was better than those of the bivalent 17-1 A and 323/A3 anti-EGP-2 antibodies, thus providing it with a preferred characteristic for tumour retention in vivo (Adams et al 1998).
  • the V-genes of MOC-31 were chosen for the rational design and generation of human anti-EGP-2 antibodies to with a view to interalia developing in vitro and in vivo diagnostics and immunotherapeutics for the treatment of solid tumours.
  • the libraries that were used in this study were prepared by PCR amplification of the V-genes from human PBL cDNA and spleen cDNA. Random priming was used for the light chain and IgM-specific priming for the heavy chain which were cloned in two steps into a phagemid vector (pCESl ; Hoogenboom et al 1998).
  • the performance of the library was tested by isolating on average 13 Fabs specific for protein antigens and even more Fab's for antigen fragments (haptens). Two human antibody fragments C2 and LD9 were isolated. These antibodies had off-rates in the order of 10 "2 to 10 "4 s "1 and affinities up to 2.7 nM. The kinetics of these phage antibodies were of the same order of magnitude as antibodies associated with a secondary immune response.
  • phage antibody selection mostly yielded antibodies that targeted the same, or closely related epitopes on the antigen as the murine antibodies obtained by immunisation of mice and hybridoma technology (Herlyn et al 1979; Edwards et al 1986; Souhami et al 1988). These results suggest that there is a 'hot spot' on the antigen that is the immunodominant target in vivo as well as the prefered target during phage antibody selection in vitro.
  • SEQ ID No 1 (C3 aa sequence) is provided in Figure 2 and Figure 4(a);
  • SEQ ID No 2 (9E aa sequence) is provided in Figure 4(b);
  • SEQ ID No 9 (LD9 VL aa sequence) is provided in Figure 9b;
  • SEQ ID No 10 (LD9 VH aa sequence) is provided in Figure 9a; SEQ ID Nos 1 1, 12 and 13 (CDRl. CDR2. CDR3 aa sequences of LD9 VL) are provided in Figure 9;
  • SEQ ID Nos 14, 15 and 16 (CDRl, CDRJ, CDR3 aa sequences of LD9 VH) are provided in Figure 9;
  • SEQ ID No 17 (LD9 VL na sequence) is provided in Figure 9b;
  • SEQ ID No 18 (LD9 VH na sequence) is provided in Figure 9a;
  • SEQ ID No 25 (C3 VL na sequence) is provided in Figure 3;
  • SEQ ID No 26 (9E VH na sequence) is provided in Figure 6;
  • Ep-CAM a human epithelial antigen is a homophilic cell-cell adhesion molecule. J Cell Biol 125: 437-446.
  • Ep-CAM Epithelial cell adhesion molecule

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Abstract

L'invention concerne des anticorps humains capables de reconnaître l'antigène de la glycoprotéine épithéliale (EGP-2). Les anticorps présentent une région variable de chaîne légère humaine et une région variable de chaîne lourde humaine. L'invention concerne également des fragments d'anticorps et des compositions pharmaceutiques comprenant ces anticorps ainsi que leurs applications in vitro et in vivo dans le diagnostic et l'immunothérapie.
PCT/GB2000/001910 1999-05-18 2000-05-18 Anticorps WO2000069914A2 (fr)

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Cited By (29)

* Cited by examiner, † Cited by third party
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WO2003025565A2 (fr) * 2001-09-21 2003-03-27 Caprion Pharmaceuticals Inc. Preparation de membranes plasmiques fortement purifiees
WO2003074566A2 (fr) * 2002-03-01 2003-09-12 Immunomedics, Inc. Anticorps rs7
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