Classifications

• • 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
  • C07K16/3007—Carcino-embryonic Antigens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

• This invention relates to a chimeric mouse-human antibody to carcinoembryomic antigen (CEA) designated T84.12.
• CEA is a widespread tumor marker. Its expression can be detected in more than 95% of all human colon cancers. It is a member of the immunoglobulin superfamily and is closely related to NCA and BGP.
• murine T84.66 antibody shows the highest specificity and affinity for CEA ( agener, et al., J. Immunology 130:2308-2315 (1985)). It has been used successfully for ii vivo tumor imaging in mice and humans. It is well suited for the immunodetection and immunotherapy of human colon cancers.
• T84.66 The jLn vivo human use of T84.66 is limited by its murine origin resulting in immune response against the heterologous immunoglobulin.
• Chimeric T84.66 was created by use of recombinant gene technology to lessen the immunogenicity in man See Neumaier, et al., Cancer Research 5_0:2128-2134 (1990) and United States Patent 5,081,235.
• the cloned antibody genes including the immunoglobulin promoter were transfected into SP2/0 myeloma cells by electroporation or CHO cells using lipofection.
• the expressed chimeric mabs were characterized in different enzyme immunoassays and a western blot.
• the sequence of the V-regions of the heavy and light chain genes were determined using the well known Sanger chain termination method.
• Murine T84.12 is another well characterized CEA specific monoclonal of the murine IgG2a isotype. It recognizes the same epitope on CEA as T84.66 but with an affinity constant which is lower by a factor of approximately ten (10). For that reason, T84.12 was selected, pursuant to this invention, to generate mouse-human chimeric antibodies for therapeutic purposes in man. cDNA clones were humanized (chimerized) by shuffling the human IgGl heavy or light chain constant domain exons, including the 5'-UT and leader peptide, to the variable regions of the heavy and light chain genes of murine T84.12.
• the resulting hybridoma produces significant quantities of chimeric T84.12 anti-CEA antibodies useful for, among other things, human therapeutic purposes.
• Production of the chimeric anti-CEA antibodies of this invention entails a series of steps including, among others, identification of the amino terminal protein sequences of murine T84.12, determination of the cDNA sequence of mouse light chain and heavy chain clones of T84.12 and of the corresponding amino acid sequences and the chimerization of murine T84.14 cDNA clones.
• One aspect of the invention entails in vitro mutagenesis of a mouse T84.12 light chain clone. Aminoterminal Sequences of Murine T84.12
• Murine T84.12 specific light (L) chain clones L1-L4 and T84.12 heavy chain clones H1-H4 were prepared and sequenced in known manner. All four heavy chain clones showed a 100% V-region homology in their V-region and therefore clone H4 was selected for the sequencing of the IgG2a heavy chain constant regions.
• the variable domains of light chain clones L2, L3 and L4 were identical.
• Clone LI was totally different, apparently representing the endogenous transcript.
• the light chain clones LI, L4 and the heavy chain clone H4 were selected.
• Table I sets forth the amino terminal sequences of the T84.12 light and T84.12 heavy chains. The reported sequences were determined using reduced (DTT) and alkylated (iodoacetic acid) purified monoclonal antibody. The heavy and light chains were separated under reducing conditions on a Sephadex G100 column using 1 M acetic acid as a running buffer. The isolated chains were subjected to amino acid sequencing.
• SEQ ID NO. 1 the light chain cDNA sequence of murine T84.12, the following regions are underlined (from the top to the bottom) : ATG start codon, start of variable region, start of C-kappa constant domain, TAG stop codon and polyadenylation signal.
• cDNA Sequence of Mouse Heavy Chain Clone T84.12 H4 The complete sequence of the full size cDNA clone T84.12 was determined in known manner (1645 bp) . This clone contained a 10 bp longer 5'-UT region than the light chain clone L4 which was also followed by the ATG start codon.
• the presence of the entire leader peptide, V-region and all three constant domain of IgG2a could be demonstrated.
• CH3 constant domain of IgG2a a TGA stop codon was present.
• the 3'-untranslated region (120 bp) contained the polyadenylation signal AATAAA.
• the entire full size cDNA clone was flanked by the destroyed Smal restriction cloning site GGG-CCC.
• the IgG2a constant domain showed a 98.7% homology to other published IgG2a constant domain sequences (Kabat) .
• the hinge region showed a 100% homology to the Kabat sequence too.
• Two different codons in the CHI domain were identical to IgG3 and three different codons in the CH3 domain identical to MOPC21.
• SEQ ID NO. 4 the heavy chain amino acid sequence of T84.12 H4 the following regions are underlined (from the top to the bottom) : ATG start codon, start of variable region, start of CHI constant domain, start of hinge region, start of CH2 constant domain, start of CH3 constant domain and TGA stop codon.
• the obtained and characterized full size cDNA murine T84.12 L4 and H4 clones were chimerized using the constant domains of human IgGl heavy chain cDNAs and the constant domains of human kappa light chain cDNAs respectively.
• the human heavy and kappa chain constant region sequences were derived from plasmids obtained from Dr. Jeffrey Schlom, National Institutes of Health.
• the plasmids contained chimeric B72.3 cDNA clones, cloned from cells expressing the chimeric B72.3 antibody (see, Hutzell, et al., Cancer Research 31:181-189 (1991)).
• Dr. Schlom's group obtained the human gamma and kappa chain genomic expression vectors from Dr.
• variable domains of T84.12 were, in known manner, fused in frame to the human constant domain(s) of chimeric B72.3 using the splice overlap extension PCR. See Ho, et al., Gene 77:51-59 (1988) and Horton, et al., Gene 77:61-68 (1989). These full size cDNA's were named CHI T84.12 L3, L6, L8, H2 and H3.
• the entire sequence of the full size cDNA clone chiT84.12 L6 was determined in known manner (956 bp) .
• the clone chiT84.12 L6 showed the correct sequence for a mouse-human chimeric T84.12 light chain.
• the clone chiT84.12 L6 was used for further subcloning into the pH ⁇ -Apr-neo vector (see Gunning, et al., Proc. Natl. Acad. Sci. 84:4831-4835 (1987)) to transfect SP2/0 myeloma cells.
• the clone chiT84.12 L6 contained a short 5 , -UT region of 9 bp which was followed by the ATG start codon. The presence of the entire leader peptide, V-region and the human Ckappa constant domain could be confirmed. At the end of the human Ckappa constant domain a TAG stop codon was present.
• the 3'-untranslated region (218 bp) contained a polyadenylation signal (AATAAA) .
• the human Ckappa constant domain showed a 100% homology to other published Ckappa constant domain sequences (Kabat) .
• the clone chiT84.12 H3 was used for further subcloning into the pH ⁇ -Apr-gpt vector to transfect SP2/0 myeloma cells which are expressing chiT84.12 kappa light chains.
• the human IgGl constant domain showed a 100% homology to other published IgGl constant domain sequences (Kabat) . The hinge region showed a 100% homology to the Kabat sequence too.
• the heavy chain cDNA sequence of chiT84.12 H3 the following regions are underlined (from the top to the bottom) : ATG start codon, start of mouse variable region, start of human CHI constant domain, start of hinge region, start of CH2 constant domain, start of CH3 constant domain, TGA stop codon and polyadenylation signal.
• the heavy chain amino acid sequence of chiT84.12 H3 the following regions are underlined (from the top to the bottom): ATG start codon, start of mouse variable region, start of human CHI constant domain, start of hinge region, start of CH2 constant domain, start of CH3 constant domain and TGA stop codon.
• cysteine residues are typically present in an immunoglobulin domain.
• the CDR3 (L3) of T84.12 light chain clone L4 contained an additional third cysteine residue in the mouse variable kappa light chain domain.
• the presence of the third cysteine is apparently related to the loss of binding activity by murine T84.12 after dissociation of both chains and chemical crosslinking using homobifunctional crosslinking agents. Therefore the cysteine (TGT) in position 364-366, see SEQ ID NO. 1, (amino acid residue 91) was changed to a serine (TCT) by site directed mutagenesis.
• the mutagenesis was carried out using the MUTA-GENE phagemid _in vitro mutagenesis kit from BioRad. The original procedure was simplified and reduced to the following eleven steps:
• PCI PEG extraction and purification
• Phosphorylation of the mutagenesis primer (represents the minus strand and binds to the single stranded plus strand phagemid DNA) .
• the insert size can be determined by restriction enzyme digest and compared to the wild type.
• T84.12 L4-12-1 One such clone, named T84.12 L4-12-1 was selected for exemplification of the invention.
• cDNA Sequence of T84.12 L4-12-1 The entire sequence of the full size cDNA clone T84.12 L4-12-1 was determined in known manner (1999 bp) .
• the clone showed the correct sequence for a mouse T84.12 light chain and the introduced cysteine to serine mutation. It was used for further subcloning into the PH / 9-Apr-neo vector (See Gunning, et al., Proc. Natl. Acad. Sci. 84:4831-4835 (1987)) to transfect SP2/0 myeloma cells.
• This T84.12 L4-12-1 clone contained a very short 5'-UT region of 10 bp which was followed by the ATG start codon. The presence of the entire leader peptide, V-region and the Ckappa constant domain could be demonstrated. At the end of the Ckappa constant domain a TAG stop codon was present.
• the 3'-untranslated region (280 bp) contained a polyadenylation signal (AATAAA) .
• the entire full size cDNA clone was flanked by the destroyed Smal restriction cloning site (GGG-CCC) .
• SEQ ID NO. 6 the light chain amino acid sequence of T84.12, the following regions are underlined (from the top to the bottom) : ATG start codon, start of variable region, start of C-kappa constant domain and TAG stop codon.
• the mutagenized TGT (cys) to TCT (ser) is underlined and in italics. All other cysteine residues are underlined.
• the mutated light chain (T84.12 L4-1) cDNA and the normal heavy chain (T84.12 H4) cDNA were transferred in a / 9-actin cDNA expression vector (Gunning, et al., supra) and cotransfor ed into Sp2/0 myeloma cells by electroporation.
• the vectors include the human -actin promoter, intervening sequence, cloning site, and a polyadenylation signal. Since the vectors contain the neomycin-resistance gene, transfectants were selected in the presence of the drug, G418. Clones were expanded and evaluated for antibody production (kappa or gamma chain) and CEA-binding activity by ELISAs. Although levels of expression were low, there was a correlation between antibody and anti-CEA activity in culture supernatants.
• GATTTCACTC TCACCATCAG CAATGTGCAG TCTGAAGACT TGGCAGAATA 350
• AAAGACAGCA CCTACAGCAT GAGCAGCACC CTCACGTTGA CCAAGGACGA 650
• MOLECULE TYPE Nucleic Acid
• HYPOTHETICAL Not Applicable
• ANTI-SENSE Not Applicable
• TTTTCCTTGT CCTTGTTTTA AAAGGTGTCC AGTGTGAAGT GAAGCTGGTG 150
• AAAAAAAAAA AAAAAAGGGG ATCCTCTAGA GTCGACCTGC AGGCA 1645
• GATTTCACTC TCACCATCAG CAATGTGCAG TCTGAAGACT TGGCAGAATA 350
• AAAGACAGCA CCTACAGCAT GAGCAGCACC CTCACGTTGA CCAAGGACGA 650
• MOLECULE TYPE Nucleic Acid
• HYPOTHETICAL Not Applicable
• ANTI-SENSE Not Applicable
• FRAGMENT TYPE Not Applicable
• MOLECULE TYPE Nucleic Acid
• HYPOTHETICAL Not Applicable
• ANTI-SENSE Not Applicable
• GATTTCACTC TCACCATCAG CAATGTGCAG TCTGAAGACT TGGCAGAATA 350
• MOLECULE TYPE Nucleic Acid
• HYPOTHETICAL Not Applicable
• ANTI-SENSE Not Applicable
• TTACGAATTC GAGCTCGGTA CCCCCTGGAT TTGAGTTCCT CACATTCAGT 50 GATGAGCACT GAACACAGAC ACCTCACCAT GAACTTCGGG TTCAGCCTGA 10 TTTTCCTTGT CCTTGTTTTA AAAGGTGTCC AGTGTGAAGT GAAGCTGGTC 15 GAGTCTGGGG GAGGCTTTGT GAAGCCTGGA GGGTCCCTGA AACTCTCCTG 20 TGCAGCCTCC GGATTCACTT TCAGTAGTTA TGCCATGTCT TGGGTTCGCC 25 AGACTCCAGA GAAGAGGCTG GAGTGGGTCG CATCCATTAG TAGTGATGGT 30 ATCACCTTCT ATGTAGACAG TGTGAAGGGC CGATTCACCG TCTCCAGAGA 35 CAATGCCAGG AACATCCTGT ACCTGCAAAT GAGCAGTCTG AGGTCTGAGG 40 ACACGGCCAT GTATTACTGT GCAAGAATCG ACTACTACGG AGGAGGGGGA 45 TTTGGTTACT
• CCCCCTGTAC ATACTTCCCG GGCGCCCAGC ATGGGAATAA AGCACCCAGC 1600
• MOLECULE TYPE Nucleic Acid
• HYPOTHETICAL Not Applicable
• ANTI-SENSE Not Applicable

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• 1993
  • 1993-06-15 WO PCT/US1993/005709 patent/WO1993025237A1/en not_active Application Discontinuation
  • 1993-06-15 JP JP6501788A patent/JPH06509947A/ja active Pending
  • 1993-06-15 EP EP93915354A patent/EP0606430A1/en not_active Withdrawn
  • 1993-06-15 CA CA002115346A patent/CA2115346A1/en not_active Abandoned
  • 1993-06-15 AU AU45366/93A patent/AU4536693A/en not_active Abandoned

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