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WO1992008801A1 - Structures de fusion d'anticorps de pontage - Google Patents

Structures de fusion d'anticorps de pontage Download PDF

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Publication number
WO1992008801A1
WO1992008801A1 PCT/US1991/008421 US9108421W WO9208801A1 WO 1992008801 A1 WO1992008801 A1 WO 1992008801A1 US 9108421 W US9108421 W US 9108421W WO 9208801 A1 WO9208801 A1 WO 9208801A1
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Prior art keywords
cell
construct
nucleic acid
acid sequence
variable region
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Inventor
Stephen D. Gillies
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Abbott Laboratories
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Abbott Laboratories
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • 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
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • This invention relates to therapies involving selective destruction of cells in vivo, and more specifically, to compositions of matter useful in the treatment of various cancers and viral infections.
  • this application relates to genetically engineered-antibody fusion constructs capable of targeting an infected cell and bringing that cell into contact with an effector cell which can kill or neutralize its detrimental activities.
  • Hormone receptors have been used as tumor-specific markers for the delivery of cytotoxic agents to tumor cells.
  • Pseudomonas exotoxin and diphtheria toxin have been coupled to peptide hormones and have been shown to be highly cytotoxic and specific for receptor-bearing cells (Astan et al. (1989) J. Biol. Chem. 264;15157-15160: Bacha et al. (1988) J. Exp. Med. 167.612-622, .
  • Antibodies have been shown to mediate the lysis of tumor cells in vitro by bridging the Fc receptor (FcR) on the cytotoxic effector cell and the antigenic site on the target cell (Henkart (1985) Ann. Rev. Immunol. 3 . :31-58. The binding is mediated by the variable (V) regions of the heavy (H) and light (L) chains of the anti-tumor cell antibody and the FcR binding site on the constant (C) region of the Ig H chain.
  • cytotoxic T lymphocytes have been targeted to cells for which they have no natural specificity
  • cross-linking agents include several hetero-bifunctional reagents that share the same mechanism; they bridge a specific marker on the tumor cell surface to a component of the T cell receptor (TCR) and in this way activate the lytic program of the cytotoxic T lymphocyte (Lui et al. (1985) Proc. Natl. Acad. Sci. USA J82.:8648-8652; Perez et al. (1986) J. Expt. Med. _16_3:166-178; Jung et al. (1986) Proc. Natl. Acad. Sci. USA £:4479-4483) .
  • TCR T cell receptor
  • heterobifunctional antibodies and chemical cross-linking reagents may not be efficient. Because of the random association of multiple H and L chains, only a fraction of the resulting antibodies usually are active. Similarly, the binding of a chemical cross-linking reagent may disrupt or inactivate the site or protein at which the reagent binds and hence may not enable the triggering of the effector cells' killing or neutralizing activities.
  • an object of the invention is to provide a construct that bridges an effector cell and a target cell, thereby enabling the killing or the neutralization of that target cell. Another object is to produce a bridging construct that will not inactivate the killing or neutralizing activities of the effector cell when it is bound thereto. Yet another object is to provide an efficient and effective method of targeting effector cells to malignant or virus-infected cells. Still another object is to provide a method of producing these bridging constructs.
  • antibody fusions constructs ' have been produced which effectively bridge a target cell, such as a malignant or virus-infected cell, and an effector cell. Such constructs enable treatment of malignancies and virus infections with accuracy and efficiency.
  • a representative antibody fusion construct includes a heavy chain variable region, a heavy chain constant region having a C H3 domain, and a non-immunoglobulin binding agent which binds a surface antigen or receptor on a target cell.
  • the heavy chain constant region may also include other domains such as a C HI domain and/or CH,2 domain.
  • the heavy chain variable region when combined with a light chain variable region, binds to a surface antigen on an effector cell.
  • SUB agent can be a ligand or a receptor.
  • nonim unoglobulin binding agent refers to a protein or polypeptide including ligands, receptors, or single chain binding sites that mimic antibody binding sites with predetermined specificity for a surface antigen on a target cell.
  • effector cell refers to any cell which can neutralize or destroy the target cell with which it has been placed in contact.
  • the invention takes advantage of the existence of particular surface proteins or antigens which are specific for a particular class of effector cells.
  • One preferred construct includes a heavy chain variable region having specificity for the CD3 antigen found on the surface of cytotoxic T lymphocytes.
  • Other constructs embraced by the invention have heavy chain variable regions with specificities for a particular surface antigen on other effector cells such as macrophages, monocutes, natural killer cells, eosinophils, and large granular lymphocytes.
  • the non-immunoglobulin binding agent includes a hormone or a growth factor which binds a receptor specific for that ligand.
  • a growth factor is an epidermal growth factor (EGF) , or an analog or fragment thereof, capable of binding the EGF receptor found on a target cell.
  • EGF epidermal growth factor
  • the non-immunoglobulin binding agent is a receptor which recognizes and binds a surface protein on a virus-infected cell such as an HIV-infected cell.
  • a virus-infected cell such as an HIV-infected cell.
  • one construct includes a CD4, or an analog or fragment thereof, which is capable of binding the gpl20 envelope protein.
  • the non-immunoglobulin binding agent is a single chain binding site, as for example a peptide sequence derived from a mammalian antibody specific for an antigen which is characteristic of a particular target cell.
  • This invention also embodies nucleic acid sequences such as DNA or RNA encoding the amino acid sequence of a bridging antibody construct, as well as cell lines transfected with such nucleic acid sequences which produce the aforementioned construct.
  • Preferred cell lines to be transfected are myeloma and hybridoma cell lines.
  • this invention encompasses methods of producing the bridging antibody constructs as well as methods of selectively killing a target cell in vivo with the use of these constructs
  • the bridging antibody constructs may be prepared as follows. Nucleic acid sequences encoding amino acid sequences of a heavy chain variable region, a heavy chain constant region, and a non-immunoglobulin binding agent, are linked. A host cell is transfected with this nucleic acid and cultured such that it expresses the construct. The host cell may be transfected concurrently with a nucleic acid sequence encoding a light chain variable region. The expressed heavy chain variable region/ligand construct and the expressed light chain variable region may then be combined to form a two or four chain construct.
  • a target cell may be selectively killed L vivo by preparing a bridging antibody construct specific for that target cell and for an effector cell capable of killing or neutralizing that target cell, and then administering the construct to the circulation of a subject harboring the target cell.
  • FIG. 1 is a schematic representation of one embodiment of the bridging antibody construct of the present invention
  • FIG. 2 is a diagrammatic representation of the construction of an antibody fusion construct including the human C7I Ig heavy chain and EGF.
  • FIG. 2A is the restriction map of a C7I gene fragment cloned in plasmid pBR322.
  • FIG. 2B shows the fusion of the C7I gene at the Sma I site to a synthetic EGF-encoding sequence.
  • FIG. 2C shows the sequence at the junction of the Ig C H3 domain and the amino terminus of EGF;
  • FIG. 3 is a graphic representation of EGF receptor binding activity of the anti-CD3/EGF conjugate. The activity is measured by comparing the abilities of the conjugate, cold EGF, and anti-EGF receptor antibody to compete with labelled EGF for EGF receptors on M-24 melanoma cells;
  • FIG. 4 is a graphic representation of anti-CD3/EGF conjugate-induced killing of tumor cell A431 epidermal carcinoma cells (FIG. 4A) , M24 etastatic melanoma cells (FIG. 4B), and IMR-32 neuroblastoma cells (FIG. 4C), by TIL 660 cells;
  • FIG. 5 is a graphic representation of anti-CD3/EGF
  • FIG. 6 is a diagrammatic representation of the preparation of an antibody fusion construct including the human C74 chain and a single chain binding site, in which FIG. 6A shows details of a V -linker-V sequence and FIG. 6B illustrates an assembled expression vector pdHL2-ocCD3/sca-X; and
  • FIG. 7 is a graphic representation of anti-CD3/single chain binding site conjugate-induced killing of M21 melanoma cells by TIL 660 effector cells, using the construct described in connection with FIG. 6.
  • the present invention concerns bridging antibody, constructs useful for homing an effector cell to a malignant or virus-infected target cell.
  • the construct includes a conjugate of an antibody portion having a specificity for a surface antigen on an effector cell, and a non-immunoglobulin binding agent complementary to receptors or ligands found on the target cell.
  • the immunoglobulin portion includes a heavy chain variable region (V ) which, when combined with a light chain variable region (V ) , binds to a surface antigen on an effector cell. It also includes at least a heavy chain C H3 domain peptide-linked to the carboxy terminus of the V H domain. C and/or C H2 domains may also be peptide-linked to the carboxy terminus of the V H domain and to the amino terminus of the CH3 domain. Without the C HI and/or the CH_ domains, the half-life of the construct decreases in vivo.
  • the immunoglobulin portion of the construct may be chimeric in that the variable region may
  • STITUTESHEET come from one species and the constant region from another.
  • FIG. 1 shows a schematic view of a representative bridging antibody construct 10.
  • ligand molecules 2 and 4 are peptide bonded to the carboxy termini 6 and 8 of C H3 regions 10 and 12 of antibody heavy chains 14 and 16.
  • V L regions 26 and 28 are shown paired with V regions 18 and 20 in a typical IgG configuration, thereby providing two antigen binding sites 30 and 32 at the amino ends of construct 10 and two receptor-binding sites 40 and 42 at the carboxy ends of construct 10.
  • the constructs need not be paired as illustrated.
  • a particularly useful specificity for the V region 26 or 28 is that for CD3, a closely associated component of the T cell receptor found on cytotoxic T lymphocytes (CTLs). CTLs lyse the cells to which they are targeted. The construct can thus induce CTLs to kill tumor cells or virus-infected cells for which they bear no specificity. Specificity for other known surface antigens found exclusively or mostly on other effector cells, such as monocytes, macrophages, natural killer cells, eosinophils, or large granular lymphocytes, also may be useful. Monoclonal antibodies to such cell surface structures are known in the art and can be generated using known techniques.
  • Binding agents include non-immunoglobulin molecules such as ligands and receptors.
  • Useful ligands include those molecules complementary to receptors or surface proteins on the chosen target cell.
  • Useful ligands include hormones such as melanocyte stimulating hormone (MSH) , among many others.
  • the ligand may be a growth factor or other non-immunoglobulin preferably single-chain polypeptide which can bind to a receptor on a target cell.
  • One particularly useful ligand includes epidermal growth
  • EGF SUBSTITUTESHEET factor
  • a particularly useful binding agent is a receptor such as a CD4 which binds the gpl20 envelope protein or HIV, and also is capable of binding the same protein expressed on the surface of HIV-infected cells.
  • binding agents include single chain binding sites which mimic the antibody binding site including V H and VL domains as disclosed in U.S. Patent No. 4,946,778 (Ladner et al.) and International Application No. PCT/US88/01737 (Creative BioMolecules, Inc.), published December 1, 1988.
  • the binding agents may be whole native or synthetic molecules or fragments which retain the ability to bind their receptor. They may have the same amino acid sequence of the native form of the ligand, or instead may be an analog of the native form of the ligand having an amino acid sequence sufficiently duplicative of the native sequence such that the analog binds the native receptor on the target cell.
  • constructs are produced by known recombinant DNA technologies including the preparation of a nucleic acid sequence encoding an amino acid sequence for the antibody/binding agent construct, transfecting a host cell line with that nucleic acid, and then culturing the transfected cell line to produce the construct.
  • a gene encoding the non-immunoglobulin ligand, or fragment or analog thereof is ligated into a plasmid capable of transfecting a preselected host cell for expression.
  • This gene fragment may be prepared by any number of known techniques.
  • DNA encoding the ligand may be synthesized from the known amino acid sequence of the ligand, or may be obtained from an established cDNA library.
  • the nucleic acid sequence of native EGF is known (see, e.g., Gregory et al. (1977) J. Peptide Protein Res. 9 . :107-118) and shown in SEQ ID NO:l.
  • the sequence of any number of known EGF analogs may be used (see, e.g., GB patent application no. 2210618; and Patent Cooperation Treaty Patent Application No. WO 89/1489A, herein incorporated as reference) .
  • CD4 also known as T4
  • T4 The nucleic acid sequence for CD4 (also known as T4) is known (see, e.g., Maddon et al. (1985) Cell .92.:93-104), and shown in SEQ ID NO:2.
  • nucleic acid sequence of any number of analogs or fragments of CD4 can be used (see, e.g., Patent Cooperation Treaty Application Nos. WO 90/01870A and WO 90/00566, herein incorporated as reference).
  • DNA encoding immunoglobulin light or heavy chain variable and constant regions is known and is readily available from cDNA libraries or is synthesized biochemically (see, e.g., Gillies et al. (1989) J. Immunol. Meth. 125:191-202; Morrison et al. (1984) Ann. Rev. Immunol. 2 :239-256; Falkner et al. , (1982) Nature 298:286-288; and Adams et al. (1980) Biochem. JL9_:2702-2710) •
  • Host cells are transfected by any number of known transfection techniques such as spheroplast fusion (Gillies et al. (1989) Biotechnol. 2:799-804), and then cultured to express the foreign DNA.
  • the host cells transfected may be prokaryotic or eucaryotic. However, if prokaryotic host cells are used, the construct produced must be processed or folded after purification from the cells. Eucaryotic host cells are preferred, as the protein produced therein may be processed by
  • Particularly useful eucaryotic host cells include myelomas and hybridomas such as non-producing hybridomas (e.g., Sp2/0) and non-producing myelomas (e.g., X63Ag8.653). These host cells may be transfected with more than one nucleic acid sequence such as a nucleic acid encoding the light chain variable region in addition to one encoding the construct. Constructs synthesized by a myeloma or hybridoma cell may be paired with a light chain variable region or an entire light chain within the cell.
  • the construct is then purified from the cytoplasm of the host cells or from the culture media, depending on the nature of the host cells used. Protein purification methods are numerous and include various chromatographic methods.
  • constructs of the invention can be used to kill selectively a target cell in vivo.
  • One prepares a construct with the specificities of choice, and then administers a therapeutically effective amount to the circulatory system of a subject harboring the target cell.
  • the construct may be administered in physiologic saline or any other biologically compatible buffered solution which will not affect the ability of the construct to bind the effector and target cells. This solution may be administered systemically via IV or by intramuscular injection. Alternatively, the construct may be administered by injection directly at the site to be treated.
  • a truncated construct not having a C HI and/or CHZ domain may be useful for this purpose as its half-life is limited in vivo.
  • the construct also may be used to treat cells .in vitro
  • SUBSTITUTE SHEET which then may or may not be returned to a subject.
  • effector cells may be removed from a subject, treated by incubation with the construct to bind thereto, and then returned to the subject where the effector cell/construct conjugate is targeted to a target cell for killing or neutralizing.
  • Constructs comprising anti-T cell antibodies and peptide hormones are useful in testing the feasibility of adoptive immunotherapy whereby a patient's tumor-infiltrating lymphocyte (TIL) cell line or peripheral blood-derived cytotoxic T lymphocyte line is given an additional target specificity.
  • TIL tumor-infiltrating lymphocyte
  • the use of conjugates containing EGF is particularly useful for many different cancers.
  • FIG. 3 shows EGF receptor binding activity of a construct including an immunoglobulin moiety with anti-CD3 specificity and EGF as the ligand moiety.
  • the ability of the construct ( ⁇ - ⁇ ) to compete with labeled EGF for its receptor was measure using M24 melanoma cells as target cell, and compared to unlabeled EGF (o - o) , unconjugated anti-CD3 antibody ( - ) and anti-EGF receptor antibody 225 (o - o) .
  • the results are normalized to the molar equivalents of EGF.
  • the anti-CD3 antibody alone showed little or no inhibition activity while the anti-CD3/EGF construct competed well with EGF for its receptor.
  • TIL cells derived from a patient with a malignant melanoma were used as a source of activated T-cells for testing a genetically engineered anti-T cell/EGF construct. These cells had little or no cytolytic activity against the tumor targets against which they were tested. In the presence of very low concentrations of the conjugate,
  • a second cytotoxic T lymphocyte line derived from peripheral blood and specific for autologous Epstein Barr Virus (EBV)-transformed cells but having no specificity for tumor cells, also can be induced to kill the tumor cells.
  • EBV Epstein Barr Virus
  • These lymphocytes have been maintained in culture for an extended time in the presence of IL-2 and stimulated bimonthly with mitomycin C-treated autologous EBV-transformed B cells. The ability of these cells to kill EGF receptor-bearing tumor cells over an extended period has not diminished, thus making this EBV-specific cytotoxic T lymphocyte system generally useful for testing hormone constructs.
  • the epidermal carcinoma cell line, A431 expresses a very high number (2 x 10 6 /cell) of EGF receptor on its cell surface, and this overexpression has been correlated with its ability to form tumors in nude mice (Santon et al. (1986) Cancer Res.4 . 6: 4701- 4705).
  • the ability of the anti-CD3/EGF construct to mediate the killing of labeled A431 cells by a human TIL cell line (TIL 660) in a 4 hour chromium release assay was tested, and the results are shown in FIG. 4A. 51 Cr-labeled targets were incubated for four hours with the indicated amount of construct and varying ratios of effector cells.
  • the amount of released radioactivity was used to calculate the percent of target cell lysis.
  • the parameters that were varied in the first studies were the effector cell-to-target cell (E:T) ratio and the concentration of the construct. No killing of the A431 targets was seen in the absence of the construct, demonstrating that the TIL 660 line has no specificity for these cells. Significant levels of lysis were seen with concentrations of construct as low as 0.1 ng/ml (6 x 10 "13 M) , and this killing increased as a function of construct concentration or effector-to-target ratio. Very little additional killing was seen at concentrations above 25 ng/ml (1.5 x 10" 10 M) .
  • Additional tumor cell lines were tested for their susceptibility to TIL cell lysis in the presence of the anti-CD3/EGF constructs. These include a human metastatic melanoma line (M24) expressing a moderate level of EGF receptor, as well as a neuroblastoma line (IMR-32) that is very sensitive to lysis in an ADCC assay (lysis by Fc receptor-bearing cells in the presence of an anti-tumor antibody) but expresses little or no detectable EGF receptor. The results are shown in TABLE 1.
  • A431 (epidermal carcinoma) 236.8
  • the killing of these cell lines by the TIL 660 effectors was found to be directly related to the expression of EGF receptor (FIGS. 4B and 4C).
  • the M24 line expresses EGF receptor, although ten-fold less than A431 cells, and is killed almost as well at low conjugate concentrations.
  • the killing of A431 cells increased at higher concentrations of the conjugate (greater than 1.5 ng/ml) whereas the killing of M24 cells did not. This difference may reflect the saturation of M24 cell receptors at the lower concentration.
  • the neuroblastoma line, IMR-32 does not express EGF receptor and was not killed by TIL 660 cells in the presence of the anti-CD3/EGF conjugate (FIG. 4C) .
  • a second cytotoxic T lymphocyte line, W-l which is derived from peripheral blood and is both CD3+ and CD8+, also killed the EGF receptor-bearing A431 (FIG. 5A) and M24 (FIG. 5B) cells very efficiently in the presence but not in the absence of the construct.
  • the specific lysis of the A431 and M24 tumor cell lines was measured in the presence or absence of the conjugate, as well as its component parts.
  • Four hour cytotoxicity assays were carried out using an effector (TIL 660 cells)-to-target ratio of 50:1 with the indicated additions. Values represent the amount of lysis obtained in a particular reaction expressed as the percentage of that obtained with the anti-CD3/EGF construct. The results are shown in TABLE 2.
  • Anti-CD3/EGF (5 ng/ml) 100 100
  • EGF alone, anti-CD3 antibody alone, nor EGF in combination with anti-CD3 antibody were able to mediate cytotoxic T lymphocyte killing of the tumor targets. Concentrations of antibody that were 100-fold higher also did not significantly increase the specific lysis above background levels. Clearly, physical linkage of the antibody and EGF is required for killing activity since only the construct was able to mediate the lysis of the EGF receptor-bearing targets. Some inhibition of killing activity is possible with a 100-fold excess of anti-CD3 antibody. Since this represents only 0.5 g/ml, it is possible that there may still be CD3 molecules available for binding. When the concentration was increased to 10 g/ml, significant inhibition was observed.
  • S UB STI TUTESHEET The invention may be better understood from the following nonlimiting Examples, in which are described the preparation of bridging antibody fusion constructs using non- immunoglobulin binding agents chosen first from ligands adapted from the proteins EGF and CD4 and then from a single chain binding site adapted from the mouse anti-human melanoma antibody 9.2.27.
  • FIG. 2 and SEQ ID NO:l shows the nucleic acid sequence synthesized and its corresponding amino acid sequence.
  • a CD4 gene fragment (nucleic acid numbers 145-1266) encoding the extracellular domain including the variable-like region (amino acid numbers 1-94) and the joining-like region (amino acid numbers 95-109) was synthesized as described in Maddon et al. (Cell (1985) .921:93-104), herein incorporated as reference.
  • the EGF or CD4 gene fragment was ligated to an engineered Smal site at the 3' end of the human C7I gene. This is shown schematically in FIG. 2.
  • An Xhol site was placed to the 3' side of the EGF coding sequence for litigation to a fragment containing the 3' untranslated region and poly A addition signal from the mouse Ig CK gene.
  • V region cassettes encoding the H and L chain variable regions of the mouse anti-CD3 antibody, 0KT3 (ATCC number CRL 8001), were constructed from cloned cDNAs as described by Gillies et al. (J. Immunol. Meth. (1989) 125:191-202), herein incorporated by reference.
  • the cassettes were inserted into the chimeric antibody expression vector pdHL2 to give pdHL2-CD3.
  • the modified H chain, to which EGF or CD4 was fused, was inserted into the pdHL2-CD3 plasmid as a HindiII to EcoRI fragment.
  • a second construct was made by replacing the Hindlll to Nsil fragment of the C7I gene with the corresponding fragment of the C74 gene. In both cases the lysine residue, normally found at the carboxy terminus of Ig H chains, was omitted from the fusion proteins.
  • Mouse hybrido a cells (Sp2/0 Agl4, ATCC No. CRL 1581) were maintained in Dulbecco's Modified Eagle's medium (DMEM) and transfected as described by Gillies et al. (Biotechnol. (1989) 1:799-804).
  • Human tumor cell lines A431 epidermal carcinoma, ATCC number CRL 1555
  • M24 metal-static melanoma, originally obtained by D.C. Morton, UCLA, and provided by Ralph Reisfeld, Scripps Clinic
  • IMR-32 nerveroblastoma, ATCC number CCL 127) were maintained in RPMI 1640 containing 10% FBS.
  • TIL tumor-infiltrating lymphocyte
  • Transfeetants secreting human antibody determinants were identified by ELISA, and their culture supernatants were tested further for anti-CD3 reactivity by their ability to stain TIL 660 cells in the presence of a fluorescenated anti-human Ig antiserum. Both the chimeric and conjugated antibody constructs were found to stain these cells as well as the original mouse antibody (OKT3, Ortho Diagnostic Systems).
  • Chimeric antibody, antibody/EGF constructs, and antibody/CD4 constructs were purified by affinity chromatography using protein A Sepharose (Repligen) . Cell culture medium was used as a source of material for the purification. Electrophoretic analyses of the purified proteins showed that they were both fully assembled into antibody molecules and that the conjugated H chain migrated as would be expected for the fusion of the Ig and EGF sequences.
  • M24 melanoma cells (2 x 10 5 cells in a final volume of 0.1 ml) were mixed on ice in Hank's balanced salt solution containing 0.1% BSA and 20 mM HEPES together with 125 I-EGF (10 ng/ml final concentration, Amersha ) and varying concentrations of cold competitor (either EGF, antibody or antibody conjugate). After a 2 hour incubation at 4°C, cells were washed three times by centrifugation, and the cell-associated radioactivity was counted. A non-specific background, determined by incubation with a 200-fold excess of cold EGF, was subtracted from all data points. The results were expressed as the percent inhibition of binding relative to the no-competitor control.
  • cells were incubated for 2.5 hours in 100 ⁇ l of buffer (HBSS, 0.1% BSA, 20 mM HEPES, pH 7.4) at 4°C with 700 pg of 125 I-EGF, washed three times with buffer and the pellet counted in a gamma counter. Non-specific binding (that obtained in the presence of a 200-fold excess of cold EGF) was subtracted.
  • buffer HBSS, 0.1% BSA, 20 mM HEPES, pH 7.4
  • Cytotoxicity assays were carried out using 51 Cr-labeled tumor targets and TIL 660 cells as effectors. A fixed number
  • T of labeled targets (10 4 per well) in 50 ⁇ l and varying numbers of effectors in 50 ⁇ l were mixed with 100 ⁇ l of diluted antibody or conjugate in the wells of a microtiter plate.
  • the plates were centrifuged and assayed for released 51 Cr following a 4 hr incubation at 37°C. Spontaneous release was subtracted from experimental values and the percent of specific lysis was determined by dividing the corrected release value by the total released with detergent lysis.
  • the assay for activity of Ig/CD4 constructs may be carried out in a manner analogous to that for Ig/EGF constructs, with the modification that the target cells used would be those expressing gpl20 on their surfaces, such as HIV-infected cells or cells that have been transfected with a gene for gpl20 and are expressing it on their surf ces.
  • SUBSTfTUTE SHEET sense primers were identical to the last six amino acids of each. Additional sequences were added for cloning purposes and for either joining purposes (for the V region) or to introduce a stop codon and a convenient Xhol restriction site (in the V H region). A carboxyl-terminal Lys was added to the end of V since all antibody H chains end with this amino acid.
  • V and V PCR products were synthesized by mixing 1 ng of template (a plasmid containing both V regions) with 50 ng of each set of primers in 100 ⁇ L standard PCR reactions (Perkin Elmer/Cetus) . These products were digested within EcoRI and Sal I (for V_)' or EcoRI and Xhol ( x for VH'). The VH product was cloned as an EcoRI-to-XhoI fragment and verified by DNA sequencing. The V L region was ligated to the 5' end of a synthetic linker fragment encoding a 5' Xhol site, a flexible peptide linker composed of Ser and Gly residues, and a 3' BamHI site:
  • 9.2.27sca The resulting 9.2.27 V Xi-linker-VH sequence, herein referred to as 9.2.27sca, was joined to the CH3 exon of the human C74 gene by first modifying the 3' end of the CH3 exon to encode a BamHI site.
  • a short oligonucleotide (GGGATCCC) was ligated to the Smal site near the end of the CH3, changing the 3' end sequence from
  • 9.2.27sca was joined to this CH3 BamHI site via its unique 5' BglH site resulting in the addition of a single Ser residue.
  • the C74-9.2.27sca fusion protein coding sequence was then inserted into a pdHL2 chimeric antibody expression vector containing the V regions of the anti-CD3 antibody, as described in Example 1 and shown in FIG. 6B.
  • a poly-A addition site (pA) was provided by the vector and, in the completed vector, was located to the 3' side of the translation stop signal in the 9.2.27 V region.
  • Figure 7 shows the results of a killing assay using varying concentrations of the anti-CD3/9.2.27sca bridging antibody and varying effector-to-target ratios of TIL 660 (effector) and M21 melanoma (target) cells. Significant killing of target cells occurred at relatively low effector-to-target ratios; this killing was seen to increase with the concentration of bridging antibody.
  • MOLECULE TYPE DNA, protein
  • MOLECULE TYPE CDNA, protein
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • ORIGINAL SOURCE
  • GAG GAC GAG AAG GAG GAG GTG GAA TTG CTA GTG 441
  • AGG AGT CCA AGG GGT AAA AAC ATA CAG GGG GGG 573
  • CAG CTC CAG AAA AAT TTG ACC TGT GAG GTG TGG 1068 Gin Leu Gin Lys Asn Leu Thr Cys Glu Val Trp 300 305

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Abstract

Cette invention concerne une structure d'anticorps de pontage comprenant une région variable à chaîne lourde qui se fixe sur un antigène de surface situé sur une cellule d'effecteur lorsqu'elle est combinée à une région variable à chaîne légère; une région constante à chaîne lourde comprenant un domaine CH3; et un agent de liaison non immunoglobuline qui fixe une protéine de surface sur une cellule cible. L'agent de liaison est lié par un peptide à l'extrémité carboxy dudit domaine CH3. L'invention concerne également une séquence d'acide nucléique codant la structure, une ligne de cellules transfectée par cet acide nucléique, un procédé de production de la structure et des procédés permettant de tuer sélectivement une cellule cible à l'aide de cette structure.
PCT/US1991/008421 1990-11-09 1991-11-12 Structures de fusion d'anticorps de pontage Ceased WO1992008801A1 (fr)

Applications Claiming Priority (2)

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US61211090A 1990-11-09 1990-11-09
US612,110 1990-11-09

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WO1992008801A1 true WO1992008801A1 (fr) 1992-05-29

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EP (1) EP0556328A4 (fr)
CA (1) CA2095842A1 (fr)
WO (1) WO1992008801A1 (fr)

Cited By (24)

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WO1996008570A1 (fr) * 1994-09-14 1996-03-21 Fuji Immunopharmaceuticals Corporation Technologie d'expression et de secretion de proteines sous forme d'immunofusines
US6617135B1 (en) 1999-08-09 2003-09-09 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US6969517B2 (en) 2001-05-03 2005-11-29 Emd Lexigen Research Center Corp. Recombinant tumor specific antibody and use thereof
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US7091321B2 (en) 2000-02-11 2006-08-15 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US7148321B2 (en) 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US7169904B2 (en) 2002-12-17 2007-01-30 Emd Lexigen Research Center Corp. Immunocytokine sequences and uses thereof
US7186804B2 (en) 2001-12-04 2007-03-06 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
US7211253B1 (en) 1999-11-12 2007-05-01 Merck Patentgesellschaft Mit Beschrankter Haftung Erythropoietin forms with improved properties
US7323549B2 (en) 2003-12-30 2008-01-29 Emd Lexigen Research Center Corp. IL-7 fusion proteins
US7432357B2 (en) 2004-01-22 2008-10-07 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US7465447B2 (en) 2003-12-31 2008-12-16 Merck Patent Gmbh Fc-erythropoietin fusion protein with improved pharmacokinetics
US7517526B2 (en) 2000-06-29 2009-04-14 Merck Patent Gmbh Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
US7589179B2 (en) 2004-12-09 2009-09-15 Merck Patent Gmbh IL-7 variants with reduced immunogenicity
WO2010051502A3 (fr) * 2008-10-31 2010-07-22 Biogen Idec Ma Inc. Molécules ciblant light et leurs utilisations
US8691952B2 (en) 2005-12-30 2014-04-08 Merck Patent Gmbh Anti-CD19 antibodies with reduced immunogenicity
US8907066B2 (en) 2009-04-22 2014-12-09 Merck Patent Gmbh Antibody fusion proteins with a modified FcRn binding site
US9029330B2 (en) 2005-12-30 2015-05-12 Merck Patent Gmbh Methods of treating cancer using interleukin-12p40 variants having improved stability
US10604576B2 (en) 2016-06-20 2020-03-31 Kymab Limited Antibodies and immunocytokines
US11753479B2 (en) 2014-03-04 2023-09-12 Kymab Limited Nucleic acids encoding anti-OX40L antibodies
US11779604B2 (en) 2016-11-03 2023-10-10 Kymab Limited Antibodies, combinations comprising antibodies, biomarkers, uses and methods
US12209128B2 (en) 2016-06-20 2025-01-28 Kymab Limited Anti-PD-L1 antibodies

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EP0394827A1 (fr) * 1989-04-26 1990-10-31 F. Hoffmann-La Roche Ag Polypeptides chimériques de CD4-immunoglobuline
ZA902949B (en) * 1989-05-05 1992-02-26 Res Dev Foundation A novel antibody delivery system for biological response modifiers
DE69127749T2 (de) * 1990-03-20 1998-04-16 The Trustees Of Columbia University In The City Of New York, New York, N.Y. Chimäre antikörper mit rezeptor-bindenden liganden anstelle ihrer konstanten region

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Nature, Volume 337, issued 09 February 1989, DANIEL J. CAPON et al, "Designing CD4 Immunoadhesions for Aids Therapy", pages 525-530, see entire document. *
Nucleic Acids Research, Volume 17, No. 24, issued 1989, CLACKSON et al, "Sticky feet' - Directed Mutagenesis and its Application to Swapping Antibody Domains", pages 10163-10170, see pages 10164-10166. *
Proceeding of the National Academy of Sciences, Volume 87, issued July 1990, SEUNG UON SHIN et al, "Expression and Characterization of an Antibody Binding Specificity Joined to Insulin like Growth Factor 1: Potential Applications for Cellular Targeting", pages 5322-5326, see entire document. *
Science, Volume 239, issued 22 January 1988, MARGARET ANN LIU et al., "Hormone Conjugated with Antibody to CD3 mediates Cytotoxic T Cell Lysis of Human Melanoma Cells", pages 395-398, see 396 and 397. *
See also references of EP0556328A4 *

Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996008570A1 (fr) * 1994-09-14 1996-03-21 Fuji Immunopharmaceuticals Corporation Technologie d'expression et de secretion de proteines sous forme d'immunofusines
US7226998B2 (en) 1997-12-08 2007-06-05 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7576193B2 (en) 1997-12-08 2009-08-18 Merck Patent Gmbh Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US6617135B1 (en) 1999-08-09 2003-09-09 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US7141651B2 (en) 1999-08-09 2006-11-28 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US7582288B2 (en) 1999-08-09 2009-09-01 Merck Patent Gmbh Methods of targeting multiple cytokines
US7211253B1 (en) 1999-11-12 2007-05-01 Merck Patentgesellschaft Mit Beschrankter Haftung Erythropoietin forms with improved properties
US7091321B2 (en) 2000-02-11 2006-08-15 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US7507406B2 (en) 2000-02-11 2009-03-24 Emd Serono Research Center, Inc. Enhancing the circulating half-life of antibody-based fusion proteins
US7517526B2 (en) 2000-06-29 2009-04-14 Merck Patent Gmbh Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
US7148321B2 (en) 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US7601814B2 (en) 2001-03-30 2009-10-13 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
US8926973B2 (en) 2001-03-30 2015-01-06 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US7459538B2 (en) 2001-05-03 2008-12-02 Merck Patent Gmbh Recombinant tumor specific antibody and use thereof
US6969517B2 (en) 2001-05-03 2005-11-29 Emd Lexigen Research Center Corp. Recombinant tumor specific antibody and use thereof
US7186804B2 (en) 2001-12-04 2007-03-06 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
US7462350B2 (en) 2001-12-04 2008-12-09 Emd Serono Research Center, Inc. Cancer treatments including administering IL-2 fusion proteins with modulated selectivity
US7169904B2 (en) 2002-12-17 2007-01-30 Emd Lexigen Research Center Corp. Immunocytokine sequences and uses thereof
US7323549B2 (en) 2003-12-30 2008-01-29 Emd Lexigen Research Center Corp. IL-7 fusion proteins
US7465447B2 (en) 2003-12-31 2008-12-16 Merck Patent Gmbh Fc-erythropoietin fusion protein with improved pharmacokinetics
US7432357B2 (en) 2004-01-22 2008-10-07 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US10633452B2 (en) 2004-01-22 2020-04-28 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US10017579B2 (en) 2004-01-22 2018-07-10 Meck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US8835606B2 (en) 2004-01-22 2014-09-16 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US9617349B2 (en) 2004-01-22 2017-04-11 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US7589179B2 (en) 2004-12-09 2009-09-15 Merck Patent Gmbh IL-7 variants with reduced immunogenicity
US10072092B2 (en) 2005-12-30 2018-09-11 Merck Patent Gmbh Methods of use of anti-CD19 antibodies with reduced immunogenicity
US8957195B2 (en) 2005-12-30 2015-02-17 Merck Patent Gmbh Anti-CD19 antibodies with reduced immunogenicity
US9029330B2 (en) 2005-12-30 2015-05-12 Merck Patent Gmbh Methods of treating cancer using interleukin-12p40 variants having improved stability
US8691952B2 (en) 2005-12-30 2014-04-08 Merck Patent Gmbh Anti-CD19 antibodies with reduced immunogenicity
US11208496B2 (en) 2005-12-30 2021-12-28 Cancer Research Technology Ltd. Anti-CD19 antibodies with reduced immunogenicity
US8734795B2 (en) 2008-10-31 2014-05-27 Biogen Idec Ma Inc. Light targeting molecules and uses thereof
WO2010051502A3 (fr) * 2008-10-31 2010-07-22 Biogen Idec Ma Inc. Molécules ciblant light et leurs utilisations
US8907066B2 (en) 2009-04-22 2014-12-09 Merck Patent Gmbh Antibody fusion proteins with a modified FcRn binding site
US11753479B2 (en) 2014-03-04 2023-09-12 Kymab Limited Nucleic acids encoding anti-OX40L antibodies
US11773175B2 (en) 2014-03-04 2023-10-03 Kymab Limited Antibodies, uses and methods
US10604576B2 (en) 2016-06-20 2020-03-31 Kymab Limited Antibodies and immunocytokines
US12209128B2 (en) 2016-06-20 2025-01-28 Kymab Limited Anti-PD-L1 antibodies
US11779604B2 (en) 2016-11-03 2023-10-10 Kymab Limited Antibodies, combinations comprising antibodies, biomarkers, uses and methods

Also Published As

Publication number Publication date
CA2095842A1 (fr) 1992-05-10
EP0556328A4 (en) 1994-06-08
EP0556328A1 (fr) 1993-08-25

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