WO1993006117A1 - Anticorps sf-25, notamment anticorps chimeriques specifiques de l'antigene de sf-25 de tumeurs humaines, leurs procedes de preparation et leur utilisation - Google Patents

Anticorps sf-25, notamment anticorps chimeriques specifiques de l'antigene de sf-25 de tumeurs humaines, leurs procedes de preparation et leur utilisation Download PDF

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WO1993006117A1
WO1993006117A1 PCT/US1992/008109 US9208109W WO9306117A1 WO 1993006117 A1 WO1993006117 A1 WO 1993006117A1 US 9208109 W US9208109 W US 9208109W WO 9306117 A1 WO9306117 A1 WO 9306117A1
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Prior art keywords
antibody
fragment
antigen
chimeric
molecule
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Jack R. Wands
Hiroshi Takahashi
Sven Ole Warnaar
John Ghayreb
Jim Looney
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General Hospital Corp
Janssen Biotech Inc
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General Hospital Corp
Centocor Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • 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
    • C07K2317/24—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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • the present invention relates to SF-25 antibodies, especially chimerized antibodies, and fragments and derivatives thereof, which have specificity for the human tumor SF-25 antigen and especially to humanized chimeric SF-25 antibodies; to methods of producing these SF-25 antibodies, fragments, and derivatives thereof, using recombinant DNA technology; to the nucleotide and protein sequences coding for these SF-25 antibodies, fragments, and derivatives; to methods of obtaining and manipulating these sequences; processes for the manufacture of pharmaceutical compositions containing these SF-25 antibodies, fragments and derivatives thereof; and also the uses thereof.
  • MAbs produced from hybridomas are already widely used in basic research, are being tested in the treatment of human diseases, including cancer, viral and microbial infections, other diseases and disorders of the immune system.
  • most MAbs have been produced by fusions of rodent spleen cells with rodent myeloma cells. They are therefore essentially rodent proteins.
  • rodent spleen cells with rodent myeloma cells. They are therefore essentially rodent proteins.
  • HAMA Human Anti-Mouse Antibody
  • MAbs directed to human tumor antigens that have been developed for the diagnosis and treatment of cancer. Since many human tumor antigens are not recognized as foreign by the human immune system, they probably lack immunogenicity in man. In contrast, those human tumor antigens that are immunogenic in mice can be used to induce murine MAbs which specifically recognize the human antigen, and which may also have therapeutic utility in humans, but have the previously referred to limitations.
  • Such chimerization procedures involve production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody is linked to constant domains derived from another antibody.
  • Some early methods for carrying out such a chimerization procedure are described in EP-A-0120694 (Celltech Limited), EP-A-0125023 (Genentech Inc. and City of Hope), EP-A-01714906 (Res. Dev. Corp. Japan), EP-A-0173494 (Stanford University), and EP-A-0194276 (Celltech Limited).
  • the latter Celltech application also shows the production of an antibody molecule comprising the variable domains of a mouse MAb, the CH1 and CL domains of a human immunoglobulin, and a non-immunoglobulin derived protein in place of the Fc portion of the human immunoglobulin.
  • Some tumors and other diseased tissues selectively express constitutive antigens which are not expressed by normal animal tissues.
  • An example is colorectal adenocarcinoma and its SF-25 constitutive antigen.
  • Colorectal cancer is one of the most common malignancies in both men and women in the Western world. More than 150,000 new cases will be diagnosed in 1991 in the United States alone (Boring et al., Cancer Statistics 41:19-36 (1991)). Despite major advances in general patient care and surgical therapy, the mortality rate associated with this disease has not changed significantly over the last forty years (Fleischer et al., JAMA 261:580-586 (1989)). Indeed, about 60,000 patients die of this disease each year in this country principally because of advanced disease or recurrence (Cancer Facts & Figures - 1990, American Cancer Society, Inc., Atlanta, Ga. (1990)).
  • Murine MAb SF-25 recognizes the SF-25 antigen which is highly expressed in human colon adenocarcinomas, their hepatic metastases, and other primary tumors of endodermal origin. Murine MAb SF-25 has been immunolocalized in tumor xenografts in nude mice" (Takahashi et al., Cancer
  • SF-25 antigen is a disulfide-bond-linked heterodimer which is composed of two glycosylated subunits termed ⁇ and ⁇ .
  • the expression of the SF-25 antigen in colon adenocarcinoma tissues is uniform in contrast to the heterogeneous expression of other tumor associated antigens (Atkinson et al., Cancer Res. 42:4820-4823 (1982); Hand et al., Id. 43:728-735 (1983)).
  • SF-25 antigen is a disulfide-bond-linked heterodimer which is composed of two glycosylated subunits termed ⁇ and ⁇ .
  • the expression of the SF-25 antigen in colon adenocarcinoma tissues is uniform in contrast to the heterogeneous expression of other tumor associated antigens (Atkinson et al., Cancer Res. 42:4820-4823 (1982); Hand et al., Id. 43:728-735 (1983)).
  • the SF-25 antigen is a constitutive antigen that is expressed on most if not all tumors of endodermal origin.
  • the SF-25 antigen has been shown by immunohistological staining to be expressed by the following human tumor types: colon adenocarcinoma; rectal adenocarcinoma; hepatocellular carcinoma; cholangiocellular carcinoma; gastric adenocarcinoma; breast adenocarcinoma; pancreatic adenocarcinoma; bladder adenocarcinoma; squamous cell carcinoma of the lung;adenocarcinoma of the lung; small cell carcinoma of the lung; large cell carcinoma of the lung; kidney carcinoma; ovary adenocarcinoma; cervix carcinoma; endometrial adenocarcinoma; choriocarcinoma; leukemia; lymphoma; and malignant melanoma.
  • the SF-25 antigen is localized on the tumor cell surface and antibody binding to the SF-25 antigen does not induce internalization therefrom.
  • the SF-25 antigen is not shed from the cell when it is examined by radioimmunoassay in culture supernatant and flow cytometric analysis (Takahashi et al. , Cancer Res. 48:6573-6579 (1988)).
  • the high number of antibody binding sites per cell (2.5 x 10 5 /colon adenocarcinoma cell) suggest that the SF-25 MAb will be bound to the tumor cells in a high density. Taken together, these properties suggest that the SF-25 MAb may be effective as an immunotherapeutic reagent (Schlom et al., in Monoclonal Antibodies in Cancer: Advances in Diagnosis and Treatment, (Roth, J.A. Ed.), Futura Publishing Company, Mount Kisco, NY, 1-65 (1986); Oldham, R.K., in Biological Response Modifiers and Cancer Therapy, (Chlao, J.K., Ed.) Marcel Dekker, Inc. New York, 3-16 (1988)). D. Monoclonal Antibodies and the Treatment of Cancer Patients
  • MAbs Possible anti-tumor mechanisms mediated by MAbs include: 1) induction of tumor cytotoxicity by effector cells such as NK-cells and macrophages (Ravetch et al. , Ann. Rev. Immunol. 9:457-492 (1991)); 2) activation of complement and induction of complement-mediated cytotoxicity (Frank, M. M., N. Engl. J. Med. 576:1525-1530 (1987)); 3) interference with cell growth or differentiation by binding growth factors or receptors on the surface of tumor cells (Sporn et al., Nature 575:745-747 (1985)); Rodeck et al, Cancer Res.
  • Cells with cytotoxic potential that bear receptors for the Fc fragment of IgG may bind and lyse target cells in the presence of antibody
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCC requires the simultaneous binding of the Fab fragment of the antibody to its antigen and the binding of the Fc fragment to Fc ⁇ R expressed on the effector cells.
  • Macrophages express the three types ofthe Fc ⁇ R which have been identified in human cells (Fc ⁇ RI, II and III). Fc ⁇ RI is found only on macrophages and is important for ADCC.
  • NK-cells only express low affinity Fc ⁇ R type III which will initiate ADCC by NK-cells upon binding to antibody (Ravetch et al., Ann. Rev. Immunol. 9:457-492 (1991); Unkeless et al., Id. 6:251-281 (1988); Adams etal., Id. 2:283-318 (1984); Perussia etal.,
  • a chimeric MAb (c-SF-25 MAb) that has the Fc fragment of human IgGl and the Fab fragment of the murine SF-25 MAb has been mentioned in Takahashi et al. ,Hepatology A12:915 (1990); Takahashi, et al. J. Cell. Biochem. Suppl. 15:Part E pg. 139 (1991); Takahashi et al., Immunocon. Radiopharm. 4:208 (1991); Id., p. 237) (Takahashi et al., Antibody Immunocon. Radiopharm. 5:86 (1990)).
  • This chimeric construct induces ADCC by both human NK-cells and macrophages in vitro, since the FC fragment of human IgGl interacts with Fc ⁇ RI and also with Fc ⁇ R type III.
  • the present invention provides a polynucleotide molecule comprising a sequence coding for the variable region of an immunoglobulin chain having specificity to the antigen bound by the murine SF-25 antibody secreted by the murine hybridoma cell line given the ATCC designation HB 9599 and which further comprises an additional sequence coding for at least part of the constant region of a human immunoglobulin chain, both said sequences in operable linkage with each other.
  • the present invention also provides a host transformed with a polynucleotide molecule comprising a sequence coding for the variable region of an immunoglobulin chain having specificity to the antigen bound by the murine SF-25 antibody secreted by the murine hybridoma cell line given the ATCC designation HB 9599 and which further comprises an additional sequence coding for at least part of the constant region of a human immunoglobulin chain, both said sequences in operable linkage with each other.
  • the present invention also provides a eukaryotic or prokaryotic host transfected with a polynucleotide molecule comprising a sequence coding for the variable region of an immunoglobulin chain having specificity to the antigen bound by the murine SF-25 antibody secreted by the murine hybridoma cell line given the ATCC designation HB 9599 and which further comprises an additional sequence coding for at least part of the constant region of a human immunoglobulin chain, both said sequences in operable linkage with each other.
  • the present invention also provides a chimeric immunoglobulin heavy chain comprising at least part of a human constant region and at least part of a variable region having specificity to the antigen bound by the murine SF-25 monoclonal antibody secreted by the murine hybridoma cell line given the ATCC designation HB 9599.
  • the present invention also provides a chimeric immunoglobulin light chain comprising at least part of a human constant region and at least part of a variable region having specificity to the antigen bound by the murine SF-25 monoclonal antibody secreted by the murine hybridoma cell line given the ATCC designation HB 9599.
  • the present invention also provides a chimeric antibody molecule comprising two light chains and two heavy chains, each of said chains comprise at least part of a human constant region and at least part of a variable region having specificity to the antigen bound by the murine SF-25 monoclonal antibody given the ATCC designation HB 9599, or a fragment or derivative of said chimeric antibody.
  • the present invention also provides a process for preparing a chimeric immunoglobulin heavy chain having at least part of a human constant region and at least part of a variable region with specificity to the antigen bound by the murine SF-25 monoclonal antibody secreted by the murine hybridoma cell line given the ATCC designation HB 9599,comprising culturing a host capable of expressing said heavy chain under culturing conditions; expressing said heavy chain; and recovering said heavy chain from said culture.
  • the present invention also provides a process for preparing a chimeric immunoglobulin light chain having at least part of a human constant region and at least part of a variable region with specificity to the antigen bound by the SF-25 murine monoclonal antibody secreted by the murine hybridoma cell line given the ATCC designation HB 9599, comprising culturing a host capable of expressing said light chain under culturing conditions; expressing said light chain; and recovering said light chain from said culture.
  • the present invention also provides a process for preparing a chimeric immunoglobulin, fragment or derivative, containing a heavy chain and a light chain, each of said heavy and light chains having at least part of a human constant region and at least part of a variable region with specificity to the antigen bound by the murine SF-25 monoclonal antibody secreted by the hybridoma cell line given the ATCC designation HB 9599 comprising culturing a host capable of expressing said light chain under culturing conditions, expressing said light chain, and recovering said light chain from said culture; separately culturing a host capable of expressing said heavy chain under culturing conditions, expressing said heavy chain, and recovering said heavy chain from said culture; and associating said recovered heavy chain and light chain, thereby preparing said chimeric immunoglobulin, fragment or derivative.
  • the present invention also provides a process for preparing a chimeric immunoglobulin, fragment or derivative, containing a heavy chain and a light chain, each of said heavy and light chains having at least part of a human constant region and at least part of a variable region with specificity to the antigen bound by the SF-25 murine monoclonal antibody secreted by the murine hybridoma cell line given the ATCC designation HB 9599, comprising co-culturing a host capable of expressing said heavy chain with a host capable of expressing said light chain under culturing conditions; expressing said heavy chain and said light chain; permitting said heavy chain and said light chain to associate into said chimeric immunoglobulin, fragment or derivative; and recovering said chimeric immunoglobulin, fragment or derivative from said culture.
  • the present invention also provides a process for preparing a chimeric immunoglobulin, fragment or derivative, containing a heavy chain and a light chain, each of said heavy and light chains having at least part of a human constant region and at least part of a variable region with specificity to the antigen bound by the SF-25 murine monoclonal antibody secreted by the hybridoma cell line given the ATCC designation HB 9599, comprising culturing a host capable of expressing said heavy chain and said light chain under culturing conditions; expressing said chimeric immunoglobulin, fragment or derivative and recovering from said culture said chimeric immunoglobulin, fragment or derivative.
  • the present invention also provides an immunoassay method for detecting an antigen capable of binding to the chimeric SF-25 monoclonal antibody in a sample comprising contacting said sample with the detectably labeled antibody, fragment or derivative thereof; and detecting said antigen by detecting the binding of said antigen to said antibody, fragment or derivative.
  • the present invention also provides an imaging method for detecting a tissue antigen capable of binding to the chimeric SF-25 monoclonal antibody, comprising contacting the detectably labeled antibody, fragment or derivative thereof with said tissue; and detecting said antigen.
  • the present invention also provides a process for the manufacture of a pharmaceutical composition for use in the killing of cells expressing an antigen, which antigen is capable of binding to the chimeric SF-25 monoclonal antibody, comprising as an active ingredient an effective dose of the antibody, fragment or derivative thereof.
  • the present invention also provides a process for the manufacture of a pharmaceutical composition for use in treating an animal having a tumor expressing an antigen, which antigen is capable of binding to the chimeric SF- 25 monoclonal antibody, comprising as an active ingredient an effective dose of the antibody, fragment or derivative thereof.
  • compositions of the present invention can be used to treat animals with the following tumors: colon adenocarcinoma; hepatocellular carcinoma; cholangiocellular carcinoma; gastric adenocarcinoma; rectal adenocarcinoma; breast adenocarcinoma; bladder adenocarcinoma; squamous cell carcinoma of the lungs; adenocarcinoma of the lungs; large cell carcinoma of the lungs; small cell carcinoma of the lungs; lymphoproliferative disease; myeloproliferative disease; lymphoma; leukemia; kidney carcinoma; ovary adenocarcinoma; cervical carcinoma; uterine endometrial adenocarcinoma; liver hepatoma; choriocarcinoma; malignant melanoma, as well as pancreatic adenocarcinoma and other pancreatic cancers.
  • the present invention also provides for the use of an effective dose of an SF-25 antibody or fragment or derivative thereof, including a chimeric
  • SF-25 monoconal antibody for killing cells expressing an antigen, which antigen is capable of binding to said SF-25 antibody.
  • the present invention also provides for the use of an effective dose of an SF-25 antibody, or fragment or derivative thereof, including a chimeric SF- 25 monoclonal antibody, for treating an animal having a tumor expressing an antigen, which antigen is capable of binding to said SF-25 antibody.
  • the present invention also provides for the use of an effective dose of an SF-25 antibody or fragment or derivative thereof, including a chimeric SF- 25 monoclonal antibody, for treating an animal which has pancreatic carcinoma or other pancreatic cancer, which expresses an antigen, which antigen is capable of binding to said SF-25 antibody.
  • the present invention also provides for the use of an effective dose of an SF-25 antibody, or fragment or derivative thereof, including a chimeric SF- 25 monoclonal antibody for treating an animal having cancer which expresses an antigen, which antigen is capable of binding to said SF-25 antibody, with the cancer selected from the group consisting of colon adenocarcinoma, hepatocellular carcinoma, cholangiocellular carcinoma, gastric adenocarcinoma, rectal adenocarcinoma, breast adenocarcinoma, bladder adenocarcinoma, squamous cell carcinoma of the lungs, adenocarcinoma ofthe lungs, large cell carcinoma of the lungs, small cell carcinoma of the lungs, lymphoproliferative disease, myeloproliferative disease, lymphoma, leukemia, kidney carcinoma, ovary adenocarcinoma, cervical carcinoma, uterine endometrial adenocarcinoma, liver hepatoma, chor
  • the present invention also provides a method of killing cells expressing an antigen, which antigen is capable of binding to an SF-25 antibody, including a chimeric SF-25 monoclonal antibody, comprising delivering to said cells an effective dose of the antibody, fragment or derivative thereof, and allowing said killing to occur.
  • the present invention also provides a method of treating an animal suspected of having a tumor expressing an antigen which is capable of binding to an SF-25 antibody, especially a chimeric SF-25 monoclonal antibody, comprising administering to said animal an effective dose of the antibody, fragment or derivative thereof.
  • the present invention also provides a method of treating a cancer in an animal which comprises administering an effective dose of an antibody specific to the SF-25 antigen, especially a chimeric SF-25 antibody, or a derivative or fragment thereof, to said animal having a cancer selected from the group consisting of colon adenocarcinoma, hepatocellular carcinoma, cholangiocellular carcinoma, gastric adenocarcinoma, rectal adenocarcinoma, breast adenocarcinoma, bladder adenocarcinoma, squamous cell carcinoma of the lungs, adenocarcinoma of the lungs, large cell carcinoma of the lungs, small cell carcinoma of the lungs, lymphoproliferative disease, myeloproliferative disease, lymphoma, leukemia, kidney carcinoma, ovary adenocarcinoma, cervical carcinoma, uterine endometrial adenocarcinoma, liver hepatoma, choriocarcinoma, malignant
  • Figure 2 Structure of the SF-25 chimeric L and H chain expression vectors.
  • the SF-25 L chain expression vector containing the xanthine-guanine phosphoribosyl (gpt) gene Figure 2A.
  • the SF-25 H chain expression vector containing the human Ig ⁇ l constant region Figure 2B.
  • Figure 3 Competitive inhibition of 125 I-labeled c-SF-25 MAb binding to LS 180 cells by unlabeled chimeric and murine SF-25 MAb.
  • the binding of 125 I-IabeIed c-SF-25 MAb was inhibited by both chimeric and murine SF-25 MAb. In contrast, this binding was not inhibited by an unlabeled non-relevant MAb (B2TT).
  • B2TT unlabeled non-relevant MAb
  • FIG 4 Antibody dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells in a 4 hour 51 Cr-release assay using LS 180 as the target cell.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • c-SF-25 MAb induced ADCC but the murine SF-25 MAb did not produce this activity by human PBLs ( Figure 4A).
  • Both purified NK- cells and macrophages mediated strong ADCC activity in the presence of c- SF-25 MAb ( Figure 4B).
  • Figure 5 All control animals developed multiple "cannon ball-like" hepatic metastases of human colon adenocarcinomas (Figure 5A). A representative liver of an animal treated with c-SF-25 MAb which is free of detectable disease ( Figure 5B). The F(ab') 2 fragment of c-SF-25 MAb had little effect on the degree of hepatic metastases ( Figure 5C) and all mice developed hepatic tumors similar to controls.
  • FIG. 6 ADCC mediated by murine effector cells in an 8 hour 51 Cr- release assay.
  • Thioglycolate-elicited murine macrophages mediated ADCC against LS 180 cells in the presence of c-SF-25 MAb (Figure 6A).
  • Murine splenocytes Figure 6B
  • FIG. 6A ADCC activity against LS 180 cells in the presence of c-SF-25 MAb.
  • Figure 7 Survival curves of mice treated with c-SF-25 MAb. All mice treated with c-SF-25 MAb survived more than six weeks, whereas all control mice died within six weeks after LS 180 cell injection. The survival rate of c-SF-25 MAb-treated animals was significantly longer than that of controls (p ⁇ 0.0002).
  • Figure 8. ADCC produced in the presence of c-SF-25 MAb (20 ⁇ g/ml) by murine splenocytes. Cytotoxicity was studied using LS 180 human colon adenocarcinoma cells at different effector to target (E:T) ratios.
  • Figure 9 Direct binding of c-SF-25 and MAb to LS 180 cells at different temperatures.
  • the specific binding of 125 I-labeled c-SF-25 MAb increased as the temperature increased from 4°C to 37 °C.
  • FIG. 11 ADCC mediated by murine NK-cells and macrophages at an E:T ratio of 50 to 1 in the presence of different concentrations of MAbs.
  • ADCC activity by murine splenocytes was induced by c-SF-25 at all antibody concentrations tested ( Figure 11 A).
  • Chimeric SF-25 MAb induced similar ADCC by macrophages ( Figure 11B).
  • an effective derivative or fragment of an antibody means a derivative or fragment of an antibody which is still capable of selectively binding to the same molecule(s) as that which the whole antibody binds to.
  • a constitutive antigen means an antigen that is produced by the majority or all of the cells of a particular tumor type or disease type.
  • imaging means the visualization or location of cells, or tumors, or other diseased tissues which express constitutive antigens and which bind detectably labeled, targeted, cytotoxic, effector cells to them.
  • a detectable label is an atom or molecule which is attached to the targeted, cytotoxic, effector cell or constituent thereof, and which is used in imaging cells or tumors or other diseased tissues.
  • labels include, but are not limited to, radioisotopic labels, non- radioactive isotopic labels, chemiluminescent labels, fluorescent labels and enzyme labels.
  • the present invention derives from the discovery of a chimerized SF- 25 antibody with specificity for the human tumor SF-25 antigen which is constitutively expressed by human colon adenocarcinoma cells and other human tumors of endodermal origin.
  • the present invention also derives from a method to produce these chimerized SF-25 antibodies and from methods to use them diagnostically and therapeutically.
  • chimeric antibody molecule is used to describe an antibody molecule having heavy and/or light chains comprising at least the variable regions of heavy and/or light chains derived from one immunoglobulin molecule linked to at least part of a second protein.
  • the second protein may comprise additional antibody constant region domains derived from a different immunoglobulin molecule or a non-immunoglobulin protein.
  • humanized chimeric antibody molecule is used to describe a molecule having heavy and light chain variable region domains derived from an immunoglobulin from a non-human species, the remaining immunoglobulin constant region domains of the molecule being derived from a human immunoglobulin.
  • an antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • hapten is intended to refer to any molecule capable of being bound by an antibody.
  • epitope is meant to refer to that portion of a hapten which can be recognized and bound by an antibody.
  • a hapten or antigen may have one, or more than one epitope.
  • An "antigen” is a hapten which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • the specific reaction referred to above is meant to indicate that the hapten will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • antibody or “monoclonal antibody” (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') 2 fragments) which are capable of binding a hapten.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl Med. 24:316-325 (1983)).
  • the antibodies of the present invention may be prepared by any of a variety of methods.
  • cells expressing, the SF-25 antigen can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding the SF-25 antigen.
  • a preparation of SF-25 antigen is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
  • the antibodies of the present invention are monoclonal antibodies (or hapten binding fragments thereof).
  • Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol 6:511 (1976); Kohler et al. , Eur. J. Immunol 6:292 (1976); Hammeriing et al, In:
  • SF-25 antigen preferably a mouse
  • SF-25-expressing cell preferably a human
  • FOCUS hepatocellular carcinoma cell line
  • Suitable cells can be recognized by their capacity to bind anti-SF-25 antibody.
  • Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at 56°C), and supplemented with 10 ⁇ g/l of nonessential amino acids, 1,000 U/ml of penicillin, and 100 ⁇ g/ml of streptomycin.
  • the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
  • Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP 2 O), available from the American Type
  • hybri doma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981), which reference is herein incorporated by reference).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the SF-25 antigen.
  • a preferred hybridoma cell line, obtained by this process, is the monoclonal antibody- producing cell line "SF-25.” This cell line produces monoclonal antibody "SF-25" which is capable of binding to the SF-25 antigen.
  • Cell line "SF-25” was deposited under the provisions of the Budapest Treaty with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, on December 8, 1987, and given the ATCC designation: HB 9599.
  • additional antibodies capable of binding to the SF-25 antigen may be produced in a two-step procedure through the use of anti- idiotypic antibodies.
  • Such a method makes use of the fact that antibodies are themselves antigens, and that, therefore, it is possible to obtain an antibody which binds to a second antibody.
  • antibodies capable of binding the SF-25 antigen are used to immunize an animal.
  • the splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce antibody whose ability to bind to anti-SF-25 antibody can be specifically blocked by the SF-25 antigen.
  • Such antibodies comprise anti-idiotypic antibodies to the anti- SF-25 antibody.
  • Such antibodies can be used to immunize an animal, and thereby induce the formation of anti-SF-25 antibodies. Since anti-idiotypic antibodies can be used to immunize an animal and thus provoke the production of anti-SF-25 antibodies, they provide one method for inducing, or enhancing, an animal's immune response to colon cancer.
  • Fab and F(ab') 2 and other fragments of the antibody of the present invention may be used according to the methods disclosed herein for the detection and treatment of colon adenocarcinoma in the same manner as intact antibody.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • hapten- binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
  • an oligonucleot ⁇ de can be constructed which is capable of encoding the c-SF-25 MAb or fragment or derivative thereof.
  • Such an oligonucleotide can be operably linked into an expression vector and introduced into a host cell to enable the expression of the c-SF-25 MAb or fragment or derivative thereof by that cell.
  • Techniques for synthesizing such oligonucleotides are disclosed by, for example, Wu et al., Prog. Nucl. Acid. Res. Molec. Biol. 21: 101-141 (1978)).
  • the identification ofthe amino acid sequence of the c-SF-25 MAb, or fragments of this MAb, also permits the cloning of the gene which encodes the c-SF-25 MAb.
  • Any of a variety of methods may be used to clone the c-SF-25 MAb gene.
  • One such method entails analyzing a shuttle vector library of cDNA inserts (derived from an c-SF-25 MAb expressing cell) for the presence of an insert which contains the c-SF-25 MAb gene. Such an analysis may be conducted by transfecting cells with the vector, and then assaying, for c-SF-25 MAb expression.
  • a preferred method for cloning the c-SF-25 MAb gene entails determining the amino acid sequence of the c-SF-25 MAb molecule. Although it is possible to determine the entire amino acid sequence of the c- SF-25 MAb molecule it is preferable to determine the sequence of peptide fragments of the molecule. If the peptides are greater than 10 amino acids long, this sequence information is generally sufficient to permit one to clone a gene such as the gene for the c-SF-25 MAb molecule.
  • c-SF-25 MAb molecules are preferably purified from producer cells by monoclonal antibody affinity chromatography and isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis ("SDS-PAGE") and electroelution.
  • the c-SF-25 MAb molecules are fragmented as with cyanogen bromide, or with proteases such as papain, chymotrypsin, trypsin, etc. (Oike et al. , J. Biol. Chem. 257:9751- 9758 (1982); Liu et al. , Int. J. Pept. Protein Res. 27:209-215 (1983)).
  • the resulting peptides are separated, preferably by reverse-phase HPLC, and subjected to amino acid sequencing.
  • the protein is preferably analyzed by automated sequencers.
  • the DNA sequences capable of encoding them are examined. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology ofthe Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357). Using the genetic code, one or more different oligonucleotides can be identified, each of which would be capable of encoding the c-SF-25 MAb peptides.
  • the probability that a particular oligonucleotide will, in fact, constitute the actual c-SF-25 MAb- encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells.
  • Such "codon usage rules" are disclosed by Lathe et al, J. Molec. Biol. 183:1-12 (1985). Using the "codon usage rules" of Lathe, a single oligonucleotide, or a set of oligonucleotides, that contains a theoretical "most probable" nucleotide sequence capable of encoding the c-SF-25 MAb peptide sequences is identified.
  • amino acid sequence may be encoded by only a single oligonucleotide
  • amino acid sequence may be encoded by any of a set of similar oligonucleotides.
  • all of the members of this set contain oligonucleotides which are capable of encoding the peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment
  • only one member of the set contains the nucleotide sequence that is identical to the nucleotide sequence of the gene.
  • this member is present within the set, and is capable of hybridizing to DNA even in the presence of the odier members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.
  • the oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding the SF-25 MAb fragment is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probable" sequence, or set of sequences.
  • An oligonucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the c-SF-25 MAb gene (Maniatis et al, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982).
  • Single stranded oligonucleotide molecules complementary to the "most probable" SF-25 peptide encoding sequences can be synthesized using procedures which are well known to those of ordinary skill in the art (Belagaje et al., J. Biol. Chem. 254:5765-5780 (1979); Maniatis et al. , In: Molecular Mechanisms in the Control of Gene Expression, Nierlich et al., Eds., Acad. Press, NY (1976); Wu et al. , Prog. Nucl. Acid Res. Molec. Biol. 27: 101-141 (1978); Khorana, R.G., Science 203:614-625 (1979)).
  • DNA synthesis may be achieved through the use of automated synthesizers. Techniques of nucleic acid hybridization are disclosed by Maniatis et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)) and by Haymes et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)), which references are herein incorporated by reference.
  • the source of DNA or cDNA used will preferably have been enriched for SF-25 MAb sequences. Such enrichment can most easily be obtained from cDNA obtained by extracting RNA from cells, such as hybridoma cells, which produce high levels of SF-25.
  • An example of such a cell is the hybridoma cell line given the ATCC designation HB 9599 which was previously described.
  • a DNA, or more preferably a cDNA, library is screened for its ability to hybridize with the oligonucleotide probes described above.
  • Suitable DNA preparations (such as human genomic DNA) are enzymatically cleaved, or randomly sheared, and ligated into recombinant vectors. The ability of these recombinant vectors to hybridize to the above-described oligonucleotide probes is then measured. Vectors found capable of such hybridization are then analyzed to determine the extent and nature ofthe SF-25 sequences which they contain. Based purely on statistical considerations, a gene such as that which encodes the c-SF-25 molecule could be unambiguously identified (via hybridization screening) using an oligonucleotide probe having only 18 nucleotides.
  • the purified cDNA is fragmentized (by shearing, endonuclease digestion, etc.) to produce a pool of DNA or cDNA fragments. DNA or cDNA fragments from this pool are then cloned into an expression vector in order to produce a genomic library of expression vectors whose members each contain a unique cloned DNA or cDNA fragment.
  • an "expression vector” is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into the vector and of thereby producing a polypeptide or protein. Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell. If aprokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Similarly, if a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequences.
  • cDNA from a cell which is capable of expressing c-SF-25 in order to produce a prokaryotic genomic expression vector library.
  • Procedures for preparing cDNA and for producing a genomic library are disclosed by Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • the expression vectors of those cells which produce a protein capable of binding to SF-25 antigen are then further analyzed to determine whether they express (and thus contain) the entire c-SF-25 MAb gene, whether they express (and contain) only a fragment of the c-SF-25 MAb gene, or whether they express (and contain) a gene whose product, though immunologically related to c-SF-25 MAb, is not c-SF-25 MAb.
  • an analysis may be performed by any convenient means, it is preferable to determine the nucleotide sequence of the DNA or cDNA fragment which had been cloned into the expression vector. Such nucleotide sequences are then examined to determine whether they are capable of encoding polypeptides having the same amino acid sequence as digestion fragments of SF-25.
  • An expression vector which contains a DNA or cDNA molecule which encodes the c-SF-25 MAb gene may, thus, be recognized by: (i) the ability to direct the expression of a protein which is capable of binding to SF-25 antigen; and (ii) the presence of a nucleotide sequence which is capable of encoding each ofthe fragments of c-SF-25 MAb.
  • the cloned DNA molecule of such an expression vector may be removed from the expression vector and isolated in pure form.
  • genomic DNA library A collection of these genomic DNA fragments is referred to as a genomic DNA library.
  • a variety of methods may be used to identify and/or isolate the genomic DNA fragments containing the SF-25 light and heavy chain antibody variable region gene segments.
  • oligonucleotides capable of encoding a fragment of the SF-25 Mab gene may be labeled and used as a hybridization probe to isolate the genomic DNA fragment containing the SF-25 light or heavy chain variable region.
  • Other nucleic acid probes that contain the mouse antibody constant region, the mouse J region, surrounding or flanking sequences, or sequences that corresponding to the SF-25 light and heavy chain antibody variable region sequences such as a cDNA may be used as a hybridization probe.
  • variable regions of both mouse and human antibodies comprise four framework residues (FRs). Within the FRs are three complementarity determining residues (CDRs) which are responsible for antigen binding.
  • CDRs complementarity determining residues
  • a human-mouse mosaic having the desired binding characteristics may be made by inserting mouse CDR sequences within human framework residues.
  • Such mosaic variants are contemplated equivalents ofthe chimeric immunoglobulins of the invention, as are partial chimeric immunoglobulins, e.g., in which only the heavy chain constant region of murine origin has been replaced by an equivalent sequence of human origin, or variants wherein one or more amino acids have been changed by directed mutagenesis.
  • the present invention therefore provides a means for obtaining a DNA molecule which encodes the c-SF-25 MAb molecule.
  • this DNA molecule or a fragment or mutated form of this DNA molecule
  • a functional promoter By operably linking this DNA molecule (or a fragment or mutated form of this DNA molecule) to a functional promoter, it is possible to direct the expression of the SF-25 MAb gene (or a fragment or derivative thereof) in a cell, or organism.
  • a DNA sequence requires that the DNA sequence be "operably linked" to DNA sequences which contain transcriptional and translational regulatory information.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.
  • the precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • Regulatory regions in eukaryotic cells will in general include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and the stability of chimeric H and L chain proteins and assembled chimeric antibodies. Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized. Known glycolytic genes can also provide very efficient transcription control signals. For example, the promoter and terminator signals of the iso-1-cytochrome C (CYC-1) gene can be utilized. A number of approaches may be taken for evaluating optimal expression plasmids for the expression of cloned immunoglobulin cDNAs in yeast.
  • CYC-1-cytochrome C CYC-1-cytochrome C
  • Plasmid vectors containing replicon and control sequences which are derived from species compatible with a host cell are used in connection with these bacterial hosts.
  • the vector carries a replication site, as well as specific genes which are capable of providing phenotypic selection in transformed cells.
  • a number of approaches may be taken for evaluating the expression plasmids for the production of chimeric antibodies or antibody chains encoded by the cloned immunoglobulin cDNAs in bacteria.
  • Other preferred hosts are mammalian cells, grown in vitro or in vivo.
  • the present invention provides "chimeric" immunoglobulin chains, either H or L, with specificity toward human tumor cell SF-25 antigen.
  • a chimeric chain contains a C region substantially similar to that present in a natural human immunoglobulin, and a V region having the desired anti-tumor specificity of the invention.
  • nuclear magnetic resonance contrasting agents examples include heavy metal nuclei such as Gd, Mn, Iron, etc.
  • the chimeric SF-25 antibodies, or fragments or derivatives thereof, of the present invention may be used to quantitatively or qualitatively detect the presence of cells which express the SF-25 antigen. Such detection may be accomplished using any of a variety of immunoassays. For example, by radioactively labeling these chimeric derivatives or fragments thereof, it is possible to detect the SF-25 antigen through the use of radioimmune assays as previously described.
  • This type of forward sandwich assay may be a simple "yes/no" assay to determine whether SF-25 antigen is present or may be made quantitative by comparing the measure of labeled c-SF-25 antibody with that obtained for a standard sample containing known quantities of SF-25 antigen.
  • Such "two-site” or “sandwich” assays are described by Wide at pages 199-206 of Radioimmune Assay Method, edited by Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970.
  • a simultaneous assay involves a single incubation step as the c-SF-25 antibody bound to the solid support and labeled c-SF-25 antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled c-SF-25 antibody. The presence of labeled c-SF-25 antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
  • stepwise addition first of a solution of labeled c-SF-25 antibody to the fluid sample followed by the addition of unlabeled c- SF-25 antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the the residue of the sample being tested and the solution of unreacted, labeled c-SF-25 antibody.
  • the determination of labeled c-SF-25 antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.
  • reporter molecules As explained above, the immunometric assays for SF-25 antigen require that the particular binding molecule be labeled with a "reporter molecule.” These reporter molecules or labels, as identified above, are conventional and well-known to the art. In the practice of the present invention, enzyme labels are a preferred embodiment. No single enzyme is ideal for use a label in every conceivable immunometric assay. Instead, one must determine which enzyme is suitable for a particular assay system.
  • Criteria important for the choice of enzymes are turnover number of the pure enzyme (the numer of substrate molecules converted to produce per enzyme site per unit of time), purity ofthe enzyme preparation, sensitivity of detection of its product, ease and speed of detection of the enzyme reaction, absence of interfering factors or of enzyme-like activity in the test fluid, stability of the enzyme and its conjugate, and the like. Included among the enzymes used as preferred labels in the immunometric assays of the present invention are peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, glycoamylase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase. Urease is among the more preferred enzyme labels, particularly because of chromogenic pH indicators which make its activity readily visible to the naked eye.
  • an effective amount of c-SF-25 MAbs is one capable of achieving the desired diagnostic discrimination.
  • the amount of c-SF-25 MAb which is typically used in a diagnostic test is generally between 1-10 mg, and preferably between 1 ⁇ g to lmg. D. Therapeutic Uses of the Present Invention
  • the c-SF-25 MAbs of the present invention also provide a means for preventing the onset of these cancers, and for treating affected animals including humans.
  • the discovery that the SF-25 antigen is constitutively expressed on colon cancer cells, and the identification of c-SF-25 MAbs capable of binding to this antigen provide a means for preventing and treating these cancers and other diseased tissues which express the SF-25 antigen.
  • the antibodies of this invention can be adapted for therapeutic efficacy by virtue of their ability to mediate ADCC and/or complement-dependent cytotoxicity (CDC) against tumor cells.
  • CDC complement-dependent cytotoxicity
  • an endogenous source or an exogenous source of effector cells (for ADCC) or complement components (for CDC) can be utilized.
  • chimeric antibodies, fragments or derivatives of this invention may be advantageously utilized in combination with other chimeric antibodies, or with lymphokines or hemopoietic growth factors, etc., which serve to increase the number or activity of effector cells which interact with the antibodies.
  • the ability to conjugate the chimeric SF-25 antibodies, or fragments or derivatives thereof, with toxin molecules provides an additional method for treating colon cancer and other cancers and diseased tissues which constitutively express the SF-25 antigen.
  • the c-SF-25 MAb, or derivatives or fragments of these antibodies, which are capable of recognizing the SF-25 antigen are conjugated with toxin molecules and administered to a patient suspected of having colon or other cancer.
  • the toxin moiety will cause the death of the cancer cell.
  • radionuclides can be conjugated to the c-SF-25 MAbs of the present invention to treat cancer patients.
  • examples of radionuclides which can be coupled to the cells of the present invention and delivered in vivo to sites of the SF-25 antigen include 212 Bi, 131 I, 125 O, 186 Re, and 90 Y, which list is not intended to be exhaustive.
  • the radionuclides exert their cytotoxic effect by locally irradiating the cells, leading to various intracellular lesions, as is known in the art of radiotherapy.
  • conjugating photo-activatable toxins with the chimeric MAbs of the present invention it is possible to direct the toxin molecule only to those cells which express the SF-25 antigen.
  • Conjugating photo-activatable toxins to antibodies has been used to provide a selective means for treating a tumor without damage to normal (i.e. non-antigen expressing) cells. Examples ofthe use of this method are provided by: Mew et al. (Cancer Res. 45:4380-4386 (1985); J. Immunol. 750:1473-1477 (1983)); by Wat et al., Prog. Clin. Biol. Res. 170:351-360, (Doiron et al. eds.), Alan R.
  • the above-described photothermolysis therapy can be accomplished using any light source which is capable of photo-activating the toxin.
  • the photo-activation of such toxins can thus be achieved using light sources other than lasers.
  • photo-activation can be achieved using light from an ordinary light bulb (Dougherty etal, J. Natl. Cane. Inst. 55:115-129 (1979); Wilson, B.C., Phys. Med. Biol. 57:327-360 (1986)).
  • Photo-activation of the toxin may alternatively be achieved by the administration of a chemiluminescent agent (i.e. a light-emitting molecule) to an individual who has recieved the photo-activatable toxin.
  • a chemiluminescent agent i.e. a light-emitting molecule
  • This embodiment of the present invention is particularly advantageous in the in situ treatment of gastric carcinomas, intestinal polyps, and Barrett's esophagus.
  • This embodiment may also be used for metastatic cancers (Phillip et al., In: Porphyrin Localization and Treatment of Tumors (Doiron et al. , eds.), Alan R. Liss, NY, pp. 563- 569 (1985)).
  • compositions which contain the c-SF-25 MAbs of the present invention, and/or their conjugates just described can be administered orally or parenterally by the intravenous, intramuscular, subcutaneous, rectal, transdermal, intrapulmonary, intraperitoneal, intrathecal, intratumoral, intranasalpharyngeal or other known routes of administration.
  • These therapeutic compositions will be manufactured by methods which are well known to those of skill in the art.
  • compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • non-aqueous solvents are propylene glycol, polyethylene gylcol, vegetable oils, such as olive oil, and injectable organic esters such as ethyl oleate.
  • Carriers or occlusive dressings can be used to increase skin permability and enhance antibody absoprtion.
  • Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form.
  • Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water.
  • inert diluents such as purified water.
  • such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.
  • Adjuvants are substances that can be used to specifically augment a specific immune response. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the animal being immunized. Adjuvants can be loosely divided into several groups based upon their composition.
  • oils adjuvants for example, Freund's complete and incomplete
  • mineral salts for example, AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH 4 (SO 4 ), silica, kaolin, and carbon
  • polynucleotides for example, poly IC and poly AU acids
  • certain natural substances for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, or Bordetella pertussis, and members of the genus Brucell ⁇ .
  • saponins such as, for example, Quil A. (Superfos A/S, Denmark). Examples of materials suitable for use in vaccine compositions are provided in Remington's Pharmaceutical Sciences (Osol, A., Ed., Mack Publishing Co., Easton, PA, pp. 1324-1341 (1980)).
  • Treatment of an individual with a tumor bearing the SF-25 antigen recognized by the c-SF-25 MAbs of this invention comprises admininstering an effective amount of this c-SF-25 MAb, or fragments or derivatives thereof in a single dose, multiple doses or an infusion of these chimeric MAbs and/or their conjugates to a patient or other animal.
  • an "effective amount" of a therapeutic composition is one which is sufficient to achieve the desired biological effect.
  • the dosage needed to provide an effective amount ofthe composition will vary depending upon such factors as the individual chimeric antibody used, the presence and nature of any therapeutic agent conjugated thereto, the animal's or patients age, condition, sex, and clinical status including extent of disease, if any, and other variables.
  • the effective dosage can vary from about 10 ng/kg body weight to about 100 mg/kg body weight.
  • Effective concentrations of the compositions of the invention can vary from 0.01-1,000 ⁇ g/ml per dose.
  • the SCID mouse model is useful because: 1) the blood supply to the tumor cells in the liver is substantially better than that to tumor cells grown in previously described models that used subcutaneous tumor xenografts; and 2) the SCID mouse lacks both T and B cells and therefore will accept xenografts of normal and tumor human tissues (Proc. Curr. Top. Microbiol Immunol. 752:1-263 (1989); Bosma et al., Ann. Rev. Immunol. 9:323-350 (1991); McCune et al. , Science 241: 1632-1639 (1988); Mosier et al, Nature 555:256-259 (1988)).
  • Applicants have produced a mouse-human c-SF-25 MAb and demonstrated that: the c-SF-25 MAb selectively binds to the SF-25 antigen; the c-SF-25 MAb induced ADCC by human PBLs of LS 180 human-derived tumor cells; the c-SF-25 MAb inhibited the development of hepatic metastases of human colon adenocarcinoma cells; mice treated with c-SF-25 MAb had a statistically significant increased survival rate compared to control mice injected with LS 180 cells; and that the stable binding of c-SF-25 MAb to the tumor cell surface by its antigen binding site at physiological temperature (37°C) is important for its anti-tumor effect.
  • the murine hybridoma cell line SF-25 secretes a monoclonal antibody of IgGl heavy chain and Kappa light chain isotype that binds to a 125 kD cell surface antigen called the SF-25 antigen which is found on a variety of human adenocarcinoma cell lines of endodermal origin. It was obtained by fusing SP2/0-AG14 cells with spleen cells from a Balb/c mouse which had been immunized with the human hepatocellular carcinoma cell line FOCUS.
  • Cell culture supernatant was adjusted to 2 mM EDTA, 140 mM NaCl, and 20 mM Tris pH 8.5 and applied to a protein A sepharose column
  • RNA and cytoplasmic RNA were isolated from SF-25 cells by standard procedures.
  • genomic DNA was digested with restriction endonucleases (New England Biolabs, Beverly, MA), fractionated by electrophoresis through a 0.8% agarose gel, and transferred to MagnaGraph (MSI, Westborough, MA).
  • dCTP 32 P-labeled probes were prepared using a random priming labeling kit (Boehringer Mannheim, Indianapolis, IN).
  • DNA fragments 3-4 kb in size were excised, electroeluted, ligated to Hindlll- digested Charon 27 and packaged in vitro.
  • the phages were screened as above using a J K probe specific for the murine J region of the kappa locus.
  • Vector pSF25kapgpt ( Figure 2 A) and pSF25 G1 apgpt ( Figure 2B) were linearized with BamHI and transfected into the non-producing mouse myeloma cell line SP2/0 by electroporation using a BioRad Gene Pulser (Bio- Rad Laboratories, Richmond, CA). 2x10 7 cells were suspended in 0.8 ml Hanks buffered saline solution with 10 ⁇ g of each expression plasmid, previously digested with BamHI, and subjected to a pulse of 200 volts.
  • Clones were assayed for antibody production by an Elisa assay using goat-anti-human IgG (Fc Fragment specific) coating antibody and goat anti- human IgG (H+L) alkaline phosphatase conjugated antibody (Jackson ImmunoResearch, West Grove, PA). Standard curves were generated using chimeric antibodies purified by protein A Sepharose chromatography. The highest producing cell lines were subcloned by limiting dilution and evaluated for antibody production in T75 flasks. Growth curves were obtained by seeding cultures at 1 x 10 5 cells/ml in triplicate T75 flasks and taking daily samples for cell counts and ELISA assays. Chimeric MAb producing cell lines were grown under mycophenolic acid selection (see above) in Iscove's media plus 5% FCS. Cloning of the SF-25 Light and Heavy Chain Genes
  • Hindlll or EcoR1 DNA fragments containing the above SF-25 hybridizing fragments were purified from preparative agarose gels, ligated to either Hindlll digested Charon 27 (light chain library) or Eco R1 -digested ⁇ gt10 (heavy chain library), and packaged in vitro. Phage clones hybridizing to the immunoglobulin light and heavy chain probes were plaque purified and characterized by restriction enzyme analysis.
  • a 3.2 Hindlll fragment hybridizing to the light chain probe was isolated from the Charon 27 library. Restriction maps of the mouse light chain K region, the light chain probe and the SF-25 light chain 3.2 Hindlll fragment are shown in Figure 1A. This 3.2 Hindlll fragment was subcloned into the human Kappa light chain expression vector pHuKapgpt, which confers resistance to mycophenolic acid when transfected into mammalian cells.
  • the SF-25 light and heavy chain expression vectors were linearized at the BamHI site and electroporated into the mouse myeloma cell line SP2/0.
  • Transfectants producing antibody were expanded from 96 well plates and were evaluated for antibody production and stability in T75 flasks over several passages.
  • the FOCUS cell line was developoed in Dr. Wand's laboratory (Lun et al, In Vitro (Rockville) 20:493-504 (1984)). All other cell lines were obtained from the American Type Culture Collection (Rockville, MD). All cell lines were maintained in Eagle's MEM (M. A. Bioproducts, Walkersville, MD) supplemented with 10% fetal calf serum inactivated at 56°C for 30 minutes, 10 ⁇ M nonessential amino acids, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin. In some experiments cells were maintained in RPMI 1640 (Hazleton Biologies, Inc., St.
  • KS Lenexa, KS supplemented with 10% heat- inactivated fetal calf serum, 10 ⁇ M nonessential amino acids, 2 mM L- glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin.
  • the monoclonal antibodies were purified by Sepharose CL-4B Staphylococcal Protein A-affinity column (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) and labeled with 125 I using the Iodogen method (Fraker et al. , Biochem. Biophys. Res. Commun. 80:849-857 (1978)). Specific activities of iodinated MAbs were calculated as a ratio of cpm of radionuclide attached per mole of antibody (cpm/mol).
  • mice Four to five week old male athymic Balb/c nude mice were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, Indiana). Throughout the experiments, these animals were maintained under specific-pathogen-free- conditions. Isolation of Human Macrophage and NK-Cells
  • Venous blood was drawn from healthy volunteers with heparin (200
  • Mononuclear cells were isolated by centrifuging at 1,200 r.p.m. for 30 minutes at 25°C and were washed three times with RPMI 1640 medium.
  • Mononuclear cells adjusted to 5 x 10 6 /ml, were incubated on a plastic plate for two hours at 37°C in a CO 2 incubator and macrophages were collected from adherent cells. The purity of the macrophage population was examined by flow cytometric analysis using anti-human monocyte/macrophage MAb, anti-Leu-M3 labeled with FITC (Dimitriu-Bona et al., J. Immunol. 130:145-152 (1983); Becton Dickinson Immunocytometry Sys., Mountain View, CA) as described below.
  • LGLs large granular lymphocytes
  • LGLs were collected from the 40.0% concentra-tion of Percoll.
  • the purity of human NK-cells was examined by flow cytometric analysis using
  • Fc receptor III Fc ⁇ RIII, CD16
  • nude mice were inoculated intraperitoneally with 1 ml of thioglycolate medium (Difco; VWR Scientific Corp., Philadelphia, PA). Four days later, peritoneal exudate cells were collected and were used as thioglycolate-elicited macrophages.
  • thioglycolate medium Difco; VWR Scientific Corp., Philadelphia, PA.
  • mice were anesthetized with ether and were sacrificed by axillary bleeding. The peritoneal cavity was exposed under sterile conditions and the spleen was excised into a plastic dish containing 5 ml of RPMI 1640 medium. Next, a small incision was made in the spleen and splenocytes were isolated by squeezing repeatedly with forceps. Splenocytes were then transferred into a plastic tube and were washed twice with RPMI 1640 medium.
  • Murine NK-cells were isolated by discontinuous density gradient centrifugation as a LGL fraction (Lafreniere et al., Cancer Res. 50: 1658-1666 (1990)).
  • Non-adherent cells were obtained from murine splenocytes after incubation in a plastic dish for 2 hours at 37 °C and were adjusted to 5 x 10 7 cells/gradient. They were separated into fractions by centrifugation at 300 X g for 1 hour at 20°C on a seven-step discontinuous density gradient of Percoll at concentrations of 20, 40, 50, 60, 70, and 100%. (The osmolarity was adjusted to 320 mOsm/kg by 10 X PBS).
  • LGLs were collected from the interfaces of 20/40% and 40/50% concentrations of Percoll.
  • the purity of murine NK-cells were examined by flow cytometric analysis using rabbit antiserum to ganglio-N-tetraosylceramide (anti-asialo GM1 ; Wako Chemicals, Dallas, TX; Kasai et al. , Eur. J. Immunol. 70:175-180 (1980)) as described below.
  • Lymphocytes were isolated from murine liver by using the method of Wiltrout et al. (J. Exp. Med. 760:1431-1449 (1984)), with some modifications. Mice were anesthetized with ether and sacrificed by axillary bleeding. The peritoneal cavity was exposed under sterile conditions and the portal vein was cannulated with a 27-gauge needle. Five ml of RPMI 1640 containing 10% FCS and antibiotics were injected to flush blood out of the liver through the portal vein. The liver was then excised and the gall bladder was removed.
  • the liver was minced into small pieces with a razor blade and incubated with 10 ml of prewarmed Mg+ + free HBSS containing 5% FCS, Pc 100 IU/ml, SM 100 ⁇ g/ml, 5 mM CaCl 2 , 0.05% collagenase type IV, and 0.002% DNAse I type II (Sigma Chemical Co. St. Louis, MO) for 30 minutes at 37°C with shaking.
  • the liver digest was then filtered through a 50-gauge stainless steel mesh using a sterile syringe plunger, and was washed twice with HBSS.
  • the packed liver digest was then resuspended in 30% metrizamide in HBSS at 4°C at a final ratio of 7 parts metrizamide to 5 parts of packed liver digest.
  • Three ml of the mixture were transferred into 15-ml conical tubes, overlain with 1.5 ml of PBS and centrifuged at 1,400 X g for 30 minutes at 4°C.
  • the lymphocyte layer was then carefully removed from the metrizamide-PBS interface and washed three times with PBS.
  • Murine cells (1 x 10 6 ) were incubated with 200 ⁇ l of rat anti-mouse macrophage MAb F4/80 producing hybridoma culture supernatant (Austyn et al, Eur. J. Immunol. 77:805-815 (1981)) for 1 hour at 4°C in a Falcon 2054 plastic tube. After washing three times with cold PBS, the cells were incubated at 4°C for 1 hour with 200 ⁇ l of biotin-conjugated anti-rat IgG antibody made in rabbits (Vector Laboratory, Buriingame, CA) adjusted to 7.5 ⁇ g/ml in PBS containing 1 % BSA.
  • the cells were washed three times with cold PBS, were incubated for another 1 hour at 4°C with 200 ⁇ l of avidin FITC (Becton Dickinson, Mountain View, CA), and were diluted 1:25 with PBS containing 1 % BSA. After washing three times with PBS, fluorescence was detected in a Becton Dickinson FACScan. In some experiments macrophages were also examined by non-specific esterase staining.
  • 1 X 10 6 LS 180 cells were incubated for 1 hour at 4°C with 200 ⁇ l of murine SF-25 MAb adjusted to 1 mg/ml in 0.01 M PBS, pH 7.2, containing 1 % bovine serum albumin. After washing three times at 4°C, the cells were further incubated at 4oC or 37°C for 15 minutes to 2 hours, and then were reacted for 1 hour at 4°C with fluorescein-conjugated goat anti- mouse IgG (Cappel Lab., CochranviUe, PA), diluted 1:100 with 1 % bovine serum albumin in PBS. Fluorescence was detected in a Becton Dickinson FACScan for single color flow cytometric analysis. Results are expressed as mean percent of positive staining cells.
  • Detection of Murine NK Cells by Flow Cvtometrv Anti-asialo GM1 rabbit serum was used to detect the NK cells in nude mice by flow cytometry.
  • the cells were collected and adjusted to 5 x 010 6 viable cells/ml in PBS containing 1 % BSA. Two hundred ⁇ l of this cell suspension and 20 ⁇ l of anti-asialo GM1, adjusted to 5 ⁇ g/ml, were incubated for 1 hour at 4°C. The cells were washed three times with cold PBS, incubated with 200 ⁇ l of FITC-labeled F(ab') 2 fragments of goat anti-rabbit IgG (Sigma Chemical Co., St.
  • LS 180 cells were used as the target cells.
  • Confluent cells were harvested with EDTA/Versene buffer and radiolabeled by incubating 1 x 10 6 cells with 100 ⁇ Ci of sodium chromate- 51 Cr (New England Nuclear, Boston, MA) for 30 minutes at 37°C. After washing, the radiolabeled cells were adjusted to 1 x 10 5 /ml of RPMI 1640.
  • One hundred ⁇ l of target cells and 50 ⁇ l of MAb (10 ⁇ g/ml in RPMI 1640) were pipetted into 96-well U bottom plates and then 100 ⁇ l of various concentrations of human PBL (1 x 10 6 - 1 x 10 7 lymphocytes/ml) were added to each well as effector cells.
  • the final concentration of MAb in ADCC was adjusted to 20 ⁇ g/ml, since this concentration had been shown to be optimal in preliminary experiments. Plates were incubated in a CO 2 incubator at 37 °C for 4 hours when human cells were used as effector cells, and for 8 hours when murine effector cells were used. After centrifugation of the U bottom plates at 1,500 rpm for 15 minutes, 200 ⁇ l of culture supernatant was collected and radioactivity was determined by a gamma well counter.
  • the spontaneous release of 51 Cr was measured after the incubation of target cells alone with RPMI 1640 and the total count was determined after the incubation of the cells in 1.0 N HCl. The spontaneous release was less than 10% of the total release in all experiments.
  • the percent specific lysis was determined by the following formula:
  • mice were anesthetized with 0.4 ml of 2% chloral hydrate (Sigma Chemical Co., St. Louis, MO) by ip. injection and placed in the decubitus position. A transverse incision was made in the left flank through the skin and peritoneum, exposing the spleen.
  • Mice were injected with 1.0 x 10 6 LS 180 cells and 20 ⁇ g of rabbit anti-serum to ganglio-N-tetraosylceramide (anti-asialo GM1; Wako Chemicals, Dallas, TX) in 0.25 ml PBS into the portal vein via the splenic hilus using a 27-gauge needle. After waiting for one minute, the portal vein was ligated, the spleen was removed and the abdominal cavity was closed.
  • 2% chloral hydrate Sigma Chemical Co., St. Louis, MO
  • mice were intravenously (iv.) injected with a single dose of c-SF-25 MAb (100 ⁇ g/mouse).
  • a single dose of c-SF-25 MAb 100 ⁇ g/mouse.
  • an equal molar amount (67 ⁇ g/mouse) of F(ab') 2 fragment of c-SF-25 MAb was injected into other mice.
  • Thirty days later (five weeks after the tumor cell injection) the mice were sacrificed and examined for metastatic spread in the liver and the presence of local abdominal tumors. Results were statistically significant when p was ⁇ 0.05 by the chi-square test with the Yates correction.
  • iota-carrageenan iota-CGN; Sigma Chemical Co., St. Louis,
  • MO is toxic to macrophages
  • this reagent was chosen as a macrophage depleting agent.
  • native iota-CGN is not water-soluble at low temperatures and the sulfate groups contained in the iota-CGN are mitogenic (Id.)
  • iota-CGN was desulfated (DS-CGN) (Ishizaka et al, J. Immunol. Method 124: 17-24 (1989)) and injected into the nude mice.
  • DS-CGN desulfated
  • 3 mg of DS-CGN dissolved in PBS was injected ip.
  • peritoneal murine macrophages were almost completely depleted by these injections and that peritoneal exudate cells positive for F4/80 decreased from 89.3% to 9.0%.
  • Macrophages detected in murine peripheral blood were also depleted by the ip injection of DS-CGN and F4/80-positive cells decreased therein from 18.0% to 1.9% .
  • mice with human colon adenocarcinoma metastases developed large hepatic tumors and all animals died within 6 to 7 weeks after the injection of tumor cells due to hepatic dysfunction.
  • Mice were injected iv with c-SF-25 MAb (100 ⁇ g/mouse once a week for 4 weeks) starting 5 days after the intraportal injection of tumor cells.
  • the survival rate was compared to the untreated control group using the algorithm of Lee and Desu (Comput. Prog. Biomed. 2:315-321 (1972)).
  • binding constants indicate that the antigen- antibody interaction is of high avidity and that the human/murine chimeric construct ofthe SF-25 MAb was identical to the murine antibody. Finally, the numbers of antibody binding sites per LS 180 cell were calculated to be approximately 2.5 x 10 5 for both the chimeric and original murine MAb.
  • ADCC produced by c-SF-25 MAb was studied using human PBL as effector cells (Figure 4A).
  • ADCC was induced at various effector to target cell (E:T) ratios by c-SF-25 but not by murine SF-25 MAb.
  • E:T effector to target cell
  • the specific lysis of colon adenocarcinoma cells by isolated subpopulations of cells was then examined.
  • the purity of macrophages and NK-cells isolated from human PBL were >95% and >90%, respectively, when examined by flow cytometry.
  • Both cell populations induced substantial ADCC against LS 180 cells in the presence of c-SF-25 MAb (Figure 4B) that was greater than that induced by the mixed cellular population of PBLs ( Figure 4A).
  • Hepatic metastases of human colon adenocarcinoma cells were established by injecting LS 180 cells into the portal vein of nude mice.
  • the administration of anti-asialo GM1 is essential to establish hepatic metastases in nude mice (Takahashi et al, Gastroenterology 96:1317-1329 (1989)).
  • anti-asialo GM1 was injected iv. one or two days before tumor cell injection, hepatic tumors developed in only 60% and 40% of the mice, respectively. Therefore, all mice were simultaneously injected iv. with LS 180 cells and anti-asialo GM1 into the portal vein. All these mice developed macroscopic tumors in their livers as well as local abdominal tumors at the site of tumor cell injection. Effect of Chimeric SF-25 MAb on Tumor Growth in vivo
  • mice developed extensive hepatic metastases 5 weeks after tumor cell injection (Group A in Table I). Large "cannon ball-like" multiple tumors developed in the livers of untreated mice ( Figure 5 A) and 74% of these untreated animals (14 of 19) developed multiple, isolated tumors larger than
  • mice with hepatic metastases were significantly reduced from 100% (19 of 19) to 22% (2 of 9) (P ⁇ 0.01: c-SF-25 treated vs controls; Table I) and most of these mice were free of detectable hepatic tumors (Figure 5B).
  • Two mice treated with c-SF-25 MAb developed hepatic tumors, but their tumor burden was substantially less than the controls and all tumors were less than 3mm.
  • c-SF-25 MAb also inhibited local abdominal tumor growth (P ⁇ 0.005 compared to controls).
  • mice treated with F(ab') 2 fragment developed multiple hepatic tumors larger than 5 mm in diameter (Group C in Table I) and were similar to control
  • mice ( Figure 5C).
  • the F(ab') 2 fragment had little effect on the local abdominal tumors (Table I).
  • Tumor size as measured by largest diameter of an individual tumor.
  • DS-CG was injected ip. to deplete murine macrophages after the injection of the LS 180 tumor cells.
  • the DS-CGN basically did not affect the development of hepatic metastases (Group A v. Group B in Table II).
  • the anti-tumor effect of c-SF-25 MAb (100 ⁇ g/mouse) on hepatic metastases was reduced by DS-CGN and hepatic metastases were present in all mice treated with c-SF-25 MAb (Group C, Table II).
  • mice treated with c-SF-25 MAb and DS-CGN were still significantly smaller than those of untreated animals (P ⁇ 0.01; Group C v. Group A in Table II).
  • the anti-tumor effect of c-SF-25 MAb on local abdominal tumors was completely blocked by DS-CGN and mice developed extrahepatic tumors similar in size to untreated mice (Group C v. Group A in Table II).
  • mice were injected iv. with c-SF-25 MAb (100 ⁇ g/mouse). The mice were sacrificed the following day. Flow cytometric analysis of liver infiltrating cells detected by anti-asialo-GM1 rabbit serum demonstrated a larger population of NK-cells in the liver (31 %) than in peripheral blood (7%) in mice with liver tumors and also in normal animals (36% vs. 5%, respectively; Table III)). There was no significant difference in the macrophage population size, as determined by non-specific esterase activity between liver (25%) and peripheral blood (15%; Table III).
  • ADCC was induced by c-SF-25 MAb using thioglycolate-elicited murine macrophages as the effector cells at various E:T ratios ( Figure 6A). Cytotoxicity was also produced when [ 3 H]methyl-thymidine labeled LS 180 cells were used as target cells in a longer term cytotoxicity assay (12-24h; data not shown ). ADCC was induced by murine splenocytes in the presence of c-SF-25 MAb ( Figure 6B).
  • NK-cells were isolated from these murine splenocytes by discontinuous density gradient centrifugation. The purity of these NK-cells, as determined by flow cytometry, was over 90%. These purified murine NK-cells demonstrated ADCC in the presence of c-SF-25 MAb. ( Figure 6C).
  • Binding studies were performed.
  • the c-SF-25 MAb association constant was 2.2 x 10 8 M -1 .
  • the number of SF-25 and binding sites per cell were 2.3 x 10 5 /cell when studied at 4°C using LS 180 cells as the antigen source.
  • ADCC was induced at various effector to target cell (E:T) ratios greater than 12.5: 1 only in the presence of chimeric MAbs (20 ⁇ g/ml; Figure 8).
  • E:T effector to target cell
  • a single intravenous injection of chimeric SF-25 MAb substantially inhibited LS-180 tumor growth.
  • the percent of mice bearing hepatic metastases was reduced from 100% to 22% (P ⁇ 0.001: vs. controls; Table 4) and most were entirely free of detectable hepatic disease.
  • Two animals in the SF-25 MAb treatment group developed hepatic tumors at 5 weeks, but the size of these tumors was considerably smaller than in the controls (Table 4).
  • Local abdominal tumors were also reduced in the SF-25 treatment group (100% to 44%, P ⁇ 0.025; Table 4). Table 4.
  • Table 4 In vivo anti-tumor effect of chimeric SF-25 MAb on human colon adenocarcinoma growth in nude mice.

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Abstract

L'invention concerne des anticorps monoclonaux SF-25, spécialement des anticorps chimériques, ainsi que leurs dérivés et fragments spécifiques de l'antigène de SF-25 de cellules tumorales humaines, leurs procédés de préparation, des compositions pharmaceutiques les contenant et leur utilisation.
PCT/US1992/008109 1991-09-25 1992-09-25 Anticorps sf-25, notamment anticorps chimeriques specifiques de l'antigene de sf-25 de tumeurs humaines, leurs procedes de preparation et leur utilisation Ceased WO1993006117A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1566639A4 (fr) * 2002-11-08 2006-10-11 Hiroshi Takahashi Procede d'examen de cellules cancereuses et reactif associe

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4735210A (en) * 1985-07-05 1988-04-05 Immunomedics, Inc. Lymphographic and organ imaging method and kit

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4735210A (en) * 1985-07-05 1988-04-05 Immunomedics, Inc. Lymphographic and organ imaging method and kit

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
A. PINCERA et al., "Monoclonal Antibodies '84: Biological and Clinical Applications", Published 1985, by EDITRICE KURTIS S.R.L., see pages 475-506. *
ANNALS NEW YORK ACADEMY OF SCIENCES, Volume 507, issued 1988, S.L. MORRISON et al., "Genetically Engineered Antibody Molecules and their Application", pages 187-198. *
BIOTECHNOLOGY, Volume 9, issued February 1991, G.T. DAVIS et al., "Single Chain Antibody (SCA) Encoding Genes: One Step Construction and Expression in Eukaryotic Cells", pages 165-169. *
CLINICAL CHEMISTRY, Volume 27, No. 11, issued 1981, E.D. SEVIER et al., "Monoclonal Antibodies in Clinical Immunology", pages 1797-1806. *
GASTROENTEROLOGY, Volume 96, No. 5, Part 1, issued 1989, H. TAKAHASHI et al., "Radioimmunolocalization of Hepatic and Pulmonary Metastasis of Human Colon Adenocarcinoma" pages 1317-29. *
METHODS IN ENZYMOLOGY, Volume 178, issued 1989, M. BETTER et al., "Expression of Engineered Antibodies and Antibody Fragments in Microorganisms", pages 476-496. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, Volume 86, issued December 1989, C. QUEEN et al., "A Humanized Antibody that Binds to the Interleukin 2 Receptor", pages 10029-10033. *
VOGELS et al., "Immunoconjugates. Antibody Conjugates in Radioimaging and Therapy of Cancer", Published 1987, by OXFORD UNIVERSITY PRESS, see pages 259-280. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1566639A4 (fr) * 2002-11-08 2006-10-11 Hiroshi Takahashi Procede d'examen de cellules cancereuses et reactif associe

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