WO2007071422A2 - Pharmaceutical antibody compositions with resistance to soluble cea - Google Patents
Pharmaceutical antibody compositions with resistance to soluble cea Download PDFInfo
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- WO2007071422A2 WO2007071422A2 PCT/EP2006/012409 EP2006012409W WO2007071422A2 WO 2007071422 A2 WO2007071422 A2 WO 2007071422A2 EP 2006012409 W EP2006012409 W EP 2006012409W WO 2007071422 A2 WO2007071422 A2 WO 2007071422A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3007—Carcino-embryonic Antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- 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
- CEA tumor associated carcinoembryonic antigen
- the human CEA gene family is clustered on chromosome 19q13.2 (Olsen et al. Genomics 23 (1994); 659-668). Its 29 genes and pseudogenes can be divided into three subgroups, i.e. the CEA subgroup containing seven expressed genes, the pregnancy-specific-glycoprotein (PSG) subgroup containing eleven expressed genes and the third subgroup which contains only pseudogenes (Hammarstr ⁇ m, Sem. Cancer Biol. 9 (1999), 67-81 ; Beauchemin, Exp. Cell Res. 252 (1999), 243-249).
- CEA and the other family members revealed that they belong to the immunoglobulin (Ig) superfamily (Williams and Barclay, Annul. Rev. Immunol. 6 (1988), 381-405). All members of the CEA subgroup are attached to the cell surface membrane: Biliary glycoprotein (CEACAM1; BGP1 ; TM-CEA; CD66a), CEA gene family member 1 (CEACAM3; CGM 1; CD66d) and CEA gene family member 7 (CEACAM4; CGM7) have hydrophobic transmembrane domains, whereas carcinoembryonic antigen (carcinoembryonic antigen-related cell adhesion molecule 5; CEACAM5; CEA; CD66e), non-specific cross-reacting antigen (CEACAM6; NCA; NCA-50/90; CD66c), CEA gene family member 2 (CEACAM7; CGM2) and CEA gene family member 6 (CEACAM8; CGM6; CD66b
- the CEA proteins are highly glycosylated with a molecular weight of up to approximately 300 kDa, depending on the number of Ig domains.
- biliary glycoprotein a glycoprotein that influences the biological activity of the CEA proteins
- CEA and non-specific cross-reacting antigen can act as homophilic and heterotypic cell adhesion molecules when expressed on the tumor cell surface (Oikawa et al., Biochem. Biophys. Res. Commun. 186 (1992), 881-887; Zhou et al., Cancer Res. 53 (1993), 3817-3822).
- CEA and non-specific cross- reacting antigen in the innate immune defense protecting colon from microbial attack has been discussed (Hammarstr ⁇ m and Baranov, Trends Microbiol. 9 (2001), p. 119- 125).
- these proteins bind and trap microorganisms preventing them from reaching and invading the epithelial cells of the microvilli.
- CEA is an oncofetal antigen which is expressed during fetal life, absent in the healthy adult and re-expressed in cancer.
- CEA is also expressed in normal adult tissue.
- biliary glycoprotein, CEA, non-specific cross-reacting antigen and CEA gene family member 2 are expressed in normal human colon, particularly in the mature columnar epithelial cells facing the gut lumen and in the highly differentiated cells at the crypt mouth (Frangsmyr et al., Cancer Res. 55 (1995), 2963-2967; Frangsmyr et al., Tumor Biol. 20 (1999), 277-292).
- Biliary glycoprotein, CEA and non-specific cross-reacting antigen are also expressed in a number of tumors of epithelial origin (Hammarstr ⁇ m, Sem. Cancer Biol. 9 (1999), 67-81 ; Shively and Beatty CRC Crit. Rev. Oncol. Hematol. 2 (1985), 355-399).
- CEA became a favored target antigen for radioimmunolocalization of colorectal and other epithelial tumors.
- CEA is overexpressed in 95% of gastrointestinal and pancreatic cancers, as well as in most small-cell and non-small-cell lung carcinomas. It is also expressed in breast carcinoma and squamous cell carcinoma of the head and neck (Primus et al., Cancer 42 (1978), 1540-1545).
- CEA is one of the most extensively used clinical tumor markers. It is used as a serum tumor marker for colorectal and some other cancers due to its stability, its fairly restricted expression in normal adult tissue and its high expression in tumors of epithelial origin. The bulk of CEA in a healthy individual is produced in colon. There it is released from the apical surface of mature columnar cells into the gut lumen and disappears with the feces.
- CEA levels in the blood of healthy individuals is less than 2 ⁇ g/l.
- CEA levels in serum from patients with colorectal and other carcinomas are increased, ranging up to more than 2000 ⁇ g/l (Thomson et al., PNAS 64 (1969), 161-167).
- progressive, malignant, or late stage epithelial tumors are frequently accompanied by high serum concentrations of soluble CEA (Fletcher; Ann. Intern. Med. 104 (1986), 66-73).
- components from the plasma membrane, including CEA are continually exfoliated from the surface as plasma membrane-derived vesicles (Taylor and Black, J. Natl.
- CEA is not only used as a tumor marker but also as a target for anti-tumor therapy.
- gastrointestinal cancers account for a large proportion of human epithelial tumors, with an estimated 21.700 new cases of gastric cancer and 135.400 new cases of colorectal cancer in the United States in the year 2001 (Greenlee; CA Cancer J Clin 51 (2001), 15-36).
- Colorectal cancer is the third most common malignancy and the third leading cause of death from cancer in both males and females (Ries; Cancer 88 (2000), 2398-2424).
- a 131 l-labeled variant of labetuzumab (labetuzumab is a humanized form of anti-CEA monoclonal antibody MN-14; Behr et al., Cancer, 94: 1373-1381 , (2002), 1559-64) has been analysed in a phase Il trial in which 30 CRC patients with small volume metastatic disease chemorefractory to 5-fluorouracil and folinic acid or in an adjuvant setting after liver metastasis have been enrolled. A single injection of 131 l-labeled labetuzumab was given. Of 19 assessable patients, 3 had partial remissions and eight showed minor responses up to 15 months in duration.
- the range of soluble CEA levels in the patients enrolled in this study was 14.8 - 1027 ng/ml (median 97 ng/ml).
- the same antibody was used in a combination therapy with 5-Fluoro-uracil (5-FU).
- 5-FU 5-Fluoro-uracil
- 21 patients with chemotherapy-refractory metastatic colorectal cancer received 5-FU and 90 Y-cT84.66. No objective responses were observed.
- the mean serum CEA level was 227,4 ng/ml, ranging from ⁇ 2,5 - 1305 ng/ml (Wong, Clin Cancer Res, 9 (2003): 5842-5852).
- one aspect of the invention relates to a pharmaceutical composition for the treatment of an epithelial tumor in a human, said pharmaceutical composition comprising an IgGI antibody specifically binding to human CEA, wherein the variable region of said IgGI antibody comprises at least the amino acid sequences selected from the group consisting of:
- DRGLRFYFDY amino acid sequence "DRGLRFYFDY" (SEQ ID NO. 27) corresponds to Kabat positions 95 - 102 of the CDR-H3 of murine monoclonal antibody A5B7 (Chester, Int. J. Cancer 57 (1994), 67-72; Harwood, Br J Cancer. 54 (1986), 75-82).
- said variable region of the IgGI antibody defined herein comprises a CDR-L 1 having the amino acid sequence "TLRRGINVGAYSIY” (SEQ ID NO. 34) and/or a CDR-L2 having the amino acid sequence "YKSDSDKQQGS” (SEQ ID NO. 33) and/or a CDR- L3 having the amino acid sequence "MIWHSGASAV” (SEQ ID NO. 32).
- CDRs Determination of CDRs is known to the person skilled in the art; see e.g. http://www.bioinf.org. uk/abs/#cdrid. Numbering of amino acid sequences in antibodies can be carried out e.g. according to the Kabat numbering scheme described in the art; see e.g. Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, and C. Foeller. 1991. Sequences of Proteins of Immunological Interest, 5th ed. Bethesda, Md.: National Center for Biotechnology Information, National Library of Medicine.
- the present invention provides means and methods particularly suited for the treatment of (an) epithelial tumor(s) in patients with high soluble CEA concentrations in their serum/plasma.
- Such high soluble CEA concentrations are found in the serum/plasma of epithelial tumor patients with progressive tumors, recurrent, metastatic, late stage tumors and for patients with high tumor load/burden.
- IgGI antibodies specifically binding to human CEA which comprise a variable region as defined above not only bind to CEA-positive target cells, but also to soluble CEA; see Example 5 of the present invention.
- the IgGI antibodies of this invention kill CEA-bearing tumor cells, even in the presence of high concentrations of soluble CEA.
- IgGI antibody constructs are not inhibited by soluble CEA in their cytotoxic activity (antibody-dependent cell cytotoxic activity; ADCC) against CEA-positive tumor cells.
- cytotoxic activity antibody-dependent cell cytotoxic activity; ADCC
- Example 5 shows cytotoxic activity against Kato III cells (CEA-positive human gastric carcinoma cell line) of CEA-reactive IgGI antibody constructs as defined above in the presence of increasing amounts of soluble CEA antigen.
- cytotoxicity mediated by the IgGI antibodies as defined herein is resistant to soluble CEA.
- the IgGI antibodies of the present invention as defined herein comprise a variable region with CDR-H regions as set forth above, including the CDR-H3 "DRGLRFYFDY" (SEQ ID NO.
- soluble CEA antigen prevents the antibody from exerting its antibody-mediated cytotoxic activity.
- T84.66-derived IgGI constructs it could not be expected that soluble CEA does not inhibit cytotoxic activity in A5B7-derived antibody constructs. More specifically, resistance to soluble CEA antigen could be found only for IgGI antibodies, the variable regions of which comprised the amino acid sequence "DRGLRFYFDY" of the CDR-H3 of murine monoclonal antibody A5B7 (Chester, loc. cit.; Harwood, Br J Cancer. 54 (1986), 75-82).
- the present invention provides means and methods particularly suited for the treatment of tumor patients with high soluble CEA concentrations in their plasma, as observed e.g. during tumor progression, for recurrent cancer, for metastasis, for patients with high tumor load/burden, or late-stage tumors.
- the IgGI antibodies in the pharmaceutical compositions of the invention in the following are referred to as being resistant to soluble CEA antigen.
- the term "resistance to soluble CEA antigen", “resistant to soluble CEA” (or related terms) as used herein refers to the fact that the cytotoxicity against CEA-positive tumor cells mediated by said IgGI antibodies is not affected by increasing concentrations of soluble CEA. In particular, the cytotoxic activity is not inhibited by even high concentrations of soluble CEA.
- CEA levels in the blood of healthy individuals is less than 2ng/ml.
- High soluble CEA concentrations in the serum/plasma of tumor patients are characteristic for progressive, recurrent, metastatic, or late stage tumors and for patients with high tumor load.
- the present invention provides means and methods particularly suited for the treatment of epithelial tumor patients with such high soluble CEA concentrations in their plasma.
- the term "high soluble CEA concentrations" as used herein denotes a soluble serum/plasma-CEA concentration higher than 10, 20, 50, 70, 80, 90 or 100 ng/ml.
- the serum/plasma CEA concentration may, inter alia, be determined by ELISA techniques.
- said soluble serum/plasma-CEA concentration is higher than 100 ng/ml, as for example determined by ELISA.
- the CEA serum concentration can be determined e.g. by CEA ELISA assays (see e.g. IBL CEA EIA, IBL Hamburg, Germany).
- the term "pharmaceutical composition” relates to a composition for administration to a human patient.
- the pharmaceutical composition comprises suitable formulations of carriers, stabilizers and/or excipients.
- the pharmaceutical composition comprises a composition for parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or by direct injection into tissue. It is in particular envisaged that said composition is administered to a patient via infusion or injection.
- Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
- the composition of the present invention may further comprise a pharmaceutically acceptable carrier.
- compositions comprising such carriers can be formulated by well known conventional methods. These compositions can be administered to the subject at a suitable dose which can be determined e.g. by dose escalating studies by administration of increasing doses of the IgGI antibodies exhibiting resistance to soluble serum CEA antigen described herein.
- a suitable dose which can be determined e.g. by dose escalating studies by administration of increasing doses of the IgGI antibodies exhibiting resistance to soluble serum CEA antigen described herein.
- the IgGI antibodies described herein with resistance to soluble serum CEA antigen can be advantageously used in the treatment of cancer patients with high CEA serum concentrations, such as progressive tumors, recurrent cancer, metastatic tumors, high tumor load/burden, or late stage tumors.
- compositions can also be administered in combination with other proteinaceous and non- proteinaceous drugs, e.g. in the form of a co-therapy.
- These drugs may be administered simultaneously with the composition comprising the IgGI antibodies as defined herein or separately before or after administration of said IgGI antibodies in timely defined intervals and doses.
- the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, and suspensions.
- non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, aqueous solutions, or suspensions, including saline and buffered media.
- Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or lactated Ringer's.
- Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases and the like.
- composition of the present invention might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin.
- co-therapy comprise, in addition to the IgGI antibodies as defined herein, further biologically active agents, depending on the intended use of the composition.
- agents might be drugs acting on the gastro-intestinal system, drugs acting as antineoplastic agents, chemotherapeutics, cytostatica, drugs preventing hyperurikemia, drugs inhibiting immunoreactions (e.g. corticosteroids), drugs modulating the inflammatory response, drugs acting on the circulatory system and/or agents such as cytokines known in the art.
- the IgGI antibody as defined herein is formulated in a buffer, a stabilizer and a surfactant.
- the buffer may be a phosphate, citrate, succinate or acetate buffer.
- the stabilizer may be (an) amino acid(s) and/or a sugar.
- the surfactants may be detergents, PEGs, or the like.
- the IgGI antibody as defined herein is formulated in citrate, lysine, trehalose and Tween 80. As a diluent for the pharmaceutical composition of the invention, isotonic saline and Tween 80 is preferred.
- an "antibody” denotes immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen, preferably human CEA. All antibodies are constructed in the same way. They form paired heavy and light polypeptide chains, and the generic term immunoglobulin is used for all such proteins. Within this general category, however, five different classes of immunoglobulins— IgM, IgD, IgG, IgA, and IgE- can be distinguished by their C regions. IgG antibodies are large molecules, having a molecular weight of approximately 150 kDa, composed of two different kinds of polypeptide chain.
- Each IgG molecule consists of two heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. In any given immunoglobulin molecule, the two heavy chains and the two light chains are identical, giving an antibody molecule two identical antigen-binding sites, and thus the ability to bind simultaneously to two identical structures. Two types of light chain, termed lambda and kappa, are found in antibodies. A given immunoglobulin either has lambda chains or kappa chains, never one of each.
- immunoglobulin M immunoglobulin M
- IgD immunoglobulin D
- IgG immunoglobulin G
- IgA immunoglobulin A
- IgE immunoglobulin E
- Their heavy chains are denoted by the corresponding lower-case Greek letter (mu, delta, gamma, alpha, and epsilon, respectively).
- IgG is by far the most abundant immunoglobulin and has several subclasses (IgGI , 2, 3, and 4 in humans). Their distinctive functional properties are conferred by the carboxy-terminal part of the heavy chain, where it is not associated with the light chain.
- the general structural features of all the isotypes are similar.
- the structure of an IgG antibody, the most abundant isotype in plasma, as a typical antibody molecule is exemplified in Figure 1.
- the antibodies as defined herein are IgG antibodies.
- an IgG comprises not only the variable antibody regions responsible for the highly discriminative antigen recognition and binding, but also the constant regions of the heavy and light antibody polypeptide chains normally present in endogenously produced antibodies and, in some cases, even decoration at one or more sites with carbohydrates.
- Such glycosylation is generally a hallmark of the IgG format, and portions of these constant regions make up the so called Fc region of a full antibody which is known to elicit various effector functions in vivo, such as e.g. antibody- dependent cellular cytotoxicity (ADCC).
- ADCC antibody- dependent cellular cytotoxicity
- the Fc region mediates binding of the IgG to an Fc receptor, hence prolonging half life in vivo as well as facilitating homing of the IgG to locations with increased Fc receptor presence.
- the IgG antibody is an IgGI antibody specifically binding to the human CEA antigen, formats which are preferred since their mechanism of action in vivo is particularly well understood and characterized. This is especially the case for IgGI antibodies.
- the IgGI antibodies referred to herein comprise the variable region(s) as defined above in combination with the entirety or a portion of the hinge region, CH1 , CH2, and CH3 domains and CL domains; see e.g. Figure 1.
- a VH domain is paired with a VL domain to provide an antibody antigen binding site.
- the VH domain comprising (i) a CDR-H 1 having the amino acid sequence "SYWMH” (SEQ ID NO. 29) and a CDR-H2 having the amino acid sequence "FIRNKANGGTTEYAASVKG" (SEQ ID NO. 28) and a CDR-H3 having the amino acid sequence "DRGLRFYFDY” (SEQ ID NO.
- the anti-CEA IgGI antibodies defined herein may be rodent antibodies (i.e. from mice or rats).
- said IgGI antibodies are humanized antibodies as set forth in more detail below.
- CDR-H3 "DRGLRFYFDY” (SEQ ID NO. 27) amino acid sequence isresistant to soluble CEA antigen, thereby allowing the treatment of tumor patients with high serum CEA concentrations in their plasma.
- human refers to the species Homo sapiens.
- a "human” molecule e.g. human CEA, is therefore the variant of that molecule as it is naturally expressed in Homo sapiens.
- epithelial tumor denotes a tumor of epithelial origin which is CEA positive (Cancer Medicine; 6th ed.; Kufe, Donald W.; Pollock, Raphael E.; Weichselbaum, Ralph R.; Bast, Robert C, Jr.; Gansler, Ted S.; Holland, James F.; Frei III, Emil, editors. Hamilton (Canada): BC Decker Inc. 2003; http://www.dkfz.de: http://www.krebsinformationsdienst.de/Krebsart/index.html).
- the epithelial tumor to be treated may be a gastrointestinal adenocarcinoma, a breast adenocarcinoma or a lung adenocarcinoma.
- Said gastrointestinal adenocarcinoma is preferably a colorectal, pancreatic, an oesophageal or a gastric adenocarcinoma.
- the pharmaceutical composition of the invention is particularly advantageous for the treatment of patients with progressive tumors, metastasis, recurrent cancer, late stage epithelial tumors, high epithelial tumor load/tumor burden, or tumor patients with a CEA serum concentration higher than 100 ng/ml (as determined e.g.
- the IgGI antibodies as defined herein may be administered in a period in which serum CEA levels decrease (due to the removal of the CEA source, i.e.
- the primary tumor in order to kill remaining tumor cells.
- the IgGI antibodies as defined herein may be useful after the removal of the primary tumor, in the case that serum CEA levels increase due to the formation of secondary tumors or metastasis.
- the CEA serum concentration can be determined e.g. by CEA ELISA assays (see e.g. IBL CEA EIA, IBL Hamburg, Germany). As set forth above, in many antibody-based therapeutic approaches, said serum CEA inhibits binding of the antibody to membrane-bound CEA on the tumor cells and blocks the activity of antibody, thereby worsening the success of the anti-tumor therapy.
- the term “specifically binds” or related expressions such as “specifically binding” or “specific reactivity with/to” etc. refer to the ability of the binding domains of the IgGI antibody as defined herein to discriminate between a first and/or second molecule to such an extent that, from a pool of a plurality of different molecules as potential binding partners, only said respective first and/or second molecule is/are bound, or is/are significantly bound.
- binding measurements can be routinely performed e.g. on a Biacore apparatus, by ELISA, FACS analysis or the like.
- the binding domain of the IgGI antibody as defined herein binds to a epithelial tumor antigen, i.e.
- human CEA (carcinoembryonic antigen, carcinoembryonic antigen-related cell adhesion molecule 5; CEACAM5; CD66e), as set forth below.
- the term "specifically binding” means in accordance with this invention that the IgGI antibody molecule is capable of specifically interacting with and/or binding to at least two, three, four, five, six, seven, eight or even more amino acids of human CEA as defined herein. Said term relates to the specificity of the antibody molecule, i.e. to its ability to discriminate between the specific regions of the human CEA antigen as defined herein.
- the specific interaction of the antigen-interaction-site with its specific antigen may result in an initiation of a signal, e.g.
- binding may be exemplified by the specificity of a "key-lock-principle".
- specific motifs in the amino acid sequence of the antigen-interaction-site and the antigen bind to each other as a result of their primary, secondary or tertiary structure as well as the result of secondary modifications of said structure.
- the specific interaction of the antigen- interaction-site with its specific antigen may result as well in a binding of said site to the antigen.
- the "specific binding" of an antibody is characterized primarily by two parameters: a qualitative parameter (the binding epitope, or where the antibody binds) and a quantitative parameter (the binding affinity, or how strongly it binds where it does).
- Which epitope is bound by an antibody can advantageously be determined by e.g. known FACS methodology, peptide-spot epitope mapping, mass spectroscopy or peptide ELISA.
- the strength of antibody binding to a particular epitope may be advantageously be determined by e.g. known Biacore and/or ELISA methodologies. A combination of such techniques allows the calculation of a signal:noise ratio as a representative measure of binding specificity.
- the signal represents the strength of antibody binding to the epitope of interest
- the noise represents the strength of antibody binding to other, non-related epitopes differing from the epitope of interest.
- a signal:noise ratio for an epitope of interest which is about 50-fold higher than for other epitopes different from the epitope of interest may be taken as an indication that the antibody evaluated binds the epitope of interest in a specific manner, i.e. is a "specific binder".
- the term "specific binding” or “specific interaction” as used in accordance with the present invention means that the IgGI antibody construct does not or essentially does not cross-react with polypeptides of similar structures.
- Cross-reactivity of a panel of antibody constructs under investigation may be tested, for example, by assessing binding of said panel of antibody constructs under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988 and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999) to the polypeptide of interest as well as to a number of more or less (structurally and/or functionally) closely related polypeptides.
- the binding domain of the IgGI antibody defined herein binds to human CEA (carcinoembryonic antigen; CEACAM5; CEA; CD66e), i.e.
- Examples for the specific interaction of an antigen-interaction-site with a specific antigen comprise the specificity of a ligand for its receptor. Said definition particularly comprises the interaction of ligands which induce a signal upon binding to its specific receptor. Examples for corresponding ligands comprise cytokines which interact/bind with/to its specific cytokine-receptors.
- Another example for said interaction is the interaction of an antigenic determinant (epitope) with the antigenic binding site of an antibody.
- binding to/interacting with may also relate to a conformational epitope, a structural epitope or a discontinuous epitope consisting of two regions of the human target molecule, i.e. human CEA, or parts thereof.
- a conformational epitope is defined by two or more discrete amino acid sequences separated in the primary sequence which come together on the surface of the molecule when the polypeptide folds to the native protein (SeIa, (1969) Science 166, 1365 and Laver, (1990) Cell 61 , 553-6).
- discontinuous epitope means in context of the invention non-linear epitopes that are assembled from residues from distant portions of the polypeptide chain. These residues come together on the surface of the molecule when the polypeptide chain folds into a three-dimensional structure to constitute a conformational/structural epitope.
- CEA denotes the carcinoembryonic antigen (carcinoembryonic antigen-related cell adhesion molecule 5; CEACAM5; CEA; CD66e), an antigen expressed in a large number of tumors of epithelial origin (Hammarstr ⁇ m, Sem. Cancer Biol. 9 (1999), 67- 81 ; Shively and Beatty CRC Crit. Rev. Oncol. Hematol. 2 (1985), 355-399).
- the amino acid sequence of human CEA is depicted in GenBank accession number NM_004363 and comprises SEQ ID NO. 37.
- the present invention it has been surprisingly found that it is possible to generate antibody-based therapeutics with specificity for human CEA, wherein the cytotoxic activity (ADCC) directed against tumor cells is resistant to even high concentrations of soluble CEA antigen.
- ADCC cytotoxic activity
- This finding is entirely unexpected in view of the fact that the IgGI antibodies defined herein bind to soluble CEA antigen.
- IgGI antibody constructs derived from monoclonal antibody T84.66 have been generated, these antibodies were highly sensitive to soluble CEA antigen, ie. their cytotoxic activity (ADCC) has been blocked in the presence of soluble CEA antigen.
- ADCC cytotoxic activity
- soluble CEA antigen prevents the antibodies from exerting their cytotoxic activity.
- the IgGI antibodies as defined herein are resistant to the presence of even high levels of soluble CEA in their cytotoxic activity towards tumor cells.
- the amino acid sequence "DRGLRFYFDY" (SEQ ID NO. 27) corresponding to Kabat positions 95 - 102 of the CDR-H3 of murine monoclonal antibody A5B7 is sufficient to mediate resistance to soluble CEA antigen when used in a human CEA-binding domain (i.e. human binding domains specifically binding to human CEA) of human IgGI antibodies.
- the pharmaceutical compositions comprising the IgGI antibodies as defined herein are particularly useful for the treatment of epithelial tumor patients with high soluble CEA concentrations in their plasma, as observed e.g. during tumor progression, for recurrent cancer, for metastasis, for patients with high tumor load/burden, or late-stage tumors.
- IgGI antibodies as defined herein can be generated by methods described in the art, e.g. by phage-display based techniques; see also the following Examples.
- the binding domain specifically binding to human CEA of the IgGI antibody defined herein comprises at least one CDR, preferably a CDR-H3, more preferably a part of or the complete CDR-H3 of murine monoclonal antibody A5B7 with the amino acid sequence "DRGLRFYFDY” (SEQ ID NO.
- the cytotoxic activity (ADCC) against tumor cells of the IgGI antibodies defined herein comprising said mAb A5B7-derived CDR-H3 "DRGLRFYFDY” (SEQ ID NO. 27) amino acid sequence in the binding domain interacting with CEA are resistant to soluble CEA antigen, thereby allowing the treatment of tumor patients with high serum CEA concentrations in their plasma. It may be desirable to further modify this A5B7-derived "DRGLRFYFDY" CDR-H3 amino acid sequence e.g.
- X 1 ", “X 2 “, “X 3 “ or “X 4 " may represent amino acid residue “R” (Arginine), “G” (Glycine), “L” (Leucine), “Y” (Tyrosine), “A” (Alanine), “D” (Aspartic acid), “S” (Serine), “W” (Tryptophan), “F” (Phenylalanine) or “T” (Threonine).
- one, two, three or all four of the indicated “X” positions may be exchanged in comparison to the original "RGLR” amino acid sequence at Kabat positions 96 to 99 in the CDR-H3 "DRGLRFYFDY” (SEQ ID NO. 27) amino acid sequence.
- soluble CEA antigen of an IgGI antibody with such a modified CDR-H3 can be tested in cytotoxicity (antibody dependent cell-mediated cytotoxicity, ADCC) assays in the presence of increasing amounts of soluble CEA, as described in the following Examples.
- variable region of the IgGI antibodies defined herein comprises at least the amino acid sequences selected from the group consisting of:
- said variable region comprises a CDR-L 1 having the amino acid sequence "TLRRG I NVGAYS IY” (SEQ ID NO. 34) and/or a CDR-L2 having the amino acid sequence "YKSDSDKQQGS” (SEQ ID NO. 33) and/or a CDR-L3 having the amino acid sequence "MIWHSGASAV” (SEQ ID NO. 32).
- amino acid sequence of the VH region of the binding domain specific for human CEA of the IgGI antibodies defined herein is preferably SEQ ID NO. 20, 22 or 24.
- amino acid sequence of the VL region of the binding domain specific for human CEA of the IgGI antibodies defined herein is preferably SEQ ID NO. 26.
- variable (V) regions of the binding domain specific for CEA of the IgGI antibodies defined herein are selected from the group consisting of: (a) the VH region consists of the amino acid sequence shown in SEQ ID NO. 22 and the VL region consists of the amino acid sequence shown in SEQ ID NO. 26; (b) the VH region consists of the amino acid sequence shown in SEQ ID NO. 20 and the VL region consists of the amino acid sequence shown in SEQ ID NO. 26; and
- VH region consists of the amino acid sequence shown in SEQ ID NO. 24 and the VL region consists of the amino acid sequence shown in SEQ ID NO. 26.
- said IgGI antibody as defined herein comprises an amino acid sequence selected from the group consisting of:
- the "IgGI antibody” to be employed in the pharmaceutical composition of the invention is a humanized IgGI antibody with human constant regions and a humanized variable region comprising the amino acid sequence "DRGLRFYFDY” corresponding to Kabat positions 95 - 102 (SEQ ID NO. 27) of the CDR-H3 of murine monoclonal antibody A5B7.
- the light chain constant region of said IgGI antibody is a lambda light chain constant region, preferably a human lambda light chain constant region.
- the IgGI antibody as defined herein may be derivatized, for example with an organic polymer, e.g. with one or more molecules of polyethylene glycol (“PEG”) and/or polyvinyl pyrrolidone ("PVP").
- PEG polyethylene glycol
- PVP polyvinyl pyrrolidone
- PEG molecules derivatized as PEG- maleimide, enabling conjugation with the antibody or fragment thereof in a site- specific manner via the sulfhydryl group of a cysteine amino acid.
- PEG-maleimide especially preferred are 2OkD and/or 40 kD PEG-maleimide, in either branched or straight-chain form.
- the IgGI antibody as defined herein may be fused to radionuclides (e.g. 131 I), cell toxins (e.g. Pseudomonas toxin A) or cytokines, such as IL-2.
- radionuclides e.g. 131 I
- cell toxins e.g. Pseudomonas toxin A
- cytokines such as IL-2.
- the resulting fusion proteins are preferably used for therapeutic purposes in the treatment of epithelial tumors.
- Antibodies of the invention fused to radionuclides may also be useful e.g. for diagnostic purposes.
- said epithelial tumor to be treated is a gastrointestinal adenocarcinoma, a breast adenocarcinoma or a lung adenocarcinoma.
- Said gastrointestinal adenocarcinoma is preferably a colorectal, pancreatic, an oesophageal or a gastric adenocarcinoma.
- said pharmaceutical composition of the invention is for the treatment of progressive tumors, late stage tumors, tumor patients with high tumor load/burden, metastatic tumors, or tumor patients with a CEA serum concentration higher than 100 ng/ml.
- Said CEA serum concentration may be determined e.g. by ELISA.
- the IgGI antibodies defined herein are humanized and/or deimmunized.
- CDR-grafted As used herein, the term "CDR-grafted”, “humanized” or “humanization” are used interchangeably to refer to a human IgGI antibody comprising in its binding domains at least one complementarity determining region ("CDR") from a non-human antibody or fragment thereof. Humanization approaches are described for example in WO 91/09968 and US 6,407,213.
- the term encompasses the case in which a variable region of the binding domain comprises a single CDR region, for example the third CDR region (CDR-H3) of the VH, from another non- human animal, for example a rodent, as well as the case in which a or both variable region/s comprise at each of their respective first, second and third CDRs the CDRs from said non-human animal.
- CDR-H3 third CDR region
- a or both variable region/s comprise at each of their respective first, second and third CDRs the CDRs from said non-human animal.
- humanized also encompasses cases in which, in addition to replacement of one or more CDR regions within a VH and/or VL of the binding domain further mutation/s (e.g. substitutions) of at least one single amino acid residue/s within the framework ("FR") regions between the CDRs has/have been effected such that the amino acids at that/those positions correspond/s to the amino acid/s at that/those position/s in the animal from which the CDR regions used for replacement is/are derived.
- FR framework
- humanized may further encompass (an) amino acid substitution(s) in the CDR regions from a non-human animal to the amino acid(s) of a corresponding CDR region from a human antibody, in addition to the amino acid substitutions in the framework regions as described above.
- humanized antibodies or related terms encompass IgGI antibodies having the amino acid sequence of a human immunoglobulin with a variable region comprising human CDR- and framework region-sequences, with exception of a CDR-H3 having the amino acid sequence "DRGLRFYFDY” (SEQ ID NO. 27) corresponding to Kabat positions 95 - 102 of the CDR-H3 of murine monoclonal antibody A5B7.
- Such antibodies can be generated as set forth in the following Examples. It is especially advantageous that the IgGI antibody as described herein be a humanized antibody. In contemplating an antibody agent intended for therapeutic administration to humans, it is highly advantageous that the major part of this antibody is of human origin.
- a humanized antibody (or fragment) thereof will most probably not elicit a strong immunogenic response by the patient's immune system, i.e. will not be recognized as being a "foreign", that is non-human protein.
- humanized antibody as used herein is to be understood as meaning that the IgGI antibody as defined herein comprises (an) amino acid sequence(s) contained in the human antibody repertoire, and a CDR-H3 having the amino acid sequence "DRGLRFYFDY” (SEQ ID NO. 27) corresponding to Kabat positions 95 - 102 of the CDR-H3 of murine monoclonal antibody A5B7.
- an antibody, or its fragment may therefore be considered humanized if it consists of such (a) human amino acid sequence(s), i.e.
- an IgGI antibody as defined herein may also be regarded as humanized if it consists of (a) sequence(s) that deviate(s) from its (their) closest human germline sequence(s) by no more than would be expected due to the imprint of somatic hypermutation.
- the (humanized) IgGI antibodies as defined herein have a human constant region and a human variable region comprising a CDR-H3 having the amino acid sequence "DRGLRFYFDY" (SEQ ID NO. 27) corresponding to Kabat positions 95 - 102 of the CDR-H3 of murine monoclonal antibody A5B7. As set forth above, said CDR-H3 mediates resistance to soluble CEA.
- the term “deimmunized” or “deimmunization” denotes modification of the binding domain vis-a-vis an original wild type construct by rendering said wild type construct non-immunogenic or less immunogenic in humans. Deimmunization approaches are shown e.g. in WO 00/34317, WO 98/52976, WO 02/079415 or WO 92/10755.
- the term “deimmunized” also relates to constructs, which show reduced propensity to generate T cell epitopes.
- the term “reduced propensity to generate T cell epitopes” relates to the removal of T-cell epitopes leading to specific T-cell activation.
- T cell epitopes means substitution of amino acids contributing to the formation of T cell epitopes, i.e. substitution of amino acids, which are essential for formation of a T cell epitope.
- reduced propensity to generate T cell epitopes relates to reduced immunogenicity or reduced capacity to induce antigen independent T cell proliferation.
- T cell epitope relates to short peptide sequences which can be released during the degradation of peptides, polypeptides or proteins within cells and subsequently be presented by molecules of the major histocompatibility complex (MHC) in order to trigger the activation of T cells; see inter alia WO 02/066514.
- MHC major histocompatibility complex
- T cells presented by MHC class Il such activation of T cells can then give rise to an antibody response by direct stimulation of T cells to produce said antibodies.
- "Reduced propensity to generate T-cell epitopes" and/or “deimmunization” may be measured by techniques known in the art.
- de- immunization of proteins may be tested in vitro by T cell proliferation assay. In this assay PBMCs from donors representing > 80 % of HLA-DR alleles in the world are screened for proliferation in response to either wild type or de-immunized peptides. Ideally cell proliferation is only detected upon loading of the antigen-presenting cells with wild type peptides.
- HLA-DR tetramers representing all haplotypes. These tetramers may be tested for peptide binding or loaded with peptides substitute for antigen-presenting cells in proliferation assays.
- binding of e.g. fluorescence-labeled peptides on PBMCs can be measured.
- deimmunization can be proven by determining whether antibodies against the deimmunized molecules have been formed after administration in patients.
- antibody derived molecules are deimmunized in the framework regions and most of the CDR regions are not modified in order to generate reduced propensity to induce T cell epitope so that the binding affinity of the CDR regions is not affected. Even elimination of one T cell epitope results in reduced immunogenicity.
- the above approaches help to reduce the immunogenicity of the therapeutic IgGI antibodies as defined herein when being administered to epithelial tumor patients.
- the invention relates to an IgGI antibody comprising an amino acid sequence selected from the group consisting of:
- the invention also relates to nucleic acids encoding the IgGI antibodies as defined above.
- said IgGI antibodies as defined herein or nucleic acids encoding the same are used as pharmaceutical compositions for the treatment of (an) epithelial tumor(s) in human.
- Said epithelial tumor(s) is (are) CEA-positive.
- the cytotoxic activity against CEA-positive epithelial tumor cells of the IgGI antibodies in these pharmaceutical compositions of the invention is resistant to even high concentrations of soluble CEA antigen in the plasma of tumor patients.
- Whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide or amino acid sequence defined herein can be determined conventionally using known computer programs.
- a preferred method for determining the best overall match between a query sequence (a sequence defined herein) and a subject sequence can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
- a sequence alignment the query and subject sequences are both DNA sequences.
- An RNA sequence can be compared by converting U's to T's.
- the invention also provides for a pharmaceutical composition
- a pharmaceutical composition comprising a nucleic acid sequence encoding an IgGI antibody as defined herein.
- Said nucleic acid can be utilized e.g. for gene therapy approaches in order to treat an epithelial tumor in a human, as set forth in more detail below.
- the invention further relates to a pharmaceutical composition
- a pharmaceutical composition comprising a vector which comprises a nucleic acid sequence as defined above.
- said vector further comprises a regulatory sequence which is operably linked to said nucleic acid sequence defined above.
- said vector is an expression vector. It is also envisaged that e.g. one expression vector encodes the heavy chains of said antibody, whereas another expression vector codes for the light chains.
- the vector of the present invention may also be a gene transfer or gene targeting vector. Gene therapy, which is based on introducing therapeutic genes or nucleic acids into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
- Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodua, Blood 91 (1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251 ; Verma, Nature 389 (1997), 239-242; Anderson, Nature 392 (Supp.
- the nucleic acid molecules and vectors as defined herein may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules as defined herein.
- the introduction and gene therapeutic approach should, preferably, lead to the expression of a functional IgGI antibody construct as defined herein, whereby said IgGI antibody construct is particularly useful in the treatment, amelioration and/or prevention of an epithelial tumor in a human.
- the invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a host transformed or transfected with a vector or a nucleic acid as defined above.
- the pharmaceutical composition further comprises suitable formulations of carriers, stabilizers and/or excipients.
- the invention in another aspect, relates to a process for the production of a pharmaceutical composition as defined above, said process comprising culturing a host as defined above under conditions allowing the expression of the IgGI antibody as defined hereinabove and recovering the produced IgGI antibody from the culture.
- a further aspect of the invention relates to a use of a IgGI antibody as defined hereinabove or as produced by the process as defined hereinabove, a nucleic acid molecule as defined hereinabove, a vector as defined hereinabove or a host as defined hereinabove for the preparation of a pharmaceutical composition for the prevention, treatment or amelioration of an epithelial tumor in a human.
- Another aspect of the invention relates to a method for the prevention, treatment or amelioration of an epithelial tumor in a human, said method comprising the step of administration of an effective amount of a pharmaceutical composition of the invention or as produced according by the process set forth above.
- the person skilled in the art in particular the attending physician can evaluate the successful treatment of the patient in need of administration of the bispecific molecule/bispecific single chain antibody of the invention. Accordingly, the administration scheme as well as the dosage and the administration time may be assessed by said person skilled in the art: A corresponding "amelioration" and/or "treatment" to be assessed is defined below.
- the most preferred mode of administration is an intravenous administration over a given time/time period. While the IgGI antibody as defined herein may be administered per alone, preferred is administration in a pharmaceutically acceptable carrier.
- suitable pharmaceutical carriers include phosphate buffered saline solutions, water, liposomes, various types of wetting agents, sterile solutions, etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors.
- dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
- Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, and suspensions.
- non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, aqueous solutions, or suspensions, including saline and buffered media.
- Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
- Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
- the composition might comprise proteinaceous carriers, like, e.g., serum albumine or immunoglobuline, preferably of human origin.
- the co-therapy might comprise, in addition to the proteinaceous IgGI antibody further biologically active agents, depending on the intended use of the pharmaceutical composition.
- agents might be agents acting on the gastro-intestinal system, agents acting as cytostatica, agents preventing hyperurikemia, agents inhibiting immune reactions (e.g. corticosteroids, FK506), drugs acting on the circulatory system and/or agents such as T-cell co-stimulatory molecules or cytokines known in the art.
- the IgGI antibody as defined herein is formulated in a buffer, a stabilizer and a surfactant.
- the buffer may be a phosphate, citrate, succinate or acetate buffer.
- the stabilizer may be (an) amino acid(s) and/or a sugar.
- the surfactants may be detergents, PEGs, or the like. More preferably, the IgGI antibody as defined herein is formulated in citrate, lysine, trehalose and Tween 80. As a diluent for said pharmaceutical composition, isotonic saline and Tween 80 is preferred.
- amelioration refers to an improvement or a moderation in the severity of a disease, i.e. an epithelial tumor.
- amelioration may be the achievement of a stable disease - or even more preferred - a shrinkage of the epithelial tumor(s), i.e. a minimal, partial response or complete response, due to the administration of the pharmaceutical compositions of the invention.
- Stable disease refers to a disease state in which no or no significant tumor progression/growth can be observed or detected by clinical and/or histological diagnostic methods. For example, a shrinkage of the tumor greater than 50% shrinkage of the sum of cross-sectional areas of index lesions may be considered as a "partial response".
- a “complete response” denotes a state in which no lesion(s) can be detected any more after treatment.
- a response with a tumor shrinkage between stable disease and partial response may be considered as a minimal response.
- a 20%, 25% or 30% shrinkage of the sum of cross-sectional areas of index lesions may be referred to as a minimal response.
- the term "amelioration” as used herein encompasses also a reduction of the number of epithelial tumors. It furthermore denotes the prevention/slowdown of tumor progression.
- an improvement of the overall survival of treated tumor patients in pomparison to non-treated tumor patients may be considered as an "amelioration" as used herein.
- the term “amelioration” can also refer to a reduction of the intensity of the symptoms of an epithelial tumor, resulting e.g. in an improvement of the quality of life of the treated tumor patients.
- prevention of an epithelial tumor is to be understood as follows: After surgical removal of the primary epithelial tumor(s) from a human patient and/or after chemotherapeutic or radiological treatment of the primary epithelial tumor(s), it may be the case that not all tumor cells could be eliminated from the body. However, these remaining tumor cells may give rise to recurrent cancer, i.e. local recurrence and/or metastases in the patient. Metastasis is a frequent complication of cancer, yet the process through which cancer cells disseminate from the primary tumor(s) to form distant colonies is poorly understood. Metastatic cancers are almost without exception uncurable raising the necessity for new therapeutic modalities.
- the pharmaceutical composition of the invention can be used to kill these disseminated tumor cells in order to prevent the formation of secondary tumors (originating from the tumor cells remaining in the body after primary therapy). In this way, the pharmaceutical composition helps to prevent the formation of local recurrence and/or metastases in tumor patients.
- the success of the anti-tumor therapy may be monitored by established standard methods for the respective disease entities, e.g. by computer-aided tomography, X- ray, nuclear magnetic resonance tomography (e.g. for National Cancer Institute- criteria based response assessment (Cheson (1999), J. Clin. Oncol.; 17(4):1244]), positron-emission tomography scanning, endoscopy, Fluorescence Activated Cell Sorting, aspiration of bone marrow, pleural or peritoneal fluid, tissue /histologies, and various epithelial tumor specific clinical chemistry parameters (e.g. soluble CEA concentration in serum) and other established standard methods may be used.
- computer-aided tomography e.g. for National Cancer Institute- criteria based response assessment (Cheson (1999), J. Clin. Oncol.; 17(4):1244]
- positron-emission tomography scanning e.g. for National Cancer Institute- criteria based response assessment (Cheson (1999), J.
- assays determining T cell activation may be used; see e.g. WO99/054440.
- Statistics for the determination of overall survival, progression-free survival or relapse-free survival of treated tumor patients in comparison to non-treated tumor patients may also be used.
- said epithelial tumor is a gastrointestinal adenocarcinoma, a breast adenocarcinoma or a lung adenocarcinoma.
- Said gastrointestinal adenocarcinoma is more preferably a colorectal, pancreatic, an oesophageal or a gastric adenocarcinoma.
- said pharmaceutical composition of the invention is for the treatment of progressive tumors, late stage tumors, tumor patients with high tumor load/burden, metastatic tumors, or tumor patients with a CEA serum concentration higher than 100 ng/ml.
- Said CEA serum concentration may be determined e.g. by ELISA.
- said pharmaceutical composition as defined hereinabove is suitable to be administered in combination with an additional drug, i.e. as part of a co-therapy.
- an active agent may be optionally included in the same pharmaceutical composition as the IgGI antibody, or may be included in a separate pharmaceutical composition.
- said separate pharmaceutical composition is suitable for administration prior to, simultaneously as or following administration of said pharmaceutical composition comprising the IgGI antibody.
- the additional drug or pharmaceutical composition may be a non-proteinaceous compound or a proteinaceous compound.
- said proteinaceous compound or non-proteinaceous compound may be administered simultaneously or non-simultaneously with a IgGI antibody as defined hereinabove, a nucleic acid molecule as defined hereinabove, a vector as defined as defined hereinabove, or a host as defined as defined hereinabove.
- said subject to be treated is a human.
- the invention relates to a kit comprising a IgGI antibody as defined hereinabove, a nucleic acid molecule as defined hereinabove, a vector as defined hereinabove, or a host as defined hereinabove.
- kit comprising a IgGI antibody as defined hereinabove, a nucleic acid molecule as defined hereinabove, a vector as defined hereinabove, or a host as defined hereinabove.
- Figure 1 Schematic representation of an IgG molecule comprising VH, CH1 , hinge, CH2, CH3, VL and CL regions. The CDRs of the VH and VL regions are indicated as black boxes.
- FIG. 2 Flow cytometric analysis of periplasmic preparations containing Flag- tagged scFv protein fragments specific for CEA from selected clones.
- Each of the scFv consists of the murine A5B7 VH region and a human VL region, as described in Example 3.
- Periplasmic preparations of soluble scFv protein fragments were added to 100,000 to 200,000 CEA-transfected CHO cells.
- For detection a monoclonal anti- Flag antibody was used followed by a PE-labeled polyclonal anti-mouse antibody. ScFvs binding to cells was measured by an increase in fluorescence intensity as compared to cells that were incubated with PBS alone.
- Fluorescence intensity is blotted on the X-axis, the number of events is blotted on the Y-axis.
- the negative control PBS and detection reagents
- the negative control is shown as filled curve, the respective scFvs are shown as grey lines. Shifting to the right indicates positive binding to the cells. All of the scFvs, i.e. A-121 , A-183, A-240, A-313, A-290, A-315, A4-35, A4-52 and MP2- A5, bind to membrane-bound CEA on CHO cells.
- FIG. 3 Flow cytometric analysis of periplasmic preparations containing Flag- tagged scFv protein fragments specific for CEA from selected clones. Each of these scFvs consists of a humanized VH region and the human VL region A240, as described in Example 4. Periplasmic preparations of soluble scFv protein fragments were added to 100,000 to 200,000 CEA-transfected CHO cells. Detection was performed by a monoclonal anti-Flag antibody followed by a PE-labeled polyclonal anti-mouse antibody. ScFvs binding to cells was measured by an increase in fluorescence intensity as compared to cells that were incubated with PBS alone.
- the humanized scFv constructs MP510_3-A5.3, MP510_3-B9.1 , and MP510_3-D8.1 bind to membrane-bound CEA on CHO cells.
- 240 Vlambda.3 is a scFv consisting of the murine A5B7 VH region and the human VL A-240 region. This construct shows also CEA-binding activity.
- Figure 4 ELISA analysis of purified human IgGI versions of the humanized antibodies A5, B9, D8 and the human IgGI versions of antibodies CEA I and CEA Il as well as antibody-containing culture supematants.
- An irrelevant human IgGI antibody was included as a negative control.
- Antibody binding was tested on immobilized CEA antigen - and for demonstration of CEA specificity also in the absence of coated CEA antigen.
- Culture supernatant, 10 ug/ml and 1 ug/ml antibody solutions were added to the +/- antigen coated wells blocked with BSA. Detection was performed by peroxidase labeled polyclonal human IgG antibody ( Jackson ImmunoResearch). The signals were measured after appropriate incubation with ABTS solution.
- FIG. 5 Flow cytometric analysis of purified CEA antibodies and the respective negative control (see Figure 4) using concentrations of 10 ug/ml of antibody.
- Antibody samples were added to 100,000 to 200,000 CEA positive Kato III cells (A), CEA-transfected CHO cells (B) and CEA-negative CHO cells (C). Detection was performed by a biotinylated polyclonal anti-human IgG antibody (DAKO) followed by PE-labeled Streptavidine (Jackson ImmunoResearch). Antibody binding to cells was measured by an increase in fluorescence intensity as compared to cells that were incubated with the respective isotype control.
- DAKO biotinylated polyclonal anti-human IgG antibody
- PE-labeled Streptavidine Jackson ImmunoResearch
- Fluorescence intensity is blotted on the X-axis, the number of events is blotted on the Y-axis.
- the negative control PBS and detection reagents
- the negative control is shown as filled curve, the respective antibodies are shown as grey lines. Shifting to the right indicates positive binding to the cells.
- Specific binding of the antibodies CEA I and CEA Il as well as the humanized antibodies IgGI A5, IgGI B9 and IgGI D8 to human CEA antigen on cells could be demonstrated in this experiment.
- FIG. 6 Cytotoxicity analysis (ADCC assay) of purified human IgGI versions of the humanized antibodies A5, B9, D8 and the human IgGI version of antibody CEA II.
- Target cells CEA transfected CHO cells
- CEA transfected CHO cells were labeled with chromium 51 and incubated with decreasing amounts of the respective antibodies in the presence of human PBMCs for 18 h and in the presence of two concentrations of soluble human CEA antigen (10 and 1 ug/ml, respectively).
- Two representative results are shown: CEA Il antibody in Figure 6A and IgGI A5 antibody in Figure 6B.
- FIG. 7 Cytolytic inhibition in the presence of soluble CEA antigen was converted into an "inhibitory factor". This factor is defined as the EC50 in the presence of 10 and 1 ug/ml soluble CEA in the ADCC assay divided by the EC50 in the absence of soluble CEA. It can clearly be shown that CEA Il has a dramatically decreased cytolytic activity in the presence of soluble CEA antigen, whereas IgGI A5-, IgGI B9- and IgGI D8-mediated cytolytic activity towards tumor cells is resistant to soluble CEA.
- EXAMPLE 1 Generation of CHO cells transfected with human CEA (carcinoembryonic antigen-related cell adhesion molecule 5; CEACAM5)
- Kato III cells human gastric carcinoma cell line; ATCC HTB-103
- CEA positive human gastric carcinoma cell line
- ATCC HTB-103 which are CEA positive were used to obtain the total RNA that was isolated according to the instructions of the kit manual (Qiagen, RNeasy Mini Kit).
- the obtained RNA was used for cDNA synthesis by random-primed reverse transcription.
- the following oligonucleotides were used: 5 ' CEACAM5 EcoRI GAATTCGCCACCATGGAGTCTCCCTCGGCCCC (SEQ ID NO. 35) and 3 ' CEACAM5 Sal I GTCGACCTATATCAGAGCAACCCC (SEQ ID NO. 36).
- a PCR (denaturation at 93 0 C for 5 min, annealing at 58°C for 1 min, elongation at 72 0 C for 1 min for the first cycle; denaturation at 93°C for 1 min, annealing at 58°C for 1 min, elongation at 72°C for 1 min for 30 cycles; terminal extension at 72°C for 5 min) was used to amplify the coding sequence.
- the PCR product was subsequently digested with EcoRI and Sail, ligated into the appropriately digested expression vector pEF-DHFR, and transformed into E.coli.
- the isolated plasmid DNA was sequenced and compared with the established nucleotide sequence of CEACAM5 (NM_004363 at the National Center for biotechnology information, http://www.ncbi.nlm.nih.gov/; SEQ ID NO. 37)
- the aforementioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (1989; 2001).
- the clone with the verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR deficient CHO cells was performed as described in Kaufmann (Kaufmann RJ. , Methods Enzymol.
- the binding of the antibody was detected with a R-Phycoerythrin-conjugated affinity purified F(ab')2 fragment, goat anti-mouse IgG, Fc-gamma fragment specific antibody, diluted 1 :100 in 50 ⁇ l PBS with 2% FCS (obtained from Dianova, Hamburg, Germany). Cells were analyzed by flow cytometry on a FACS-Calibur (Becton Dickinson, Heidelberg). FACS staining and measuring of the fluorescence intensity were performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-lnterscience, 2002). As a result, the transfectants demonstrated a clearly positive staining for the human CEA antigen.
- EXAMPLE 2 Generation and characterization of IgGI antibodies derived from murine monoclonal antibodies (mAb) A5B7 and T84.66
- the full IgG molecules have been derived from murine monoclonal antibodies A5B7 (Chester, Int. J. Cancer 57 (1994), 67-72; Harwood, loc. cit.) and T84.66 (Neumaier, Cancer Res. 50 (1990), 2128-34). The respective sequences were extracted from the literature and the corresponding V-regions were gene-synthesized at Entelechon, Germany. These synthesized DNA fragments were used as templates in the following PCR steps.
- the DNA fragment encoding the VL region of antibody CEA I was reamplified by PCR, resulting in Vkappa fragments with a Bsu36l-site at the 5'-end and a Xho l-site at the 3'-end.
- the primers 5'-VL CEA I Bsu36l ( ⁇ '-TTCTCTCCTTAGGTGTCCACTCCGACATTGAGCTCACC CAGTCTCC-3') (SEQ ID NO. 81) and 3'-VL CEA I Xho I (5'-CATGCACTCGAG CTTGGTCCCTCCACCGAACGTC-3') (SEQ ID NO. 82) were used for this purpose.
- CEA I From the murine VH regions of CEA I (A5B7) and CEA Il (T84.66, the variable region of the heavy chain was reamplified by PCR, generating Bsu36l restriction sites at both ends.
- CEA I the combination of the following two primers was used: 5'-primer 5'-CEA I VH-Bsu36l (5'-
- TTCTCTCCTTAGGTGTCCACTCCCAGGTCCAACTGCAGGAGTCAGG-S 5'-primer 5'-CEA Il VH-Bsu36l (5'- TTCTCTCCTTAGGTGTCCACTCCGAGGTTCAGCTGCAGCAGTCTGG-S') (SEQ ID NO. 89) and 3 ' -primer 3'-CEA Il VH-Bsu36l (5'-GACTCACCTGAGGAGACGGTGAC TGAGGTTCCTTGACC-3') (SEQ ID NO. 90).
- a plasmid encoding for one (murine) light chain and a plasmid encoding for one heavy chain (murine VH/human IgGI constant region) generated as set forth above were co-transfected into HEK cells according to standard protocols for transient protein expression and the cells were cultured to allow the expression and production of the immunoglobulins into the culture medium.
- hulgGI CEA I derived from antibody CEA I (A5B7) and hulgGI CEA Il derived from antibody CEA Il (T84.66) were produced.
- Said hulgGI antibody constructs consist of a murine variable (V) region derived from CEA I (A5B7) or CEA Il (T84.66), a murine constant (C) light chain (Ckappa) region and human constant heavy chain CH1 , CH2, CH3 and hinge regions (the human IgGI heavy chain constant region is described in Griffin, Cancer Immunol Immunother. 50 (2001): 141- 50).
- V murine variable
- C constant
- CH1 , CH2, CH3 and hinge regions the human IgGI heavy chain constant region is described in Kunststoff, Cancer Immunol Immunother. 50 (2001): 141- 50.
- ADCC Antibody dependent cellular cytotoxicity
- PBMCs peripheral blood mononuclear cells
- effector cells were isolated from healthy donors.
- the PBMCs were separated by Ficoll density gradient centrifugation with a subsequent 100 x g centrifugation step.
- Unstimulated PBMCs (5 x 10 5 cells) were added in a volume of 100 ⁇ l of RPMI 1640 medium with 10 % FCS to each well of a flat bottomed microtiter plate and incubated overnight at 37 0 C.
- target cells CEA-transfected CHO cells have been used.
- Target cells were labeled for 2 h with 51 Cr.
- Target cells (50.000 cells) and antibodies in different concentrations (10pg/ml -10 ⁇ g/ml) were added to the PBMCs and incubated for 18 h at 37 0 C. This assay has been carried out in the absence of soluble CEA (sCEA) antigen. Corresponding non-binding isotypes were used as negative controls.
- sCEA soluble CEA
- soluble CEA sCEA
- Soluble CEA (sCEA) antigen has been used in different concentrations, i.e. 1 ⁇ g/ml and 10 ⁇ g/ml.
- hulgd CEA Il derived from antibody CEA Il showed reduced cytotoxic (ADCC) activity.
- hulgd CEA I derived from antibody CEA I A5B7
- cytotoxic activity A5B7
- cytotoxicity against CEA positive tumor cells mediated by hulgGI CEA I derived from antibody CEA I is resistant to soluble CEA.
- cytotoxic activity directed against tumor cells of hulgd CEA I derived from antibody CEA I (A5B7) is resistant to even high concentrations of soluble CEA antigen.
- This finding is entirely unexpected in view of the fact that this IgGI antibody binds to soluble CEA antigen.
- this antibody was highly sensitive to soluble CEA antigen, ie. the cytotoxic activity (ADCC) has been blocked by soluble CEA antigen.
- This antibody has also been found to be capable of binding to soluble CEA.
- soluble CEA antigen prevents hulgGI CEA Il derived from antibody CEA Il (T84.66) from exerting its antibody-mediated cytotoxic activity.
- IgGI CEA I antibodies derived from antibody CEA I (A5B7) as defined herein are resistant to the presence of even high levels of soluble CEA in their cytotoxic activity towards tumor cells.
- humanized IgGI antibodies with resistance to soluble CEA antigen have been generated.
- human VL regions with resistance to soluble CEA have been isolated.
- the aim of this experiment is the selection of human VL regions which can pair with the maternal, murine VH of monoclonal antibody (mAb) A5B7.
- soluble CEA antigen was biotinylated. Biotinylation was accomplished in PBS containing 5% DMSO (Sigma) with a fifteenfold molar excess of EZ-Link Sulfo NHS-LC-LC-Biotin (Pierce) for 1 hour at room temperature in a sample mixer (Dynal). For the separation of free Biotin and biotinylated CEA antigen, the assay was excessively dialized against PBS according to standard protocols. The retained bioactivity of the biotin-labeled CEA was confirmed in ELISA binding experiments.
- PBMCs Peripheral blood mononuclear cells
- RNA was isolated from the isolated cells using the RNeasy® Midi Kit (QIAGEN) following the manufacturer's instructions.
- cDNA was synthesized according to standard methods (Sambrook, Cold Spring Harbor Laboratory Press 1989, 2001).
- VL-regions variable light chain regions
- RT-PCR was carried out using V- kappa- (5'-huVK1-Sacl-2001 (5'-GAGCCGCACG AGCCCGAGCT CCAGATGACC CAGTCTCC-3') (SEQ ID NO. 38), 5'-huVK2/4-Sacl-2001 (5'-GAGCCGCACG AGCCCGAGCT CGTGATGACY CAGTCTCC-3') (SEQ ID NO. 39), 5'-huVK3-Sacl- 2001 (5'-GAGCCGCACG AGCCCGAGCT CGTGWTGACR CAGTCTCC-3') (SEQ ID NO.
- V lambda 5 ' -huVL1a-Sacl-2001 (GAG CCG CAC GAG CCC GAG CTC GTG TTG ACG CAG CCG CCC TC) (SEQ ID NO. 47), 5 ' -huVL1 b-Sacl- 2001 (GAG CCG CAC GAG CCC GAG CTC GTG CTG ACT CAG CCA CCC TC) (SEQ ID NO. 48), 5 ' -huVL2-Sacl-2001 (GAG CCG CAG GAG CCC GAG CTC GCC CTG ACT CAG CCT SCC TCC GT) (SEQ ID NO.
- RNA from human B-cells was transcribed into cDNA (as described above) and used as template DNA in PCR reactions. Per PCR reaction, one 5'-primer was combined with one 3'-primer. The number of different PCR reactions was determined by the number of possible combinations of 5'- and 3'-primers.
- PCR-program was used for amplification: Denaturation at 94 0 C for 15 seconds, primer annealing at 52°C for 50 seconds and primer extension at 72°C for 90 seconds were performed over 40 cycles, followed by final extension at 72 0 C for 10 minutes. Light chain DNA V-fragments were then isolated according to standard protocols.
- a phage display library was generally constructed based on standard procedures, as for example disclosed in "Phage Display: A Laboratory Manual”; Ed. Barbas, Burton, Scott & Silverman; Cold Spring Harbor Laboratory Press, 2001.
- the primers chosen for PCR amplification gave rise to 5'-Sacl and 3'-Spel recognition sites for the light chain V-fragments.
- Four ligation reactions were set up, each consisting of 400 ng of light chain fragments (Sacl-Spel digested, 2 x kappa and 2 x lambda) and 1400 ng of the phagemid pComb3H5BHis (Sacl-Spel digested; large fragment; this vector is described in the thesis dissertation of Dr.
- the four resulting antibody V-light chain pools were then each transformed into 300 ⁇ L of electrocompetent Escherichia coli XL1 Blue by electroporation (2.5 kV, 0.2 cm gap cuvette, 25 mF, 200 Ohm, Biorad gene-pulser) resulting in library sizes of kappal : 2 x 10 8 kappa2: 6 x 10 7 lambdal : 9 x 10 7 Iambda2: 6 x 10 7 independent clones.
- Kappa (light chain) DNA-fragments from the different PCR amplifications were weighted for each ligation as follows: Each 5'-primer defines a specific group. Within these groups the 3'-primers define the subgroups. The kappa subgroups were weighted 1 :2:1 :1 corresponding to the primers 3'-hu-Vk-J1-Spel-BsiWI : 3'-hu-Vk- J2/4-Spel-BsiWI : 3'-hu-Vk-J3-Spel-BsiWI : 3'-hu-Vk-J5-Spel-BsiWI.
- the groups were weighted according to their germline distribution 1 :1 :1 :0.2:0.2 corresponding to the primers 5'-huVK1-Sac-2001 : 5'-huVK3-Sac-2001 : 5'-huVK2/4-Sac-2001 : 5'- huVK5-Sac-2001 : 5'-huVK6-Sac-2001.
- Lambda (light chain) DNA-fragments from the different PCR amplifications were weighted for each ligation as follows: Each 5'-primer defines a specific group. Within these groups the 3'-primers define the subgroups. The lambda subgroups were weighted 3:1 corresponding to the primers 3 ' -hu-Vlam-Blnl-Spel-2001 : 3 ' -hu-Vlam2- Blnl-Spel-2002.
- the groups were weighted according to their germline distribution 1 :1 :2:2:3 corresponding to the primers 5 ' -huVL1a-Sacl-2001 : 5 ' -huVL1 b-Sacl-2001 : 5 ' - huVL2-Sacl-2001 : 5 ' -huVL4-Sacl-2001 + 5 ' -huVL5-Sacl-2001 : 5 ' -huVL6-Sacl-2001 + 5 ' -huVL7/8-Sacl-2001 : 5 ' -huVL3/9-Sacl-2001.
- each transformed E.coli culture was incubated in SOC broth (Fluka) for phenotype expression.
- the two kappa cultures were combined as well as the two lambda cultures.
- the resulting kappa culture and the resulting lambda culture were then each incubated in 500 ml_ of SB selection medium containing 50 ⁇ g/mL carbenicillin and 2 % w/v glucose overnight.
- cells were harvested by centrifugation and plasmid preparation was carried out using a commercially available plasmid preparation kit (Qiagen).
- PCR was performed to amplify the maternal VH of mAb A5B7 from a vector containing said maternal VH.
- a PCR protocol according to standard procedures was followed using the 5'-primer 5'-AVH-Xhol (5'-GTC ACA CTC GAG TCA GGA GGA GGC TTG GTA C-3') (SEQ ID NO. 57) and the 3'-primer 3'-AVH- BstEII (5'-GTC ACA GGT GAC CGT GGT CCC TTG GCC CCA G-3' (SEQ ID NO. 58).
- the DNA fragment was cut with the restriction enzymes BstEII and Xhol.
- the phagemid pComb3H5BHis (this vector is described in the thesis dissertation of Dr. RaIf Lutterb ⁇ se) was digested accordingly and the large fragment was ligated with the above mentioned fragment.
- a single clone was cultivated in 100 mL SB medium (containing 50 ⁇ g/mL carbenicilline) and the plasmid was prepared according to standard protocols. The successful cloning was confirmed by sequencing the insert (Sequiserve, Kunststoff).
- This vector pComb3H5BHis/maternalVH of mAb A5B7 was restricted with the restriction enzymes Sacl and Spel.
- the large vector fragment was isolated.
- Plasmid- DNA containing the Vkappa- and the Vlambda library was restricted with the restriction enzymes Sacl and Spel.
- the small Vkappa - and the respective Vlambda fragment (each approximately 350 bp) were isolated according to standard protocols.
- 1200 ng of the vector fragment were ligated with a mix of each 200 ng of both the Vkappa and the Vlambda fragments.
- the ligation reaction was transformed into 300 ⁇ l_ of electrocompetent E. coli XL1 Blue by electroporation (2.5 kV, 0.2 cm gap cuvette, 25 mF, 200 Ohm) resulting in a total scFv library size of 1.2 x 10 8 independent clones.
- the antibody library was transferred into SB-Carbenicillin (50 ⁇ g/mL) selection medium.
- the antibody library was then infected with an infectious dose of 1 x 10 12 particles of helper phage VCSM 13 resulting in the production and secretion of filamentous M 13 phage, wherein each phage particle contained single stranded pComb3H5BHis-DNA encoding a half-human scFv-fragment and displayed the corresponding scFv-protein as a translational fusion to phage coat protein III.
- the phage particles carrying the scFv-repertoire were harvested from the culture supernatant by PEG8000/NaCI precipitation and centrifugation. Then approximately 1 x 10 11 to 1 x 10 12 scFv phage particles were resuspended in 0.5 ml_ of TBS/1 % BSA and incubated with biotinylated soluble CEA, that was immobilized in Streptavidin coated wells of an ELISA plate (Nunc) for 1 h.
- binding entities were eluted by using HCI-glycine, pH 2.2. Following neutralization with 2 M Tris, pH 12, the eluate was used for infection of a fresh uninfected E. coli XL1 Blue culture.
- E. coli XL1 blue culture (OD600 ⁇ 0.5) were added to the wells and incubated for 15 minutes. Both cultures were then mixed and cells successfully transduced with a phagemid copy, encoding a human scFv-fragment, were again selected for carbenicillin resistance and subsequently infected with VCMS13 helper phage to start the second round of antibody display and in vitro selection.
- Plasmid DNA corresponding to 4 rounds of panning was isolated from E. coli cultures.
- VH-VL-DNA fragments were excised from the plasmids (Xhol-Spel), and cloned via the same restriction sites in the plasmid pComb3H5BFIag/His, in which the expression construct (e.g. scFv) includes a Flag-tag (TGDYKDDDDK) (SEQ ID NO. 59) between the scFv and the His6-tag and the additional phage proteins are deleted.
- each pool (different rounds of panning) of plasmid DNA was transformed into 100 ⁇ l_ heat shock competent E.
- scFv production was induced by adding 20 ⁇ L LB carb 6 mM IPTG to each well.
- cells were lysed in a 1 h incubation at room temperature with 40 ⁇ L lysis buffer (400 mM boric acid, 320 mM NaCI, 4 mM EDTA pH 8, 2.5 mg/mL lysozyme). Residual cells and cell debris were separated by centrifugation for 12 minutes at 1 ,900 x g (Hettich). The supernatants containing scFv molecules were then tested for binding in flow cytometric binding assays.
- CHO cells transfected with human CEA were used as CEA-positive cell line.
- Cell binding assays were carried out by initially incubating between 100,000 and 200,000 cells with periplasmic preparation containing human scFv or relevant controls. After incubation the cells were washed in PBS/1 % FCS (fetal calf serum) and further incubated with 5-10 ⁇ g/ml of anti-FLAG M2 antibody (Sigma). After the cells had again been washed, they were incubated with polyclonal, PE-labeled anti-mouse antibodies (Dianova) and subsequently analyzed by flow cytometry. Approximately 600 clones were tested for binding signals on CEA-positive CHO cells. 27 positive clones were obtained.
- Figure 2 depicts binding of the nine different half-human scFv (i.e. murine A5B7 VH- human VL) constructs to the CEA-transformed CHO cell line as measured by flow cytometric analysis.
- Said Figure contains multiple diagrams, one for each construct tested. In any given diagram, the black distribution shows fluorescence intensity for cells incubated only with PBS alone in the absence of any construct but with all appropriate detection agents as used for detection of scFvs. In this way, any fluorescence shift observed can be definitely attributed to scFv construct rather than detection agents or buffer.
- Shifts in fluorescence which are indicative of construct binding to the respective cell line are depicted by a gray line in each diagram.
- a shift of higher magnitude away from, i.e. further to the (black) control indicates stronger binding
- a shift of lower magnitude away from, i.e. closer to the (black) control indicates weaker binding.
- the constructs A-121 , A-183, A-240, A-313, A-290, A-315, A4-35, A4-52, MP2-A5 show clearly discernable shifts in fluorescence intensity as compared to the respective control, indicative of binding of the scFvs to membrane-bound CEA on the CHO target cells.
- the human VL region of scFv A-240 has been selected and used for the isolation of a human VH region. Said human A-240 VL region is encompassed e.g. in SEQ ID NO. 2.
- EXAMPLE 4 Construction of the antibody libraries and phage display selection of humanized VH regions resistant to soluble CEA antigen
- the aim of the following experiments is the selection of a set of humanized VH regions resistant to soluble CEA antigen that pair with the human VL region of scFv A-240, selected as described in Example 3.
- Said human A-240 VL region is encompassed e.g. in SEQ ID NO. 2.
- PBMCs peripheric blood mononuclear cells
- PBMCs Peripheral blood mononuclear cells
- RNA was isolated from PBMCs using the RNeasy® Midi Kit (QIAGEN) following the manufacturer's instructions.
- cDNA was synthesized according to standard methods (Sambrook, Cold Spring Harbor Laboratory Press 1989, 2001). 2. PCR-Amplification of variable heavy chain regions (VH-reqions) The VH library was constructed and named Lib 134-VH.
- This VH-library consists of the human repertoire of FR1-CDR1-FR2-CDR2-FR3 from the PCR amplified VH- regions of the above described PBMC pool, linked operatively to the VH CDR3 of the maternal antibody followed by a human FR4 germline sequence.
- RT-PCR was carried out using a 5'- VH-specific primer set (5'-huVH1 ,3,5-Xhol-2001 (5'-AGG TGC AGC TGC TCG AGT CTG G-3 1 ) (SEQ ID NO. 60), 5'-huVH4-Xhol-2001 (5'-CAG GTG CAG CTG CTC GAG TCG GG-3') (SEQ ID NO. 61), 5'-huVH4B-Xhol-2001 (5'-CAG GTG CAG CTA CTC GAG TGG GG-3') (SEQ ID NO.
- PCR-program was used for amplification: Denaturation at 94°C for 15 seconds, primer annealing at 52°C for 50 seconds and primer extension at 72°C for 60 seconds was performed over 40 cycles, followed by final extension at 72°C for 10 minutes.
- the amplification products with a size of approximately 350 bp were isolated according to standard methods.
- RT-PCR was carried out in two steps.
- the human heavy chain VH-segments (FR1-CDR1-FR2-CDR2-FR3) were PCR- amplified from the isolated template VH fragments using the same 5'-VH-specific primer set as described above (5'-huVH1 ,3,5-Xhol-2001 , 5'-huVH4-Xhol-2001 , 5'- huVH4B-Xhol-2001 ; SEQ ID NOs.
- PCR reaction Per PCR reaction, one 5'-primer was combined with the 3'-primer; the number of different PCR reactions was determined by the number of possible combinations of 5'- and the 3'-primer.
- the following PCR-program was used for amplification: Denaturation at 94 0 C for 15 seconds, primer annealing at 52 0 C for 50 seconds and primer extension at 72°C for 90 seconds was performed over 40 cycles, followed by final extension at 72°C for 10 minutes.
- the human VH segments are prolonged for a part of the maternal VH CDR3, which then in turn is the priming site for the second step PCR 3'-primer.
- VH-(FR1-CDR1-FR2-CDR2-FR3) DNA-fragments were then used as templates in a second PCR reaction using again the respective 5'VH-specific primer and a universal 3' primer matching to the universal 3'-terminus of the amplified DNA- fragments (3' A134-JH6-BstEII, 5 1 - CGA GAC GGT GAC CGT GGT CCC TTG GCC CCA GTA GTC AAA GTA GAA CCG TAG CC -3') (SEQ ID NO. 70).
- the following PCR-program was used for amplification:
- a human VL of scFv A-240 identified in the first, previous selection was chosen, and subsequently combined with the library of human VH fragments with the aim of generating a human scFv.
- a phage display library was generally constructed based on standard procedures, as for example disclosed in "Phage Display: A Laboratory Manual”; Ed. Barbas, Burton, Scott & Silverman; Cold Spring Harbor Laboratory Press, 2001.
- One ligation reaction was set up consisting of 400 ng of human Lib 134-VH fragment pool (Xhol-BstEII digested) and 1200 ng of the plasmid pComb3H5BHis/A-240 VL (the DNA encoding the VL region of scFv A-240 was cloned via the restriction sites Sacl and Spel into pComb3H5BHis according to standard procedures).
- the resulting antibody human VH pool was then transformed into 300 ⁇ L of electrocompetent Escherichia coli XL1 Blue by electroporation (2.5 kV, 0.2 cm gap cuvette, 25 mF, 200 Ohm, Biorad gene-pulser) resulting in a library size of 0.8 x 10 8 independent clones in total. After electroporation the assay was incubated in SOC broth (Fluka) for phenotype expression. The cultures were then each incubated in 500 mL of SB selection medium containing 50 ⁇ g/mL carbenicillin and 2 % v/v glucose overnight.
- the antibody library was then infected with an infectious dose of 1 x 10 12 particles of helper phage VCSM 13 resulting in the production and secretion of filamentous M 13 phage, wherein each phage particle contained single stranded pComb3H5BHis-DNA encoding a human scFv-fragment and displayed the corresponding scFv-protein as a translational fusion to phage coat protein III. 4. Phage display selection of a human VH
- the phage particles carrying the human scFv-repertoire were harvested from the culture supernatant by PEG8000/NaCI precipitation and centrifugation. Then approximately 1 x 10 11 to 1 x 10 12 scFv phage particles were resuspended in 0.5 ml_ of TBS/1 % BSA and incubated with biotinylated soluble CEA, that was immobilized in Streptavidin coated wells of an ELISA plate (Nunc) for 1 h.
- E. coli XL1 blue culture (OD600 > 0.5) were added to the wells and incubated for 15 minutes. Both cultures were then mixed and cells successfully transduced with a phagemid copy, encoding a human scFv-fragment, were again selected for carbenicillin resistance and subsequently infected with VCMS 13 helper phage to start the second round of antibody display and in vitro selection.
- Plasmid DNA corresponding to 4 rounds of panning was isolated from E. coli cultures.
- VH-VL-DNA fragments were excised from the plasmids (Xhol-Spel), and cloned via the same restriction sites in the plasmid pComb3H5BFIag/His, in which the expression construct (e.g. scFv) includes a Flag-tag (TGDYKDDDDK; SEQ ID NO. 59) between the scFv and the His6-tag and the additional phage proteins are deleted.
- each pool (different rounds of panning) of plasmid DNA was transformed into 100 ⁇ L heat shock competent E.
- scFv production was induced by adding 20 ⁇ l_ LB carb 6 mM IPTG to each well.
- cells were lysed in a 1h incubation at room temperature with 40 ⁇ L lysis buffer (400 mM boric acid, 320 mM NaCI, 4 mM EDTA pH 8, 2.5 mg/mL lysozyme). Residual cells and cell debris were separated by centrifugation for 12 minutes at 1 ,900 x g (Hettich). The supematants containing scFv molecules were then tested for binding in flow cytometric binding assays.
- CHO cells transfected with human CEA were used as CEA-positive cell line.
- Cell binding assays were carried out by initially incubating between 100,000 and 200,000 cells with periplasmic preparation containing human scFv or relevant controls. After incubation the cells were washed in PBS/1 % FCS (fetal calf serum) and further incubated with 5-10 ⁇ g/ml of anti-FLAG M2 antibody. After the cells had again been washed, they were incubated with polyclonal, PE-labeled anti-mouse antibodies (Dianova) and subsequently analyzed by flow cytometry. 46 clones were tested for binding signals on CEA-positive CHO cells. All of them showed positive signals.
- the humanized constructs MP510_3-A5.3 (MP510-A5; SEQ ID NO. 2), MP510_3-B9.1 (MP511-B9; SEQ ID NO. 4), MP510_3-D8.1 (MP511-D8; SEQ ID NO. 6) have been selected for further characterization.
- the humanized VH region in these constructs contain the amino acid sequence "DRGLRFYFDY" (SEQ ID NO. 27) corresponding to Kabat positions 95 - 102 of the CDR-H3 of murine monoclonal antibody A5B7.
- the corresponding amino acid sequences of the scFvs are shown in Table 1.
- Periplasmic extracts of said humanized constructs MP510-A5, MP511-B9, MP511- D8 as well as the half human construct A-240 Vlambda.3 were further analyzed in flow cytometric experiments with CEA-positive and -negative cell lines. It can be seen from Figure 3, that the humanized constructs MP510-A5 (SEQ ID NO. 2), MP511-B9 (SEQ ID NO. 4), MP511-D8 (SEQ ID NO. 6) show clearly discernable shifts in fluorescence intensity as compared to the respective half-human control A-240 Vlambda.3 (murine VH A5B7/human VL A240).
- the human scFv constructs show stronger binding activity to membrane-bound human CEA than the half human construct A-240 Vlambda.3.
- all of the human constructs showed distinct binding to CEA-positive human KATO III cells (human gastric cancer cell line), whereas none of them showed binding to CEA- negative, untransfected CHO cells as well as to CEA-negative human NALM 6 cells (human B cell line) (data not shown).
- EXAMPLE 5 Generation and characterization of humanized IgGI antibodies with resistance to soluble CEA antigen
- VL region of scFv A-240 and different VH regions of scFv molecules selected in Example 4 were subcloned into mammalian expression vectors.
- the human VL of clone A-240 contained a Bsu36l restriction site in its nucleotide sequence. Therefore a variant was generated (A-240delBsu) using standard protocols that had a nucleotide exchange in the restriction motif that did not result in an amino acid exchange.
- VL A-240 For cloning of the VL A-240 into a suitable mammalian expression vector, suitable terminal restriction sites had to be inserted. Therefore the DNA fragment encoding the VL region of scFv A240 was reamplified by PCR using the primers 5'-A240- Bsu36l (TTCTCTCCTTAGGTGTCCACTCC CAG GCC GTG CTG ACT CAG CCG GC) (SEQ ID NO. 93) and 3'-A240-overlap (GCCTTGGGCTGACCTAGGACGGTC AACTTGGTCC) (SEQ ID NO. 94).
- 5'-A240- Bsu36l TTCTCTCCTTAGGTGTCCACTCC CAG GCC GTG CTG ACT CAG CCG GC
- 3'-A240-overlap GCCTTGGGCTGACCTAGGACGGTC AACTTGGTCC
- a human lambda constant region was amplified from a human cDNA pool using the 5'-primer 5'-Clam-overlap (GTTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCG) (SEQ ID NO. 95) and the 3'-primer 3'-Clam-Notl (GACGTA GCGGCCGC GTCGAC CTATGAACATTCTGTAGGGGC) (SEQ ID NO. 96) according to standard methods.
- the approximately 330 bp DNA products of both PCRs had an identical 3' (A-240) or 5' (C lambda) overlap in sequence.
- the two fragments were used in a fusion PCR (according to standard protocols) in combination with the 5'- primer 5'-A240-Bsu36l and the 3'-primer 3'-Clam-Notl to generate a full length light chain product coding for the human VL A-240 fused to a human lambda constant region (corresponding amino acid sequence shown in SEQ ID NO: 80).
- This DNA-fragment contained a Bsu36l-site at the 5'-end and a Sail restriction site at the 3'-terminus followed by a Notl restriction site.
- This fragment was then subcloned into the pBS-derived plasmid (as described above) by Bsu36l and Not I thus adding a human leader sequence.
- A-240 VL-Clambda DNA was excised from said construct and subcloned into the eukaryotic expression vector pEF-ADA derived from the expression vector pEF-DHFR (Mack et al. (1995) Proc. Natl. Acad. Sci. USA. 92, 7021-5) by replacing the cDNA encoding murine dihydrofolate reductase (DHFR) with that encoding murine adenosine deaminase (ADA).
- DHFR murine dihydrofolate reductase
- ADA murine adenosine deaminase
- variable region was reamplified by PCR, generating Bsu36l restriction sites at both ends.
- primers 5'-primer 5'-CVH-Bsu36l (5'-
- amino acid sequence of the A5 heavy chain is shown in SEQ ID NO. 77
- amino acid sequence of the B9 heavy chain is shown in SEQ ID NO. 78
- amino acid sequence of the D8 heavy chain is shown in SEQ ID NO. 79. 3. Expression of humanized full IgG proteins
- Plasmid encoding for one light chain and plasmid encoding for one heavy chain were cotransfected into HEK cells according to standard protocols for transient protein expression and the cells were cultured to allow the expression and production of the immunoglobulins into the culture medium.
- IgGI A5 derived from scFv A5 IgGI D8 derived from scFv D8 and IgGI B9 derived from scFv B9 were produced.
- the supernatants were harvested and the humanized immunoglobulins were isolated via Protein A chromatography according to standard protocols for the purification of immunoglobulins. Culture supernatants, as well as purified immunoglobulins were then used for further characterization experiments.
- the amino acid sequence of the A5 heavy chain is shown in SEQ ID NO. 77
- the amino acid sequence of the B9 heavy chain is shown in SEQ ID NO. 78
- the amino acid sequence of the D8 heavy chain is shown in SEQ ID NO. 79.
- the amino acid sequence of the A240 light chain is shown in SEQ ID NO. 80.
- IgGI A5, derived from scFv A5, comprises the A5 heavy chain shown in SEQ ID NO. 77 and the amino acid sequence of the A240 light chain shown in SEQ ID NO. 80.
- IgGI B9, derived from scFv B9 comprises the amino acid sequence of the B9 heavy chain shown in SEQ ID NO. 78 and the amino acid sequence of the A240 light chain shown in SEQ ID NO. 80.
- IgGI D8, derived from scFv D8, comprises the amino acid sequence of the D8 heavy chain shown in SEQ ID NO. 79 and the amino acid sequence of the A240 light chain shown in SEQ ID NO. 80.
- the (humanized) VH region in these IgGI antibody constructs contains the amino acid sequence "DRGLRFYFDY” (SEQ ID NO. 27) corresponding to Kabat positions 95 - 102 of the CDR-H3 of murine monoclonal antibody A5B7.
- the corresponding amino acid sequences of the above mentioned heavy and light chains are shown in Table 1.
- the humanized antibodies IgGI A5, IgGI D8 and IgGI B9 as well as the hu IgGI -CEA I and -CEA Il constructs of Example 2 showed distinct binding to the immobilized soluble CEA (sCEA) antigen as compared to the negative control - and no binding in an equivalent setting with the difference that no sCEA antigen was coated.
- sCEA immobilized soluble CEA
- the humanized IgGI A5, IgGI D8 and IgGI B9 constructs as well as the hulgG-CEA I and -CEA Il constructs of Example 2 showed distinct binding to the CEA positive cells ( Figures 5A, B and C). None of the antibodies showed binding to untransfected CHO cells. IgG-controls were negative on Kato-cells, CHO/CEA-cells and untransfected CHO cells.
- ADCC Antibody dependent cell-mediated cytotoxicity
- PBMCs peripheral blood mononuclear cells
- effector cells were isolated from a healthy donors.
- the PBMCs were separated by Ficoll density gradient centrifugation with a subsequent 100 x g centrifugation step.
- Unstimulated PBMCs (5 x 10 5 cells) were added in a volume of 100 ⁇ l of RPMI 1640 medium with 10 % FCS to each well of a flat bottomed microtiter plate and incubated overnight at 37 °C.
- target cells the CEA-positive gastric cancer cell line KATO III has been used.
- Target cells (50.000 cells) were labelled for 2 h with 51 Cr.
- the humanized anti-CEA antibodies IgGI A5, IgGI D8 and IgGI B9 proved to mediate cytotoxicity to the CEA positive gastric cancer cell line KATO III as compared to the negative control.
- the same result has been observed for the IgG versions of the antibodies CEA I and CEA Il (hulgG1-CEA I and -CEA Il constructs) as set forth in Example 2.
- the antibody samples were preincubated with soluble CEA (sCEA) antigen for 20 min under agitation and then mixed with the labeled target cells and the human PBMCs. Otherwise, the assay has been performed as described above. Soluble CEA (sCEA) antigen has been used in different concentrations, i.e. 1 ⁇ g/ml and 10 ⁇ g/ml.The assay was measured and the respective cytotoxic values plotted.
- EC50 values were determined by the analysis software. The EC 50 values are depicted in Table 2.
- inhibitory factor Cytolytic inhibition in the presence of soluble CEA antigen was converted into an "inhibitory factor". This factor is defined as the EC50 in the presence of 10 and 1 ug/ml soluble CEA in the ADCC assay divided by the EC50 in absence of soluble CEA.
- the inhibitory factors are depicted in Table 3.
- FIG. 7 A graphic presentation of the inhibitory factors is illustrated in Figure 7.
- This Figure shows clearly, that CEA Il has a dramatically decreased cytolytic activity in the presence of soluble CEA antigen at 1 ug/ml, showing a more than 37 times reduced cytolytic activity in the presence of soluble CEA antigen, whereas IgGI A5- and IgGI D8-mediated cytolytic activity towards tumor cells is completely resistant to soluble CEA.
- IgGI B9-mediated cytolytic activity towards tumor cells is only slightly affected by sCEA.
- Said antibody constructs show inhibitory factors below 10. This effect is even more pronounced at the 10 ug/ml concentration of soluble CEA in the ADCC assay.
- the cytolytic curve of antibody CEA Il is reduced to baseline (no cytolytic activity even at the highest concentrations could be detected). Therefore the inhibitory factor for antibody CEA Il was estimated to be 74 at it's best, probably being far underestimated.
- hulgG-CEA Il derived from mAb T84.66
- the humanized CEA antibodies IgGI A5, IgGI D8 and IgGI B9 showed no significant decrease of cytolytic activity as compared to the activity in the absence of soluble CEA.
- IgGI A5-, IgGI B9- and IgGI D8-mediated cytolytic activity towards tumor cells is resistant to soluble CEA.
- the amino acid sequence "DRGLRFYFDY" SEQ ID NO.
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AU2006328882A AU2006328882B2 (en) | 2005-12-21 | 2006-12-21 | Pharmaceutical antibody compositions with resistance to soluble CEA |
JP2008546269A JP5463036B2 (en) | 2005-12-21 | 2006-12-21 | Pharmaceutical antibody composition having resistance to soluble CEA |
NZ569144A NZ569144A (en) | 2005-12-21 | 2006-12-21 | Pharmaceutical antibody compositions with resistance to soluble CEA - comprising IgG1antibodies that bind to CEA |
CA2633756A CA2633756C (en) | 2005-12-21 | 2006-12-21 | Pharmaceutical antibody compositions with resistance to soluble cea |
US12/095,952 US20090226444A1 (en) | 2005-12-21 | 2006-12-21 | Pharmaceutical antibody compositions with resistance to soluble cea |
EP06841100.8A EP1976885B1 (en) | 2005-12-21 | 2006-12-21 | Pharmaceutical antibody compositions with resistance to soluble cea |
US13/324,823 US8901278B2 (en) | 2005-12-21 | 2011-12-13 | Pharmaceutical antibody compositions with resistance to soluble CEA |
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US20120121600A1 (en) | 2012-05-17 |
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