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WO1996022310A1 - Anticorps utilises dans le diagnostic et la therapie du cancer - Google Patents

Anticorps utilises dans le diagnostic et la therapie du cancer Download PDF

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Publication number
WO1996022310A1
WO1996022310A1 PCT/SE1996/000029 SE9600029W WO9622310A1 WO 1996022310 A1 WO1996022310 A1 WO 1996022310A1 SE 9600029 W SE9600029 W SE 9600029W WO 9622310 A1 WO9622310 A1 WO 9622310A1
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Prior art keywords
antibody
cells
antibodies
cancer
binding
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Roland Carlsson
Bo Jansson
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Bioinvent International AB
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Bioinvent International AB
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Priority to AU44991/96A priority Critical patent/AU4499196A/en
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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
    • C07K16/3046—Stomach, Intestines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • the present invention relates to the area of cancer therapy and diagnosis, and in particular to antibodies for use in cancer therapy and diagnosis.
  • Monoclonal antibodies have a great potential in cancer therapy as they can bind to tumour antigens with great selectivity and guide cytotoxic activities to the cancerous cells.
  • cytotoxic compounds such as radioisotopes, drugs or toxins
  • naked antibodies can be used utilising natural defence mechanisms such as antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) against the tumour cells (Mujoo et al.1987).
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • the degree of success in therapies using the antibodies depends on many factors, but an important factor is the restricted expression of the antigen recognised by the antibody to the tumour cells.
  • tumour associated antigens are expressed both on normal as well as tumour cells.
  • tumour associated antigens are expressed both on normal as well as tumour cells.
  • the ability of any given monoclonal antibody to discriminate between tumour and normal tissue can to a large extent be determined by in vitro tests or animal model tests.
  • the present invention provides antibodies having substantially the same binding specificity as monoclonal antibody PA1-3F10 produced by the hybridoma cell line deposited at BCCM LMBP Culture Collection, Belgium, the 4 January 1995, under reference [LMBP1322CB].
  • the present invention provides antibodies as obtainable from the hybridoma cell line deposited at BCCM under reference [LMBP1322CB].
  • the present invention provides a mutant, derivative, functional equivalent or fragment of the antibody PA1-3F10 or an antibody having substantially the same binding specificity as the PA1-3F10 antibody.
  • modified antibodies are discussed at greater length below.
  • the antibodies according to the present invention are closer defined by their aminoacid sequences and their corresponding DNA-sequences encoding the complementary determining regions (CDR's) or alternatively by their aminoacid sequences and their corresponding DNA sequences encoding the variable heavy and light chains.
  • the present invention provides the hybridoma cell line deposited at BCCM under reference [LMBP1322CB].
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody as described above, in combination with a pharmaceutically acceptable carrier.
  • the present invention provides an antibody as described above for use in methods of medical treatment, prophylaxis or diagnosis.
  • the present invention relates to cancer therapy and diagnosis, and more especially the therapy or diagnosis of colorectal cancer.
  • the present invention provides a method of detecting cancer cells using an antibody described above.
  • One method of detecting cancer cells is to label antibodies as described above, expose them to the cells to be tested and detecting the binding of the antibody to the cells using the label.
  • the present invention relates to isolated DNA encoding any of the antibodies above.
  • the invention also includes expression vectors comprising DNA encoding the above antibodies, the DNA being operably linked to control sequences and host cells transformed by the expression vectors.
  • these targeting therapies are well known in the art and include so called ADEPT or VDEPT techniques in which an active agent is administered as a precursor, for conversion to an active form by an activating agent targeted to or produced in the cells to be treated.
  • the activating agent can be targeted to the cells by conjugation to antibodies of the present invention.
  • the antigen to which the PA1-3F10 antibody binds has not yet been fully identified but is most probably a membrane integral glycoprotein.
  • the antigen is not shed from the tumour cells since it can not be found in patients blood or in spent culture medium from antigen expressing cells.
  • the unique binding profile of the antibody also indicates that it has not been described earlier.
  • PA1-3F10 Compared to an array of other monoclonal antibodies PA1-3F10 demonstrates a more selective binding to tumour cells. In particular, tumours of the colon are intensely stained.
  • the antibody also mediates ADCC in vitro towards tumour cells expressing the antigen despite it being of murine IgGl isotype which generally mediates this effect poorly.
  • the antibody has also been demonstrated to target to human tumours transplanted into immunodeficient mice and to cause retardation of tumour outgrowth in such model systems.
  • the PA1-3F10 monoclonal antibody thus, shows unique features in binding activity and selectivity of human tumours, useful for the diagnosis and therapy of human tumours, such as lung cancer, ovary cancer or breast cancer and in particular colorectal cancer.
  • the PA1-3F10 antibody of the invention has a number of in vivo therapeutic applications.
  • the antibody may be used in conjunction with an appropriate therapeutic agent to treat human cancer.
  • the antibody may be linked to a therapeutic drug, such as cytostatica or a cytokine a radioisotope, for example 99 Tc or 125 I or toxin, such as abrin or ricin for delivery of the therapeutic or toxic agent to the tumour cells according to well-known techniques.
  • the PA1-3F10 may be administered in vivo or ex vivo alone or in combination with one or more other antibodies.
  • compositions for use in the treatment or prophylaxis of human tumours include compositions comprising a therapeutically effective amount of PA1-3F10, either unmodified or a mutant, derivative, functional equivalent or fragment thereof, it may be conjugated or a part of an antibody cocktail for the purpose of treating cancer.
  • the antibody composition of the invention can be administered using conventional modes including intravenous, intraperitoneal, oral, intralymphatic or directly into the tumour. Preferably the antibody is administered parenterally.
  • the dosage forms may include Uquid solutions, or suspensions, tablets, microcapsules, liposomes and injectable solutions.
  • the preferred form is dependent upon the mode of administration and the therapeutic application.
  • the pharmaceutical compositions preferably include conventional pharmaceutically acceptable carriers and adjuvants known in the art such as human serum albumin, ion exchangers, alumina, lecithin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate and salts of electrolytes.
  • the dosage of the compositions should be adjusted to the individual patient, but the effective dose of the antibody composition of this invention may be in the range of from 1 to 50 mg per kg body weight.
  • novel antigen of the present invention i.e. the antigen reactive with PA1- 3F10 may also be used for therapeutic applications such as in vaccination.
  • the antibodies described above can be altered in a variety of ways using recombinant DNA technology coupled with advances in the field of monoclonal antibody and protein engineering. This has enabled access to a large selection of antibodies and antibody fragments with different properties and structures to natural antibodies.
  • Monoclonal antibodies can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or EP-A- 239400.
  • a hybridoma producing a monoclonal antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • an antibody specific for a protein may be obtained from a recombinandy produced library of expressed immunoglobulin variable domains, eg using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • the library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with the target, or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest (or a fragment thereof).
  • Antibodies may be modified in a number of ways. Indeed the term “antibody” should be construed as covering any specific binding substance having a binding domain with the required specificity. Thus tins term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimaeric antibodies are described in EP-A-0120694 and EP-A-0125023.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S.
  • VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993).
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (eg by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol.
  • bispecific antibody fragments eg prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al, Embo Journal, 10, 3655-3659, (1991).
  • Bispecific diabodies as opposed to bispecific whole antibodies, are also particularly useful because they can be readily constructed and expressed in E.coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • bispecific antibodies incorporating a specificity for the T-cell co-receptor CD3 have been shown to inhibit tumour growth (Titus, J. A. et al., J. Immunol.
  • humanised antibodies comprise framework regions derived from human immunoglobulins (acceptor antibody) in which residues from one or more complementary determining regions (CDR's) are replaced by residues from CDR's of a non-human species (donor antibody) such as mouse, rat or rabbit antibody having the desired properties, eg specificity, affinity or capacity.
  • CDR's complementary determining regions
  • donor antibody such as mouse, rat or rabbit antibody having the desired properties, eg specificity, affinity or capacity.
  • Humanised antibodies may be prepared according to well known techniques in the art and are illustrated herein by the references Jones P.T., Dear P.H., Foote J., Neuberger M.S and Winter G. "Preparing the complementarity-determining regions in a human antibody with those from a mouse", Nature, 321: 522-525 (1986) and Verhoeyen M.E., Saunders J.A.,
  • Figure 1 shows the cloning steps of the PA1-3F10 antibody leading to the master working cell bank
  • Figure 2 is a copy of an outprint from a computer coupled to the FACS machine and shows the staining of the colon carcinoma cell line COLO 205 with a) PBS-BSA, b) PA1-3F10 antibody and the teratoma cell line PA-1 incubated with c)
  • FIG 3 shows ADCC activity of PA1-3F10 on chromium labelled antigen expressing leukaemia (MOLT-4) cells and antigen negative melanoma cells (M24-MET);
  • Figure 4 shows a Lindmo plot of the binding of labelled PA1-3F10 antibody in antigen excess
  • Figure 5 shows the binding of PA1-3F10 analysed on MOLT-4 cells
  • Figure 6 a and b shows the uptake of radiolabelled PA1-3F10 in different tissue types
  • Figure 7 shows the concentration of radiolabelled PA1-3F10 in sera
  • Figure 8 shows the effect of PA1-3F10 antibody on the outgrowth of COLO 205 cells transplanted to SCID mice;
  • Figure 9 shows the rapid intemalization in MOLT-4 cells of the radiolabelled antibody PA1-3F10;
  • Figure 10 shows the DNA sequence and the corresponding amino acid sequence of the variable light chain (VL) of PA1-3F10;
  • FIG 11 shows the DNA sequence and the corresponding amino acid sequence of the variable heavy chain (VH) of PA1-3F10.
  • Immune spleenocytes were obtained from Balb/c mice (Charles River UK. Ltd, Margate Kent). The mice which were negative for a number of pathogenic virus and bacteria were inoculated intraperitoneally (i.p.) with human tumour cell lines in accordance with the following schedule.
  • PBS 8 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ,137 mM NaCl, 3mM KCl, pH 7.3
  • T-47D (ATCC HTB 133) is a breast carcinoma cell line isolated from a pleural effusion obtained from a patient with an infiltrating ductal carcinoma of the breast (Sher et al., 1981).
  • PANC-1 (ATCC CRL 1469) is an epitheloid carcinoma cell line established from a patient with pancreatic cancer (Lieber et al., 1975). The cell lines were both originally from the American Type Culture collection (ATCC, Rockville, MD).
  • the cell lines were cultivated without addition of antibiotics in RPMI 1640 medium (Gibco, No 041-08170) with addition of L-glutamine (Gibco, No 043-05030), "non essential amino acids” (Gibco, No 043-01140) and foetal calf serum (Gibco, No 011-06290).
  • the cells were regularly checked for contaminating mycoplasma with the fluorescent dye Hoechst 33258 (Chen, 1972), an ultramicrochemical test (Uitendaal et al., 1978) and with a filter hybridization technique using 32 P labelled restriction fragment H900 as a radioactive DNA-probe specific for mycoplasma (SVA) and were always found negative.
  • the cells were kept in serial cultures and removed from the tissue culture flasks using a trypsin EDTA-solution (Gibco No 45300-019). The cells were washed in PBS before being inoculated into the mice for immunisation.
  • HAT sensitive SP2/0-Ag-14 myeloma cells using polyethyleneglycol 1540 (Polyscience Inc. PA).
  • the fusion mixture was distributed into Costar 96-well tissue culture plates at a cell density of 129 ⁇ 10 5 /200 ⁇ l/well.
  • Hybridomas were selected in DMEM-HAT medium (Gibco, No 041-01965 and No 043-01060) supplied with HECS (Human Endothelial Cell Supernatant, Costar, No C6110) used according to the manufacturers recommendation and with 10% foetal calf serum (Gibco, No 011-6290).
  • the wells were supplied with fresh medium each week until supernatants from wells with growing hybrids were screened for binding activity against tumour cell lines utilising cell-ELISA techniques (see below).
  • Six different wells were classified as positive and were then cloned through limiting dilution.
  • One of the wells was cloned four times seeding 2 cells/well, for the two first cloning steps and 0.4 and 0.5 cells/well for the two last cloning steps respectively as illustrated in figure 1.
  • the various steps were characterised by the following:
  • the PA1-3F10-1C1-3B1-4E2-2A5 monoclonal hybridoma cell line produces a monoclonal antibody called 3F10 or more extensively PA1-3F10, which is a murine IgGl kappa antibody as determined by immunodif fusion and ELISA assays using isotype-specific (ICN Biomedicals Inc., USA) and light chain specific (Southern Biotechnology Associated Inc., USA) antibodies respectively.
  • the hybridoma was found to produce 11.5 ⁇ g antibody/ml/10 6 cells/24h under exponential growth in tissue culture flasks.
  • cDNA sequence of PA1-3F10 m-RNA was prepared from the PA1-3F10 hybridoma and isolated with oligo-dT beads and turned into cDNA by reversed transcriptase. The material was amplified using PCR. The PCR-products were purified and cloned into pCRTMII. Colonies were tested for inserts using PCR. Positive clones were propagated and plasmid DNA was isolated and DNA sequencing was performed according to Sanger.
  • FR 1: 56-124, CDR 1: 125-175, FR 2: 176-220, CDR 2: 221-241, FR 3: 242-337, CDR 3: 338-364, FR 4: 365-394 of PA1-3F10 VL are shown in figure 10 and FR 1: 33-122, CDR 1: 123-137, FR 2: 138-179, CDR 2: 180-230, FR 3: 231-326, CDR 3: 327-362, FR 4:
  • RNA preparation 200 000 hybridoma cells were pelleted for 5 minutes at 6500 rpm. The cells were washed once in PBS (137mM NaCl, 2.7mM KCl, 4.3mM Na 2 HPO 4 x7H 2 O, 1.4mM KH 2 PO 4 pH 7.4.), pelleted again and resuspended in 100 ⁇ l lysis buffer (10mM Tris-HCl, 0.14M NaCl, 5mM KCl, 1 % Triton X- 100 pH 7.4 and RNAsin (PROMEGATM) was added to a final concentration of 100 U/ml prior to use. The lysed cells were put on ice for 1 minute and centrifugated at 6500 rpm for
  • the supernatant was transferred to an eppendorf tube containing 100 ⁇ l of binding buffer (20mM Tris-HCl, 2mM EDTA, 1.0 LiCl pH 7.5) and 80 ⁇ g oligo-dT beads (DYNALTM). Hybridization took place for 5 minutes and the RNA beads were fixed using a magnet. The sample was washed twice with 200 ⁇ l washing buffer (lOmM Tris-HCl, ImM EDTA, 0.15M LiCl, pH 7.5),
  • RNA precipitate was centrifugated at 12500 rpm for 20 minutes, washed once with 1 ml 70% ethanol, dried for 30 minutes in 37°C and resolved in 4.6 ⁇ l DEPC water (0.1% DEPC, added to water and incubated at 37°C for 1 hour, autoclaved at 120°C for 30 minutes. 0.5 U RNAsin/ ⁇ l DEPCwater was added prior to use).
  • the cDNA mix (1.0 mM dNTP (Boehringer-Mannheim), 1 ⁇ cDNA synthesis buffert (GIBCO BRL), 0.1 ⁇ g/ ⁇ l BSA, 40 ng/ ⁇ l oligo-dT, 10 mM DTT, 1.0 U/ ⁇ l RNAsin, 10 U/ ⁇ l M-MLV reverse transcriptase, (Boehringer-Mannheim), 4.6 ⁇ l poly A RNA) was added and the sample was incubated for 1 hour at 37°C. The reverse transcriptase was inactivated by incubation for 5 minutes at 65°C.
  • the material was amplified for 30 cycles using PCR (1x PCR buffer containing 2.5 mM MgCl 2 (Perkin-Elmer Cetus), 0.2 mM dNTP, 1 ⁇ M of each primer (5 'GGGAATTCATGGGCWTCAARATGRARWCWCAT3 ' and5 ' CAAGCTTACTGGATGGTGGGAAGATGGA3 ' for the VL region and 5 'GGGGAATTCCACCATGRAATGSASCTGGGTYWTYCTCT3 ' and
  • PCR-products were run on an 2% agarose gel, extracted from the gel using JETsorbTM (SAVEEN biotech AB) and cloned into pCRTMII (Original TA Cloning Kit, Invitrogen) according to the manufactures protocol. Colonies were tested for insert using PCR (1x PCR buffer (Perkin-Elmer Cetus) containing 2.5 mM MgCl2 , 0.2 mM dNTP, 1 ⁇ M of each primer 5 'TCACACAGGAAACAGCTATGAC3 ' and
  • DNA sequencing was performed according to Sanger using the Sequenase Version 2.0 Kit (USB Biochemical) and 35S-dATP. The sequencing reactions were run on 8% polyacrylamide gels and autoradiography was typically over night.
  • tumour cell lines were tested for reactivity with hybridoma supematants using an indirect immunofluoresence assay.
  • Adherent cell lines were treated with trypsin and washed in PBS containing 1% bovine serum albumin (PBS- BSA) while nonadherent cells were incubated directly with antibody containing culture supernatant.
  • the cells (0.5 -1 ⁇ 10 6 ) were incubated with 100 ⁇ l diluted hybridoma cell culture supernatant for 45 min on ice, washed as above, and re-incubated for 45 min on ice with a F1TC conjugated goat anti-murine Ig (Dako A/S). The cells were then washed and analysed on a
  • FACStar plus (Beckton-Dickinson, San Jose, CA) flow cytometer as illustrated in figure 2.
  • Protease treatment necessary to obtain single cell suspensions and to avoid clogging of adherent cells, was shown to reduce the level of expression of the antigen as revealed after analysis performed on MOLT-4 cells.
  • most of the adherent cell lines although treated with trypsin for 5 min at 37°C, were stained to various degrees with the PA1-3F10 antibody. Also a variety of tumour cell lines with hematopoietic origin were stained, and some of them to a high intensity (Table 2).
  • Tissue specimens used for immunostaining were removed from patients during surgery or autopsy and snap frozen in isopentane within one hour after removal.
  • the tissues were sliced into four ⁇ thick sections using a cryotome.
  • the sections were dried over night at room temperature and fixed in acetone at -20°C for 10 min. After fixation the sections were soaked for 30 min at +20°C in TRIS buffer containing 10% FCS and then incubated at room temperature for 1 hour with antibody containing supematants, diluted to give a final Ig-concentration of 5-10 ⁇ g/ml.
  • the sections were then washed 3 times and biotin labelled goat anti-murine Ig was added.
  • a horseradish peroxidase avidin biotin complex (VECTASTAIN ABC-kit, Vector Laboratories Inc., Burlingame, CA) was added and incubated for 30 min. The sections were then washed and supplied with a solution containing diaminobenzidine (DAB) to visualise bound antibody. After a final washing step the sections were dried and mounted in mounting media (PERTEX, Histolab, Sweden) before examined in a microscope. Tumours were considered positive if more man 10% of the tumour cells were clearly stained. In many tumours all neoplastic cells were found to express the antigen. The staining was associated with the plasma membrane but the antibody also bound to the cytoplasm of many cells.
  • DAB diaminobenzidine
  • PA1-3F10 antibody bound a high frequency of a variety of human carcinomas. However, PA1-3F10 also bound to some normal tissue, although to a low frequency and often with a low intensity. Since it is well known that most, if not all, monoclonal antibodies to tumour associated antigens also bind to normal tissue to varying degrees the staining pattern of the PA1-3F10 antibody was compared to that of four other murine monoclonal antibodies already used in clinical trials for immunolocalisation and therapy of tumours (Table 4).
  • the following antibodies were evaluated against a non-binding IgGl antibody and cell culture medium controls; 17-1 A (Reitm ⁇ ller et al.1994) and KS 1/4 (Labus et al.1992) monoclonal antibodies which probably recognise different epitopes on a tumour associated antigen present on e.g.
  • the PA1-3F10 When compared with the four monoclonal antibodies described above the PA1-3F10 showed to be as broad in its tumour interaction as they were, but it had much less cross reactivity with normal tissue.
  • the 17-1A and KS1/4 antibodies showed a more homogeneous binding to the colon tumours tested. However, their binding to normal tissue was also very pronounced.
  • the KS 1/4 Besides binding to epithelial cells in kidney, colon, stomach, pancreas, oesophagus and breast the KS 1/4 also bound to peripheral nerve and to endothelium in small blood vessels in brain and placenta. In the liver both the bile collecting ducts and some arteries were stained. The intensity of the staining was as strong in normal tissue as in tumour tissue.
  • 17-1A showed a similar pattern, but stained weaker than KS 1/4. Staining with both HMFG-1 and B72.3 was associated with mucin producing tissue and stained the apical parts of tumour as well as normal cells. Both antibodies also stained kidney tubule, normal epithelia in ducts of the breast and the ascini of pancreas. HMFG-1 stained most colon carcinomas but the intensity of the staining was similar in normal colon and tumour tissue. In the results presented here, the HMFG-1 antibody stained all normal striated muscle tested with a low intensity. The B 72.3 antibody also stains cell nucleus and some normal blood vessels in the brain, this staining was weak but reproducible and found in most brain tissue tested. A staining of blood vessels in the brain was also observed with KS 1/4.
  • the PA1-3F10 showed both less frequent and less intense cross reactivity with normal tissue as compared to the other monclonal antibodies.
  • the PA1-3F10 and the KS 1/4 antibodies titrated similarly on tumour tissue originating bom from patients (colon tumour) and SCID mice transplanted with a human colon carcinoma cell line (COLO 205) and stained these tissues still at a concentration of 150 ng antibody/ml.
  • the binding of KS1/4 to tumour was more intense than that of the PA1-3F10 antibody but was also corresponded by an equally or more intense staining of normal human colon epithelium.
  • the PA1-3F10 antibody only faintly stained this tissue and only when used at the higher concentrations.
  • the PA1-3F10 antibody stain normal epithelium of tonsil and kidney tubules only when added at the higher concentrations while the KS 1/4 antibody stained these structures more intensely and at lower concentrations.
  • ADCC Antibody dependent cellular cytotoxicity
  • PA1-3F10 antibody (5 ⁇ g/ml) and with 5, 2.50 or 0.50 ⁇ 10 5 effector cells in RPMI 1640 with 10% FCS.
  • Triplicate cultures were set up at each effector to target cell ratio in round bottomed 96-well tissue culture plates. After the incubation period the culture supematants were collected and the amount of released radioactivity was determined in a gamma counter. Spontaneous release of 51 Cr was estimated after incubation of labelled cells with medium only, whereas maximum release was estimated after lysing labelled cells with addition of 1 % SDS.
  • PBL from blood donors were used as effector cells.
  • PBL comprises cells such as monocytes and NK cells with a potential to mediate ADCC.
  • the results obtained demonstrated that the PA1-3F10 antibody had the ability to mediate ADCC activity towards antigen expressing MOLT-4 cells but not towards the antigen negative MET 24 cells. Therefore, the antigen recognised by the antibody fulfils the criteria for being a target for antibody dependent cellular cytotoxicity.
  • the ADCC effect might be of importance in vivo should the antibody be used for therapy of tumours. No lytic effect was observed when the antibody was investigated for ability to mediate complement dependent cytoxicity (CDC).
  • mice which had been tested and found to have a serum-concentration of murine antibodies less than 1 ⁇ g/ml were used in these studies.
  • the mice were inoculated subcutaneously with 0.1 ml cell suspensions containing 1-2 ⁇ 10 6 viable tumour cells in each shoulder.
  • Each animal were supplied with one PA1-3F10 antigen negative (M24 MET) and one PA1-3F10 antigen positive tumour (COLO 205).
  • the tumours were allowed to grow to the size of approximately 8x8 mm when the animals were injected i.p. with 500, 250 or 125 ⁇ g of purified unlabelled PA1-3F10 antibody dissolved in sterile PBS.
  • the animals were sacrificed 1, 2, 3, 4 or 5 days after injection of antibodies and both tumours as well as liver and kidney were snap frozen, sectioned and analysed in immunohistochemistry for the presence of antibody utilising staining with a secondary HRPO labelled rabbit anti-mouse Ig antibody.
  • Purified PA1-3F10 antibody was labelled with 125 I using the lodogen method (Fraker et al.1978, Buchsbaum et al.1993).
  • 220 ⁇ l of PA1-3F10 in PBS (2.7 mg/ml) and 20 ⁇ l of Na 125 I (82 MBq) were added to the reaction vial precoated with 50 ⁇ g lodogen.
  • the reaction mixture was incubated for 60 seconds at room temperature.
  • the radiolabelled antibody was men separated from free iodine through gel filtration on a Pharmacia PD-10 column equilibrated with 0.1 M phosphate buffer, pH 7.2 and pretreated with a 1 % solution of BSA.
  • the labelling efficiency (the percentage of added iodine adhering to the protein) was estimated to be 77.5% and the specific activity of the antibody was determined to be 120 MBq/mg protein.
  • the amount of free 125 I in the protein fraction after separation on the PD-10 column was less than 1.5% of the total activity in the protein fraction as determined by TCA precipitation.
  • MOLT-4 cells were allowed to react with a fixed amount of radiolabelled antibody. After incubation for 60 minutes at 4°C the cells were centrifuged through a cushion of 200 ⁇ l of 40% of Ficoll to separate bound from free antibody. Samples of 50 ⁇ l of the supematants were removed and analysed in a gamma counter for determination of the amount of unbound antibody. The tubes were then frozen, the cell pellet containing the bound antibody were cut off and analysed in a gamma counter giving the amount of bound radioactivity (Table
  • the fraction of immunoreactive antibody could be estimated with the aid of a Lindmo plot (Lindmo et al.1984). Briefly, the fraction of reactive antibody was determined under assay conditions with infinite antigen excess. Plotting the quotient between the total and bound amount of antibody against the inverse of the concentration of cells a straight line was formed, and an intercept with the abscissa defining the inverse of the fraction of active antibody could be obtained after linear extrapolation. The results from a Lindmo plot of the data presented in table 6 is shown in figure 4.
  • the affinity constant could be determined to be 2 ⁇ 10 8 M -1 and the number of epitopes per cell on MOLT-4 cells to be 166 000 antibody epitopes /cell.
  • mice Different tissues from mice were analysed at different time points after injection of radio labelled antibody. Five mice were analysed at each time point. The dark bars indicate the values obtained after correction for radioactivity contributed by blood.
  • the mouse sera were also tested for its content of radioactive antibody with intact specific binding capacity to MOLT-4 cells. This was done after 96 h and 120 hours to estimate how large fraction of the circulating labelled antibody that was still biologically active.
  • the sera from the animals were incubated with MOLT-4 cells and the bound fraction of antibody were compared to the binding of the same batch of labelled antibody stored in vitro at 4°C. Remaining active antibody after 96 and 120 hours were estimated to
  • the antigen to be a glycoprotein associated with the plasma membrane of tumour cells.
  • proteolytic enzymes such as thermolysine, papaine or bromeline totally eliminated the epitope whereas treatment with trypsin only marginally reduced the binding.
  • the cell membrane associated epitopes are destroyed after 10 minutes treatment with a glycine buffer at pH
  • the protein nature of the antigen has also been indicated by other observations.
  • the antigen formed a cap on the surface of MOLT-4 cells when incubated at 37°C after cross-linking with the PA1-3F10 antibody followed by FITC labelled rabbit anti-mouse antibody.
  • Such cap-formation is characteristic for integral membrane proteins.
  • the antigen- antibody complex also seemed to internalise.
  • Incubation of MOLT-4 cells with FITC labelled PA1-3F10 antibody at 37°C for 15 to 30 min partly protected the antibody from being removed from the cell surface using treatment of the cells with an acidic (pH 2) buffer. From this observation and from experiments with radiolabelled antibody it is shown that most of the antibody bound to the cellmembrane is rapidly internalized.
  • MOLT-4 cells were mixed with 125 I labelled antibody and placed in 37°C, every 2 minutes two identical samples were removed and washed on 40% Ficoll (Pharmacia, Sweden). One of the individual samples was eluted with an acidic (pH2) buffer, which removes >90% of the surface bound antibody and the
  • Soluble forms of the antigen has never been found neither in spent cell culture medium from e.g. MOLT-4 cells nor in serum or plasma from patients with tumours positive for the antigen in immunohistochemistry.
  • the binding of the antibody was not affected by different lectins such as jaccalin, PNA, Viva Villosa, Lens Culinaris or DBA. Neither did small carbohydrates such as glucose, galactose, N-acetyl-D-glucoseamine, ⁇ -L-fucose, D-mannose or cellobiose inhibit the binding.
  • the PA1-3F10 antibody did not bind to neutral glycolipids extracted from the highly PA1- 3F10 positive cell lines PANC-1 or COLO 205. The lipids were tested both in ELISA and on immunostaining of thin layer chromatography (Magnani et al.1980).
  • the PA1-3F10 antibody differs from other antibodies recognising tumour associated antigens on colorectal carcinomas.
  • the best documented anti-colon carcinoma antibodies: HH8 (anti-Gal ⁇ (1-3) GalNac- ⁇ -O-Ser/Thr) (Kjeldsen et al.1974), MLS-128 (anti Tn ⁇ GalNac- ⁇ -O- Ser/Thr) (Nakada et al.
  • the antibody shows ADCC activity when measured on MOLT-4 cells with PBL as effector cells.
  • the presence of antibody also reduces the growth rate of transplanted human carcinomas in SCID mice.
  • PA1-3F10 antibody localised to the COLO 205 explanted tumours but not to antigen negative melanoma in SCID mice, PA1-3F10 looks like a promising antibody for diagnostic and therapeutic use in patients suffering from cancer, especially colorectal carcinoma.
  • Antibody-guided irradiation of malignant lesion Three cases illustrating a new method of treatment. Lancet, 2, 1441-1443.
  • Kalofonos H.P. Sackier J.M. et al., (1989). Kinetics, quantitative analysis and radioimmunolocalization using indium-1U-HMFG1 monoclonal antibody in patients with breast cancer. Br. J. Cancer, 59, 939-942.
  • the antitumour monoclonal antibody MOv2 recognizes the Lewis A hapten. Hybridoma, 7, 129-139.
  • Disialoganglioside GD2 on human neuroblastoma cells target antigen for monoclonal antibody mediated cytolysis and suppression of tumor growth. Cancer Res., 47, 1098-1104.
  • TAG-72 Tumour associated glycoprotein
  • O-linked mucin-type glycoproteins in normal and malignant colon mucosa lack of T antigen expression and accumulation of Tn and sialosyl-Tn antigens in carcinomas. Int. J. Cancer,

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Abstract

La présente invention concerne l'anticorps monoclonal PA1-3F10 ainsi qu'une lignée cellulaire d'hybridome permettant de le produire. Il est apparu que cet anticorps se lie à divers carcinomes et cellules malignes d'origine hématopoïétique, ceci avec une grande intensité et un très haut niveau de sélectivité, comme l'ont démontré les analyses d'immuno-histochimie et les dosages de liaison cellulaire. Cette invention concerne également des anticorps possédant sensiblement les mêmes capacités de liaison que l'anticorps monoclonal PA1-3F10, ainsi que les mutants, les dérivés, des équivalents fonctionnels ou des fragments de cet anticorps. Les applications de ces anticorps comprennent la thérapie ou le diagnostic du cancer, par exemple en associant des composés cytotoxiques à des cellules cancéreuses. Cette invention s'applique tout particulièrement à la thérapie contre le cancer et au diagnostic du cancer colorectal.
PCT/SE1996/000029 1995-01-18 1996-01-15 Anticorps utilises dans le diagnostic et la therapie du cancer Ceased WO1996022310A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7264805B2 (en) 2000-04-05 2007-09-04 Scintec Diagnostics Gmbh Proteins with a high immunoreactivity and a method for the production thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0199586A2 (fr) * 1985-04-22 1986-10-29 Hybritech Incorporated Antigène associé aux tumeurs
EP0272113A2 (fr) * 1986-12-18 1988-06-22 Kyowa Hakko Kogyo Co., Ltd. Anticorps monoclonaux contre le cancer humain
US4892933A (en) * 1988-04-20 1990-01-09 New England Deaconess Hospital Corporation Monoclonal antibody for colorectal carcinoma
WO1991004490A1 (fr) * 1989-09-15 1991-04-04 Genetic Systems Corporation Hybridome ct43 produisant un anticorps monoclonal contre un epitope de mucine du cancer colorectal
EP0519866A1 (fr) * 1991-06-18 1992-12-23 Ciba-Geigy Ag Anticorps contre V.I.H.
WO1994005690A1 (fr) * 1992-09-09 1994-03-17 Smithkline Beecham Corporation Nouveaux anticorps destines a conferer une immunite passive contre des infections dues a un germe pathogene chez l'homme
WO1994012214A1 (fr) * 1992-12-01 1994-06-09 Protein Design Labs, Inc. Anticorps humanises reagissant avec la cd-18

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0199586A2 (fr) * 1985-04-22 1986-10-29 Hybritech Incorporated Antigène associé aux tumeurs
EP0272113A2 (fr) * 1986-12-18 1988-06-22 Kyowa Hakko Kogyo Co., Ltd. Anticorps monoclonaux contre le cancer humain
US4892933A (en) * 1988-04-20 1990-01-09 New England Deaconess Hospital Corporation Monoclonal antibody for colorectal carcinoma
WO1991004490A1 (fr) * 1989-09-15 1991-04-04 Genetic Systems Corporation Hybridome ct43 produisant un anticorps monoclonal contre un epitope de mucine du cancer colorectal
EP0519866A1 (fr) * 1991-06-18 1992-12-23 Ciba-Geigy Ag Anticorps contre V.I.H.
WO1994005690A1 (fr) * 1992-09-09 1994-03-17 Smithkline Beecham Corporation Nouveaux anticorps destines a conferer une immunite passive contre des infections dues a un germe pathogene chez l'homme
WO1994012214A1 (fr) * 1992-12-01 1994-06-09 Protein Design Labs, Inc. Anticorps humanises reagissant avec la cd-18

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 157, AIDSLINE, Dialog Accession No. 00109894, Aidsline Accession No. 95128080, THURNHER M. et al., "Carbohydrate Receptor-Mediated Gene Transfer to Human T Leukaemic Cells"; & GLYCOBIOLOGY, (England), August 1994, 4(4), p429-35. *
DIALOG INFORMATION SERVICES, File 34, SCISEARCH, Dialog Accession No. 13388651, KUROSAKA A. et al., "Production of Monoclonal-Antibodies Recognizing Cancer-Associated Antigens Expressed on Mucin-Type Sugar Chains"; & BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1994, V203, N3, (Sep. 30), p1828-1834. *
GENE, Volume 121, 1992, HYO JEONG HONG et al., "Cloning and Characterization of cDNAs Coding for Heavy and Light Chains of a Monoclonal Antibody Specific for Pre-S2 Antigen of Hepatitis B Virus", pages 331-335. *
NATIONAL LIBRARY OF MEDICINE, FILE MEDLINE, Medline Accession No. 94208950, GOLD D.V. et al., "Characterization of Monoclonal Antibody PAM4 Reactive With a Pancreatic Cancer Mucin"; & INT. J. CANCER, 15 April 1994, 57(2), 204-10. *
NATIONAL LIBRARY OF MEDICINE, FILE MEDLINE, Medline Accession No. 95187393, O'BOYLE K.P. et al, "Expression of Human Tumor Mucin-Associated Carbohydrate Epitopes, Including Sialylated Tn and Localization of Murine Monoclonal Antibodies CC49 and B72.3 in a Syngeneic Rat Colon Carcinoma Model"; & J. IMMUNOTHER. EMPHASIS TUMOR IMMUNOL., *
STRAND, EMBL DATABASE, Accession No. M95574, ABERGEL C. et al., "Crystal Data and Primary Structure of the Anti-Tumor CC49 Fab", 18 June 1992, 29 July 1995. *
STRAND, GENESEQ, Geneseq Accession No. R76087, ZENECA LTD., "Antigen Binding Structures Containing CDRs Recognising the CA55.1 Antigen - Produced by Hybridomas and Host Cells, for Use in the Diagnosis and Therapy of Cancer"; & WO,A,95 15382, (08 June 1995). *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7264805B2 (en) 2000-04-05 2007-09-04 Scintec Diagnostics Gmbh Proteins with a high immunoreactivity and a method for the production thereof
US7820410B2 (en) 2000-04-05 2010-10-26 Scintec Diagnostics Gmbh Proteins with high immunoreactivity and method for their production
US9005574B2 (en) 2000-04-05 2015-04-14 Scintec Diagnostics Proteins with high immunoreactivity and method for their production

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