EP0473651A1 - Monoclonal antibodies which recognise malignant cells from bladder carcinomas - Google Patents
Monoclonal antibodies which recognise malignant cells from bladder carcinomasInfo
- Publication number
- EP0473651A1 EP0473651A1 EP90908164A EP90908164A EP0473651A1 EP 0473651 A1 EP0473651 A1 EP 0473651A1 EP 90908164 A EP90908164 A EP 90908164A EP 90908164 A EP90908164 A EP 90908164A EP 0473651 A1 EP0473651 A1 EP 0473651A1
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- European Patent Office
- Prior art keywords
- antibody
- antigen
- blca
- cells
- cell line
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
- A61K51/106—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from kidney or bladder
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
- A61K47/6861—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from kidney or bladder cancer cell
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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
- C07K16/3038—Kidney, bladder
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2121/00—Preparations for use in therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to antibodies, single domain antibodies and portions or fragments of the antibodies directed against malignant cells in the urine of patients suffering from bladder carcinoma, to the cell lines
- antibodies The antibodies, single domain antibodies, or portions or fragments of the antibodies have both diagnostic and therapeutic utility.
- Urinary cytology with specificity and sensitivity of 50-80% (Murphy et al., 1984) is hampered by subjective observer error, the
- tumour markers have been identified by using monoclonal antibodies raised against bladder carcinomas. These have been used to detect bladder cancer cells in bladder washings (Chopin et al., 1985).
- the present inventors have discovered a family of novel antigens detectable in urine and found in patients with bladder carcinoma.
- washings is non-invasive and does not require any surgical procedure. It is a relatively simple, cost effective, highly specific technique, not requiring a high level of cytopathological skill for interpretation, and can be completed on the day of receipt of the sample. Moreover, the percentage of positive results is much higher than that which can be obtained by conventional cytological analysis of the urine.
- carcinoma which antigen is found in voided urine of bladder carcinoma patients.
- a method for the detection of bladder carcinoma comprises detecting the presence of an antigen recognised by an antibody, portion or fragment thereof or a single domain antibody of the second embodiment in voided urine from a patient suspected to be suffering from bladder carcinoma.
- kit for the detection of bladder carcinoma which kit comprises:
- At least one antibody, or antigen binding fragment or portion thereof, or single domain antibody according to the second embodiment together with a positive and/or negative control.
- a method of tumour therapy which method comprises the administration of an antibody, or antigen binding fragment or portion thereof, or single domain antibody of the second embodiment, or an antibody composition of the third embodiment to a host in need of such treatment where the antibody, or antigen binding fragment or portion thereof, or single domain antibody is labelled with a radiolabel, immunotoxin or a cytotoxic drug.
- the antibody, or antigen binding fragment or portion thereof, or single domain antibody is used to target the radiolabel, immunotoxin or cytotoxic drug to the tumour.
- the cell line of the present invention is preferably an immortal cell line, such as a hybridoma cell line.
- hybridoma cell lines are produced by the fusion of an antibody-producing cell with a myeloma cell.
- the cell line may also be a recombinant cell line such as a bacterial or eukaryotic cell line expressing a foreign gene encoding a single domain antibody directed against an antigen of malignant cells associated with bladder
- carcinoma which antigen is found in voided urine of bladder carcinoma patients.
- the malignant cells with which the antigen is associated are cells from a transitional cell carcinoma (TCC) of the bladder.
- TCC transitional cell carcinoma
- the antigen found in the voided urine of the patient is stable in the voided urine of the patient.
- the antigen is a neutral glycolipid.
- the antigen is a protein.
- the antibody is an IgG 3 antibody.
- the antibody, antigen-binding fragment or portion thereof, or single domain antibody is directed against a TCC cell line selected from the group consisting of UCRU-BL-17-CL, UCRU-BL-23/3, UCRU-BL-13/0 and 5637 as herein described.
- the cell line is a hybridoma cell line selected from BLCA-8, BLCA-30 and BLCA-38 as herein
- hybridoma cell line is BLCA-8.
- the antibody, or antigen-binding fragment or portion thereof, or single domain antibody is typically used for diagnostic or therapeutic purposes in labelled form.
- Typical labels include fluorescent labels, radiolabels and enzyme labels.
- Labelling may also be by means of a second antibody against the antibody, fragment or portion thereof, or single domain antibody.
- detectable label such as an enzyme, radiolabel or
- amplification systems such as biotin-avidin associated labelling systems to achieve amplification of the signal to be detected is also encompassed by the present invention.
- a preferred fluorescent label of the present invention is fluorescein isothiocyanate.
- diluents used in the preparation of antibody compositions of the present invention include isotonic buffered saline solutions such as PBS or TBS, and tissue culture media.
- any suitable detection procedure for detecting labelled cells may be employed.
- Preferred procedures include: fluorescent immunoassay; enzyme immunoassay, such as ELISA; radioimmunoassay,
- the procedure may involve the use of a second antibody and may involve the use of an amplification system such as a biotin-avidin system.
- the preparation of bladder cells is
- kit comprises an enzyme labelled antibody, portion or fragment thereof, or single domain antibody, the kit also preferably includes a substrate for that enzyme.
- Cytotoxic drugs suitable for labelling an antibody, fragment or portion thereof, or single domain antibody for use in therapy include Vinblastine, Iadarubicin,
- Aminopterin Methotrexate or 5-Fluorodeoxyuridine.
- Immunotoxins include Pseudomonas exotoxin or ricin. Radiolabels include 131-Iodine, 153-Samarium, 90-Yttrium, 186-Rhenium, 211-Astatine and 67-Copper.
- the labelled antibody, fragment or portion thereof, or single domain antibody may be administered systemically or directly into the bladder through its orifice.
- the antigens encompasses those fragments and portions capable of binding to the antigen recognised by the antibody. It includes for instance, Fab and F(ab') 2 fragments.
- the antigens include for instance, Fab and F(ab') 2 fragments.
- antibodies of this invention are antigens of malignant cells associated with bladder carcinoma, and found in the voided urine of bladder carcinoma patients.
- Figure 1 Shows immunofluorescence profiles (solid lines) obtained by flow cytometric analysis of UCRU-BL-17-CL cells stained with BLCA-8 (A), BLCA-30 (B) or BLCA-38 (C) and SaMIg-FITC. The dotted lines indicate the profiles obtained with the control antibody and SaMIg-FITC.
- Figure 2 Shows photomicrographs of BL-23 cells stained with A) biotinylated-BLCA-8 or B)
- biotinylated-K-1-21 (control) and SA-HRP.
- the bar indicates 10 ⁇ m.
- Figure 3 Shows the effect of exposure to urine on antigen expression in UCRU-BL-17-CL cells.
- the cells were incubated in urine at 36°C for 2 hours and then at 20°C for a further 16 hours. At selected time points, the cells were stained with BLCA-8 ( ⁇ ), BLCA-30 ( ⁇ ) or with BLCA-38 ( ⁇ ) followed by SaMIg-FITC.
- Figure 4 Shows the appearance of inner layer (A and B) or outer layer (C and D) urothelial cells by either normal light microscopy (A and C) or after immunofluorescent staining with BLCA-8 and SaMIg-FITC (B and D).
- Figure 5 Shows reactivity of agarose-embedded cells from voided urine samples of patients with TCC of the bladder (solid histograms) or normal controls (open
- Figure 6 Shows the percentage of outer ( ⁇ ) and inner ( ⁇ ) urothelial cells from voided urine reactive by surface immunofluorescence in an agarose assay with BLCA-8. Sample were obtained from normal controls and from patients with biopsy-proven TCC of the bladder.
- Figure 7 Shows immunofluorescence profiles obtained after flow cytometric analysis of BL-23 cells stained with BLCA-8 (clear histogram) or K-1-21 (control, shaded
- Figure 8 Shows binding of BLCA-8 by ELISA to antige present in spent media from BL-23 cells (----), in spent media after boiling (___), and in normal culture media (_ _), (control).
- Fiqure 9A shows the binding of BLCA-8 (solid line) or BLCA-7 control MAb (interrupted line) in an ELISA. MAbs were tested either against a lipid extract prepared from BL-23 cells ( ⁇ ) or against ganglioside
- Fiqure 9B Shows the binding of these same test and control MAbs against a lipid extract prepared from BL-17 cells ( ⁇ ) or against ganglioside G - (A)
- the horizontal axis indicates the concentration of antigen in ⁇ g per ml from which 50 ⁇ l per well was added to the plate.
- Fiqure 10 Shows immunoblotting of control MAb, K-1-21 (A,B) or test MAb, BLCA-8 (C,D) to a TLC separation from a BL-23 lipid extract. Six ⁇ l(A,C) or 9 ⁇ l (B,D) of
- (E) indicate components specifically bound by BLCA-8.
- Track F indicates lipids visualised with ⁇ -naphthol. Components with equivalent migration to the BLCA-8 reactive components shown in E are in solid colour.
- Fiqure 11 Shows images from tumour bearing mice injected with 131 I-BLCA-38 or control MAb. Nude mice
- FIG. 12 Shows the growth of BL-17 human bladder cancer xenografts (A and B) or Jo. N. human ovarian cancer xenografts (C and D) after injection of a
- Figure 13 Shows the change in the log percent initial tumour volume after a single injection of:
- the monoclonal antibodies of the present invention are prepared by standard techniques. These are described in (Walker et al 1986 ) .
- the monoclonal antibodies are either present in the supernatant taken from hybridoma cell culture or they are purified by standard techniques.
- Antibody compositions of the present invention are prepared by admixing purified antibodies, antigen-binding fragments or portions thereof, or single domain antibodies of the invention with a pharmaceutically acceptable carrier or diluent using standard methods of pharmaceutical
- diagnostic kits are prepared by formulating antibodies, antigen-binding fragments or portions thereof, or single domain antibodies at appropriate concentration to the antigen to be detected with a pharmaceutically acceptable carrier or diluent.
- a positive control standard of a known concentration of the antigen to be detected is prepared similarly.
- the negative standard comprises carrier or diluent alone or an antibody of irrelevant specificity diluted with a pharmaceutically acceptable carrier or diluent.
- the antibody, antigen-binding fragment or portion thereof, or single domain antibody is provided in labelled form or alternatively the label is provided separately in the kit.
- antigen-binding fragments or portions thereof, or single domain antibody or antibody compositions for targetting tumours is in accordance with standard techniques.
- the antibodies, antigen-binding fragments or portions thereof, or single domain antibody are used to target radioisotopes, immunotoxins or cytotoxic drugs with which they are
- This method is particularly suited for use with bladder cancers since the bladder has an
- Human bladder cancer UCRU-BL-17-CL (Russell et al., 1988), UCRU-BL-13 (Russell et al 1989), UCRU-BL-23/3 (Russell et al unpublished) and 5637 (obtained from The American Type
- the BL-23/3 bladder cancer cell line was established in the Urological Cancer Research Unit, Royal Prince Alfred Hospital by Dr Pam Russell and
- the cell line is tumourogenic in nude mice and has 62-66 human chromosomes. Clonal abnormalities include deletion 1p, derivative 3q, translocation between 1q and 6q, deletion 6q, insertion 11p, +7, --16, and 2 metacentric markers;
- the bladder lines were maintained at 37°C in 5% O 2 , 7.5% CO 2 and 87.5% N 2 , whilst all other lines were
- FCS foetal calf serum
- EXAMPLE 1 Murine monoclonal antibodies from mice immunised with the UCRU-BL-17CL human bladder cancer cell line.
- mice were immunised with multiple intraperitoneal (i.p.) injections of the human bladder TCC line, BL-17 and derived xenografts.
- the immune spleen cells were fused with NSI-1 Ag4.1 mouse myeloma cells (Kohler et al 1976) and cultured as described previously (Walker et al., 1986).
- MAb K-1-21 of irrelevant specificity was used as a negative control while the MAb BB7.7 (from the American Type Culture Collection) reactive against human
- HLA-A,B,C antigens acted as a positive control.
- Monoclonal antibodies were used either as supernatants from the
- BLCA-8 and BLCA-30 are of the IgG- subclass while BLCA-38 is an IgG 1 antibody.
- EXAMPLE 2 Reactivity of murine monoclonal antibodies with normal and malignant cells.
- BL-23 cells grown in chamber slides (Lab-tek Products, Miles Laboratories, Napierville, IL) or frozen sections from biopsy specimens were fixed for 5 min in acetone.
- the slides were washed in TBS for 10 min, biotinylated BLCA-8 (5 mg/ml) diluted 1/50 in PBS/Az was added and slides were then incubated for 1 h and washed 3 times in TBS.
- SA-HRP Amersham, Sydney, NSW, Australia
- slides were again washed twice in TBS.
- Developing solution (3% H 2 O 2 diluted 1/50 in 0.025% diaminobenzidine (Sigma) in TBS) was applied and when the colour had appeared, slides were counterstained with Hematoxylin. All incubations were at 37°C.
- a portion of normal bladder tissue was also obtained from a transplant donor.
- Cell suspensions were prepared mechanically from these tissues, and were stained for surface immunofluorescence, or after a period of up to 2 weeks in short term tissue culture.
- Cell lines were
- the antibodies also reacted with short term cultures of TCC of the bladder, but did not react with normal bladder cells (Table 1).
- One MAb, BLCA-38 also showed reactivity with human ovarian and colonic cancer lines, and some melanoma lines. None of BLCA antibodies showed reactivity with lymphoid or leukaemic cell lines.
- the BLCA-8 antigen was found to be poorly resistant to formalin fixation but was well preserved after treatment with acetone.
- Examples of BL-23 cells fixed in acetone and stained with biotinylated BLCA-8 and SA-HRP are shown in Figure 2. Strong staining was observed both on the cell membrane and within the cytoplasm, whereas no cell membrane or cytoplasmic staining was observed with a control
- BLCA-8 showed no reactivity with normal bladder, breast or testis. It was however, detected in sections of 17 week but not 14 week kidney and stomach.
- the BLCA-8 antigen may have some prognostic value. Positive reactivity was found in biopsy specimens from some patients with a previous history of TCC but with no known disease at the time of sampling. It was also found in some samples from patients with recurrent infection or haematuria but not in patients with chronic inflammation.
- BLCA-8 showed no reactivity with biopsy specimens from patients with benign conditions of the ovary or prostate but showed reactivity in 1/2 bladder conditions of benign origin. BLCA-8 reacted with all biopsy specimens from patients with TCC of the bladder (grades I-III) but showed no reactivity with breast or prostate adenocarcinoma. EXAMPLE 3 - Stability of antigen expression in cells exposed to urine.
- tumour stages included in this patient group were T IS , T 1 , T 2 , T 3 , and T 4 and the majority of
- tumours (22) were grade 3.
- the slides were incubated in a humidified chamber at 4°C for 30 min, before being washed in PBS/Az for 30 min at 4°C. Excess PBS was wiped from the slides, and 10 ⁇ l of second antibody, SaMIg-FITC diluted 1/50, was added over each aliquot of agarose-embedded cells. After further incubation at 4°C for 30 mins, the slides were again washed in PBS/Az before fixation in
- immunofluorescent staining was assessed by fluorescence microscopy using a Zeiss photomicroscope.
- outer cells Figure 4c and d
- inner cells Figure 4a and b
- BLCA-8, -30 and -38 in urine samples from cancer patients and in controls are shown in Figure 5.
- Each MAb showed a significantly higher reactivity against patients' cells than against normal control urothelial cells.
- the greatest difference was observed with BLCA-8 where the percentages of positive cells in patients' urine were 24.3, ⁇ 5.8 and 27.0 ⁇ 4.6 for outer and inner layers, respectively, as compared with 2.9 ⁇ 1.0 percent and 0.8 ⁇ 0.5 percent in cells from control urines (P ⁇ 0.05 and P ⁇ 0.002, respectively).
- EXAMPLE 6 Relation between the expression of the BLCA-8 reactive antigen on cells in voided urine and the stage of invasiveness of the tumour.
- tumour stages represented in this patient group were as follows (patient numbers are given in brackets): T. (carcinoma in situ) (1); T 1 (invasion of the lamina propria) (2); T 2 (superficial muscle invasion) (9); T 3
- T 3 deep muscle invasion
- T 4 invasion of surrounding organs
- the increased expression of the BLCA-8 antigen on tumours of higher stage may be the result of either an increase in the numbers of malignant cells shed into the urine or it may indicate an interesting new marker of neoplastic invasion.
- urothelial cells was minimal except in the case of one person, a female, heavy smoker aged 50, who was found on two separate occasions to have a high reactivity with BLCA-8 (78-81% outer cells positive) ( Figure 5 and Table 4).
- cytopathologist revealed the presence of "atypical" cells in her voided urine, but cytoscopy revealed no evidence of malignancy.
- BLCA-8 expression remained low in 2 other subjects who were also heavy smokers (2 cases) and also in 3 women who were pregnant. Other cases of interest examined are shown in Table 4.
- Urothelial cells were examined from two patients who had undergone cystectomy but no BLCA-8 reactivity was detected.
- the first patient developed carcinoma in situ four months after sampling; the second was found to have a papillary tumour, grade I-II, five months later.
- the cytoplasmic expression of the BLCA-8 antigen on BL-23 cells was determined by fixation of the cells in 50% ethanol/PBS at 4°C for 5 min and immunofluorescence staining with BLCA-8 and SaMIg-FITC followed by flow cytometric analysis.
- BLCA-8 antigen is thus expressed both on the cell surface and in the cytoplasm.
- pronase protease
- neuraminidase (Clostridium perfringens) , (Sigma) 0.1 U/ml, 1 x 10 7 cells in 0.05M sodium acetate buffer, p HH 5.5, with 1% CaCl 2 for 45 min; (c) EDTA, 0.1M, 5 x 10 6 cells in 1 ml PBS for 1 h; (d) 2-ME (Sigma), 5 x 10 6 cells in 1 ml PBS for 1h at 4°C; (e) fucosidase (bovine epididymus, Sigma) 0.031 U/ml, 1 x 10 6 cells in 500 ⁇ l PBS for 30 min (f) sodium periodate (Sigma) ImM, 5 x 10 6 cells in 1 ml PBS for 1 h at 4°C. In addition, cells were incubated with Limulus Polyphemus lectin (Sigma),
- BLCA-8 antigen was unaffected by pronase or endoglycosidase F (Table 5) which suggests that the antigen is unlikely to be protein or glycoprotein in nature. Moreover, surface expression of the antigen does not appear to be dependent on divalent cation bridges or disulfide bonds since EDTA or 2-ME had no effect on antigen expression. In addition, the epitope does not appear to involve sialic acid since treatment of the cell surface with mild periodate oxidation or with neuraminidase to cleave sialic acid groups did not affect antibody binding. Neither was binding blocked by Limulus lectin which has specificity for sialic acid residues,
- the BLCA-8 antigen can be detected in spent medium from BL-23/3 bladder cancer cells by ELISA. This assay was used to determine whether the BLCA-8 antigen was stable to heat treatment.
- Spent medium was obtained from confluent cultures of BL-23 cells overgrown for 7 days in 25 cm 2 tissue culture flasks (Costar, Delta Packaging, Cambridge, MA). Duplicate 400 ⁇ l aliquots of spent medium, were serially diluted in PBS in the wells of a 96 well plate (Flow Laboratories Inc., McLean VA). After O/N incubation, wells were washed in PBS and blocked with 2.5% BSA (Miles Diagnostics, Sydney,
- FIG. 8 The BLCA-8 antigen appeared to be stable after treatment at 100°C for 90 seconds and was detectable in UCRU-BL-23/3 spent medium at dilutions of less than 1/128. c). Expression of the BLCA-8 antigen in lipid extracts from human bladder cancer cells.
- Lipid extraction from BL-17 or BL-23 cells was carried out as described by Magnani and co-workers (1987).
- a lipid extract from BL-17 cells was further purified as follows. The extract was dissolved at 10 mg/ml in chloroform:
- Extracts were solubilized in absolute ethanol at 20 ⁇ g/ml (BL-17 extract) or 2
- the plate was then incubated for 2 h at 37°C and washed 3 times in PBS and once with 1% BSA in PBS. SaMIg-B, A-AP and enzyme substrate were then added.
- BLCA-8 showed good reactivity with lipid extracts from both BL-23/3 ( Figure 9A) and BL-17 ( Figure 9B) although clearly more antigen was present in the BL-23 extract. No reactivity with either BL-17 or BL-23 lipid extracts or the standard gangliosides was observed with a control antibody of matched IgG 3 isotype.
- the BLCA-8 reactive bladder cancer cell lines were known to also react by surface immunofluorescence with BG-8
- BLCA-8, BLCA-30 and BLCA-38 all fail to cross-react with group A human red blood cells either by surface immunofluorescence staining or by serology.
- the extract was dissolved in absolute ethanol and 60 ⁇ l of a 10 ⁇ g/ml solution was added to the wells of a 96 well plate (Flow) and evaporated to dryness. Wells were then blocked with 2.5% BSA in PBS with ImM CaCl 2 for 2 h before incubation at 37°C with sodium periodate (Sigma) at 1.0 to 100 mM for 1-2 h or with various enzymes diluted in PBS/CaCl 2 at the following
- ⁇ -galactosidase 240 ⁇ l at 0.5U/ml for 2 h or 350 ⁇ l at 0.7 U/ml O/N; ⁇ -galactosidase, 200 ⁇ l at 25 U/ml O/N;
- ⁇ -glucosidase 350 ⁇ l at 2.5 U/ml O/N; ⁇ -glucosidase,
- Biotinylated lectin solution Biotinylated lectins (E-Y Laboratories, San Mateo, CA) were used as indicators of enzyme activity. The activity of ⁇ - and ⁇ -galactosidase was confirmed with GS-I, which binds ⁇ -gal and ⁇ -galNac end groups; DBA, which binds terminal non-reducing
- ⁇ -fucosidase was confirmed with UEA-I, which recognises ⁇ -L-fucose.
- Con A was used at a dilution of 1/1250 from a 2.5 mg/ml solution in TBS/CaCl 2 while all other lectins were used at a dilution of 1/500 from a 1 mg/ml solution in PBS/CaCl 2 .
- biotinylated reagents After incubation with biotinylated reagents for 1 h, plates were washed as above before addition of a 1/500 dilution of A-AP in PBS/CaCl 2 to each well.
- Glycolipids are ⁇ -galactose and ⁇ -glucose residues. Glycolipids are
- X-ray film (XRP-1 Kodak Australasia, Sydney) was then exposed to the air-dried plates using cassettes with intensifying screens (X-omatic, Kodak).
- BL-23 extracts were solubilized at 10 mg/ml in 1 : 1 chloroform: methanol and 60 or 90 ⁇ g was applied to 6 replicate TLC plates.
- Ganglioside G D1 (1 ⁇ g) was applied as a standard. Plates were run as above. Four plates were visualised with TLC spray reagents (see below) while the 2 remaining plates were immunoblotted (as above) with BLCA-8.
- BLCA-8 was found to bind specifically to three components (2 major and 1 minor) in the BL-17 extract while no staining occurred with the control MAb (data not shown). BLCA-8 also bound to 4 components (2 major and 2 minor) in the BL-23 extract which had a pattern of migration approximating those components specifically stained in the BL-17 extract ( Figure 10A).
- the BLCA-8 reactive components were not visualised with resorcinol and thus did not appear to be gangliosides or to contain sialic acid.
- the BL-23 extract was found to contain several phospholipids after staining with Molybdenum Blue (1.3% molybdenum oxide in 4.2 M sulphuric acid) but these did not include the BLCA-8 reactive components.
- Molybdenum Blue (1.3% molybdenum oxide in 4.2 M sulphuric acid
- the BLCA-8 reactive moieties are either neutral glycolipids or sulphatides which stain with ⁇ -napthol
- glycolipid fraction The glycolipid fraction.
- EXAMPLE 9 Localisation of radiolabelled BLCA-38 to human bladder cells cancer xenografts in nude mice.
- mice Athymic BALB/c nu/nu mice were bred at the Australian Nuclear Science and Technology Organisation
- mice were kept under sterile conditions, being housed in cages fitted with filter tops, and were fed sterilized standard mouse food and water ad libitum.
- Antibodies were concentrated to 1 mg/ml for iodination and to approximately 20 mg/ml for chelation with 153 -Sm or
- Antibodies were labelled with 131 I as described previously (Walker fit al 1986). In (Amersham, Bucks, UK) or 153 Sm (Australia Nuclear Scientific and Technology Organisation, ANSTO, Lucas Heights, NSW, Australia) were chelated to antibodies using the bicyclic anhydride of diethylenetriaminepentaacetic acid (DTPA) as a bifunctional chelating agent using methods developed by Boniface and co-workers (1989). Briefly, the bicyclic anhydride of DTPA (Sigma, St Louis, MI) at 1 mg/ml in chloroform was dried under nitrogen onto the sides of an acid washed Reactivial (Pierce Chemical Co., Rockford, IL). Antibody (20 mg/ml) was added at a ratio of
- mice Groups of 5-6 nude mice were implanted subcutaneously on each flank with a BL-17 human bladder cancer xenograft.
- mice were injected with 131 I-, 111 In- or
- mice 153 Sm-labelled BLCA-38 or a labelled control MAb were injected with 153 Sm-BLCA-38. Each mouse received 80-120 ⁇ g of protein labelled with 300-500 ⁇ Ci of isotope.
- mice were anaesthetised and placed prone beneath the pinhole collimator of a large field of view
- mice 153 Sm-labelled MAbs were exsanguinated by cardiac puncture and killed by
- Biodistribution studies were carried out on nude mice bearing s.c. BL-17 human bladder cancer xenografts, seven days after injection of 131 I-, 111 In- or 153 Sm-BLCA-38 or 131 I-L7 or 111 In- and 153 Sm control MAbs.
- conjugates in particular in relation to liver uptake.
- liver uptake was 7.27 ⁇ 1.39%ID/g and 5.27 ⁇ 0.61%ID/g
- liver uptake demonstrated with the Sm-BLCA-38 conjugate appears to be a consequence both of a predilection of intact MAb to localise to liver following Fc receptor and lectin binding, together with our observation of colloid formation from the dissociation of 153 Sm ions from the conjugate during incubation in plasma (see below).
- a characteristic feature of the biodistribution of Sm-labelled conjugates was also noted in these studies, namely, the relatively high uptake in bone.
- bone uptake was 2.11 ⁇ 0.37 and 2.70 ⁇ 0.32%ID/g after injection of either 153 Sm-BLCA-38 or Sm control respectively
- indicating label deposition is mainly in osseous bone.
- EXAMPLE 10 Therapeutic effects of 153 Sm-BLCA-38 on human cancer xenografts in nude mice.
- mice Athymic BALB/c "nude" mice were maintained as in
- Example 9 Human cancer xenografts derived from the human bladder cancer BL-17 line described above or from Jo N, an ovarian cancer line developed as described by van
- tumour volumes were determined from the formula for an ellipsoid (Russell et al 1986).
- mice Seven groups of 3 or 4 mice were killed from 30 min to seven days after the injection of 1 53 Sm-BLCA-38 (150-175 ⁇ Ci on 100 ⁇ g per mouse).
- Tissues were weighed and counted as described in Example 9.
- A is the cumulated activity to the organ in ⁇ Ci.h; m is the mean organ mass; ⁇ i is the absorbed dose constant for the ith emission and ⁇ i is the absorbed fraction for the ith emission.
- 153 Sm is a beta emitter with E ma x -640
- ⁇ i was taken as 0.5687 g rad/ ⁇ Ci.h (Myers et al
- penetrating radiations in the mouse amounted to less than 2% of total body dosage based on the absorbed fractions and was therefore neglected.
- Total blood mass was estimated at 5.85% of body mass and total skin, muscle, and bone masses were determined by
- mice bearing human cancer xenografts of 200-1000 mm 3 were given a single i.p.
- tumours were assessed for necrosis. As some patchy necrosis occurs spontaneously in large tumours, a "necrotic" tumour was defined as one which had become completely necrotic throughout its mass with virtually no intact xenograft tissue observable macroscopically.
- mice bearing BL-17 human bladder cancer xenografts were treated in this way, the tumours grew 54% in geometric volume after injection of the control antibody. Three of the tumours (30%) showed no growth, while two had some necrosis. In contrast, mice injected with 153 Sm-BLCA-38 showed an increase of only 25% geometric mean volume in the same period. Eight of the 15 tumours (53%) showed no growth while 7 (47%) had become completely necrotic. (Figure 12A).
- BLCA-38 either alone, or as a conjugate labelled with a beta emitting isotope, had any long term effects upon the growth of well established (200-1000 mm 3 ) BL-17 xenografts.
- mice injected with cold BLCA-38 had a mean log percent initial tumour volume of 3.4 ⁇ 0.53 which was not significantly different (Student's t test) from that seen in animals injected with PBS (mean log percent initial tumour volume, 3.97 ⁇ 0.14).
- tumour growth in mice receiving 131 I-BLCA-38 antibody there was a significant retardation in tumour growth in mice receiving 131 I-BLCA-38 antibody compared with controls, with a dramatic fall in tumour volume at day
- mice receiving 131 I-control MAb there was also a decreased growth rate in mice receiving 131 I-control MAb.
- mice At day 20 the mean log percent initial tumour volume for treated mice was 1.44 ⁇ 0.38, compared with 2.72 ⁇ 0.10 in controls, (P ⁇ 0.02, Student's t test). After this time there was a rapid recovery in growth rate so that by the end of the 36 day period of observation there was no difference in the mean log percent initial tumour volume of tumours in mice injected with 131 I-labelled test (3.15 ⁇ 0.41) or control antibodies (3.26 ⁇ 0.16).
- mice were injected with 250 ⁇ Ci of 153 Sm labelled BLCA-33 or control MAb (these mice were given only half the dose given to mice receiving iodinated MAbs)
- the radiation dosage to mouse organs and a BL-17 human bladder cancer xenograft was determined in nude mice injected i.p. with 153 Sm-BLCA-38. Cumulative activities calculated are given in Table 11.
- the whole body dosage per mCi injected was determined from the total activities of all measured elements according to the MIRD dosage formula.
- Total cumulated activity to the body was 29,830 ⁇ Ci.h, whilst body radiation dose from beta radiation was 850 rads, whilst whole body radiation dose from gamma radiation (calculated from absorbed dose
- Radionuclides radiation dose and effects. ORNLC
- H - A monosialoganglioside is a monoclonal antibody-defined antigen of colon carcinoma.
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Abstract
L'invention se rapporte à une famille de nouveaux antigènes associés aux cellules malignes de carcinomes cystiques ainsi qu'à des anticorps, à des parties ou à des fragments de ces anticorps, ou à des anticorps monodomaines qui reconnaissent ces antigènes. L'invention propose des procédés de détection des carcinomes cystiques au moyen de ces anticorps, de ces fragments ou de ces parties d'anticorps, ou de ces anticorps monodomaines, des kits destinés à être utilisés dans ces procédés, ainsi que des procédés de traitement des carcinomes cystiques au moyen de ces anticorps, de ces fragments ou de ces parties d'anticorps, ou de ces anticorps monodomaines.The invention relates to a family of new antigens associated with malignant cells of cystic carcinomas as well as antibodies, parts or fragments of these antibodies, or monodomain antibodies which recognize these antigens. The invention provides methods of detecting cystic carcinomas using these antibodies, fragments or parts of antibodies, or these single domain antibodies, kits for use in these methods, as well as methods of treatment cystic carcinomas using these antibodies, fragments or parts of antibodies, or these single domain antibodies.
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU4351/89 | 1989-05-24 | ||
AUPJ435189 | 1989-05-24 | ||
AU57306/90A AU642419B2 (en) | 1989-05-24 | 1990-05-24 | Monoclonal antibodies which recognise malignant cells from bladder carcinomas |
Publications (2)
Publication Number | Publication Date |
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EP0473651A1 true EP0473651A1 (en) | 1992-03-11 |
EP0473651A4 EP0473651A4 (en) | 1992-10-14 |
Family
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Application Number | Title | Priority Date | Filing Date |
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EP19900908164 Withdrawn EP0473651A4 (en) | 1989-05-24 | 1990-05-24 | Monoclonal antibodies which recognise malignant cells from bladder carcinomas |
Country Status (3)
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EP (1) | EP0473651A4 (en) |
AU (1) | AU642419B2 (en) |
WO (1) | WO1990014433A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1994018562A1 (en) * | 1993-02-05 | 1994-08-18 | Epigen, Inc. | Human carcinoma antigen (hca), hca antibodies, hca immunoassays, methods of imaging and therapy |
EP0710362B1 (en) * | 1993-07-23 | 1998-11-04 | Bard Diagnostic Sciences, Inc. | Methods for determining the invasiveness of a bladder tumor |
KR102249981B1 (en) | 2014-10-23 | 2021-05-11 | 미노믹 인터내셔널 리미티드 | Monoclonal anti-gpc-1 antibodies and uses thereof |
CN107847593B (en) | 2015-04-20 | 2021-12-14 | 美侬米克国际有限公司 | Therapeutic antibodies and uses thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0118891A2 (en) * | 1983-03-11 | 1984-09-19 | Sloan-Kettering Institute For Cancer Research | Monoclonal antibodies to human bladder and ureter cancers and method |
-
1990
- 1990-05-24 EP EP19900908164 patent/EP0473651A4/en not_active Withdrawn
- 1990-05-24 WO PCT/AU1990/000218 patent/WO1990014433A1/en not_active Application Discontinuation
- 1990-05-24 AU AU57306/90A patent/AU642419B2/en not_active Ceased
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0118891A2 (en) * | 1983-03-11 | 1984-09-19 | Sloan-Kettering Institute For Cancer Research | Monoclonal antibodies to human bladder and ureter cancers and method |
Non-Patent Citations (3)
Title |
---|
JOURNAL OF CELLULAR BIOCHEMISTRY, vol. 0, no. 12, part E, 1988, page 147, abstract no. T314, New York, US; P.J. RUSSELL et al.: "Development and characterization of monoclonal antibodies to human bladder cancer" * |
PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, ANNUAL MEETING, San Francisco, California, 24th - 27th March 1989, vol. 30, page 285, abstract no. 1136; K.Z. WALKER et al.: "A new murine monoclonal antibody (BLCA-8) with potential for clinical application in the management of bladder cancer" * |
See also references of WO9014433A1 * |
Also Published As
Publication number | Publication date |
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EP0473651A4 (en) | 1992-10-14 |
AU5730690A (en) | 1990-12-18 |
WO1990014433A1 (en) | 1990-11-29 |
AU642419B2 (en) | 1993-10-21 |
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