AU609939B2 - Tumour imaging - Google Patents
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- AU609939B2 AU609939B2 AU29547/89A AU2954789A AU609939B2 AU 609939 B2 AU609939 B2 AU 609939B2 AU 29547/89 A AU29547/89 A AU 29547/89A AU 2954789 A AU2954789 A AU 2954789A AU 609939 B2 AU609939 B2 AU 609939B2
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- 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
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- 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
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- A61K49/00—Preparations for testing in vivo
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- 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
- A61K51/1018—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 material from animals or humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Description
00 *~0 4 4 To: THE COMMISSIONER OF PATENTS (a member of the firm of DAVIES COLLISON for and on behalf of the Applicant).
Davies Collison, Melbourne and Canberra.
iin.. U EMil mimE. ml EU III III. COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION (original) FOR OFFICE USE 60~93 Class Int. Class Application Number: Lodged: Complete Specification Lodged.
Accepted: Published: :Prio rity: 0 elated Art: 0 0 0 0o 0 0 0000 0 0 0 00 0 Thsdocument contains the amendnif.nts made unde ~cion 49 LJa"Id is correct for printing.
00 0 0 0 0 Name of Applicant: 000 0 0 a a 00 0 0 0 0 0 00 Address for Service: THE UNIVERSITY OF' MELBOURNE Grattan Street, Parkville 3052, in the State of Victoria, Commonwealth of Australia IAN FARQUHAR CAMPBELL McKENZIE JOE JANWAR TJjANJDRA DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street,-Melbourne, 3000.
Complete specification for the invention entitled: M006295 02 2 ~IJ0UR IMAGING" including the best method of performing it known to us 1- Insert place and date of signature.
Signature of declarant(s) (no aftstation required) Note Initial all alterations.
4 The basic application......... refer ed to in paragraph 3 of this Declaration was were the first application made in a Convent on country in respect of the invention the subject of the application.
Declared at ?tf'L200lUPAJ this day ofj f I James Baildon Potter,Registrar DAVI:S COLLISON. MELBOURNE and CANBERRA.
L: r b i 11 rI i .I 0 0 0 0 0 00 o 0 0 0 0 00 o00 00 0 0 0 0 o O TUMOUR IMAGING 23 The present invention relates to tumour identification and localisation utilizing antibodies 0° 25 specific for tumour antigens. [Note: References are 0 00 oo0 collected at the end of the specification.] 27 Since the advent of monoclonal antibodies with a o0 0 Spreferential activity for cancer tissue, radiolabelled 29 antibodies have been used in radioimmunodetection of Scancer and its metastases. Administration of 31 radiolabelled antibodies combined with nuclear imaging (hereinafter referred to as immunoscintigraphy) has 33 allowed detection of a wide range of tumours in different sites. Imaging procedures-using intravenously administered radiolabelled antibodies are limitec by background radioactivity in the blood pool and 37 extravascular spaces and, furthermore, antibodies may be catabolised before reaching their target, resulting in 39 only a very small tumour uptake.
The concept of immunoscintigraphy has been extended r I~on 2 1 to using subcutaneously injected radiolabelled tumour-associated antibodies to delineate metastatic 3 deposits in regional lymph nodes. These earlier studies have shown that non-specific uptake by normal lymph glands and other tissues has been a major problem which severely hampers the effectiveness of the imaging techniques.
7 It has previously been proposed to reduce non-specific or non-target antibody binding, and thus 9 enhance the resolution of tumour imaging, by the following VA techniques: 11 Injecting patients intravenously with an antibody 00.. preparation containing tumour specific antibody, and 0 000o 13 an excess of unlabelled antibody.
0000 This technique has been largely unsatisfactory in 0 0 0o°0 15 reducing non-specific antibody binding. As tumours 0 00 are bathed in extracelluar fluids containing 17 antibodies and large amounts of other proteins such ooo 0O o as albumin, it is not surprising that the 0 00o0 19 administration of additional antibody is largely ineffective in reducing background.
00 o 21 (ii) The administration of tumour specific antibody labelled with a radionuclide, and labelled human 23 serum albumin or a second radionuclide (usually o 9 9 mTc) capable of imaging normal tissue. Specific i binding of tumour specific antibody is detected by subtracting the signal produced by the second 27 radionuclide.
The disadvantage with this approach is that a 29 patient is exposed to increased levels of radiation, with attendant side affects. Further, independent 31 detection devices may be required to detect the signal from different radionuclides, thus making 33 this proceducure costly and cumbersome. In addition, the isotopes used in substraction (e.g.
3 1 technetium) do not distribute in exactly tle same way as normal IgG.
3 (iii) Injecting a subject intravenously with a tumour specific antibody labelled with a radionuclide, and, after sufficent time to permit antibody-tumour binding injecting the patient with a second 7 non-labelled antibody or fragment thereof, specific_ against the tumour specific antibody. The second 9 antibody clears non-bound antibody from the o circulation thus improving the resolution of tumour 0O9 11 imaging. Such a technique is described in U.S.
o Patent No.4,624,846.
0 0 o6 0o a 13 The above technique has the disadvantage that 0009 °o0 binding of the second antibody to the tumour o 0 o 15 specific antibody results in the formation of large o 00 antibody aggregates, which may cause immune complex 17 disease (as antibody aggregates are much more 0400 "o o0 immunogenic than monomeric molecules), damage to the 0 00 0 o a 19 reticuloendothelial system, and kidney overload, with its associated complications.
0 0 a o 21 If the tissue distribution of the tumour specific antibody, and the second antibody is different, a 23 poor result will be obtained on imaging.
o 00 Additionally, this technique is not suitable for subcutaneous tumour imaging, and in particular, the imaging of tumours associated with the lymphatic 27 system. Labelled tumour specific antibody would be taken up specifical-ly by tumours, and r 29 non-specifically-by normal lymph nodes and other tissues. Such antibody would not be accessible for 31 clearance by a second anti-tumour antibody.
33 The present invention is based on the surprising finding that the background non-specific antibody binding 7 4 1 of isotopically labelled tumour specific antibodies normally associated with tumour imaging, is significantly 3 reduced if a patient is injected with both a tumour specific antibody labelled with a radioisotope of an element and an antibody which is not specifically tumour-reactive and which is labelled with a 7 non-radioactive isotope of the same or other element. The identification and location of tumours is thus facilitated.
9 According to the present invention, there is o provided a method for identifying and/or determining the 0 0 S 11 location of a tumour which produces or is associated with 0o0 a a cytoplasmic, intra-cellular or cell surface marker 0 00 o" 13 substance, the method comprising the steps of: o injecting a human or animal subject with tumour o"o 15 specific antibody or a fragment thereof specific for the 0 00 marker and labelled with a radioactive isotope of an 17 element or a paramagnetic conjugate, and an irrelevant 0000 "o 0 (non-tumour reactive) antibody which has been labelled 0o °o 19 with a non-radioactive isotope of the same or other element, or a non-paramagenetic conjugate; and o0 0 0o o0 21 after a period of time sufficient foT selective binding of the labelled tumour specific antibody to the 23 tumour scanning the human or animal subject with a 0o 0 0o 0 detector to locate the site or sites of uptake of labelled antibody or fragments thereof.
The present invention has particularly utility in 27 the identification and location of subcutaneous tumours, and in particular breast cancer, and associated lymph node 29 metastases. Currently the best predictor of occult metastases is the involvement of axillary lymph nodes by 31 the tumour, the detection of which involves axilliary dissection with its associated morbidity. Clinical 33 assessment of axillae is notoriously unreliable, and of patients with breast cancer who have no palpable lymph i I 5 1 nodes in the axillae, about one third have histological evidence of lymph node invasion. The detection of lymph 3 node metastases will be greatly facilitated by the present invention which avoids the complications of surgery and axillary dissection. It is to be understood however, that the invention is not limited to such specific applications.
7 Tumour associated markers may be associated with the cytoplasmic, intracellular, or cell-surface regions of 9 tumour cells. Advantageously, the tumour associated O markers are cell-surface antigens. Tumour associated 0 0 o 11 markers are generally associated only with tumours and are not associated with other non-tumour tissues such as 13 muscle, fat, blood vessels and eryithrcy'ces. Small o °0 amounts of reactivity with non-tumour tissue may however 0o 15 be tolerated where this reactivity is weak compared with a 00 tumour reactivity.
17 Irrelevant antibody is generally non-tumour 0 reactive. However, small amounts of tumour reactivity may o00 19 be tolerated.
o 00 Human or animal subjects may be injected o0 0o 21 intravenously, intraperitonelly or subcutaneously with antibody or antibody fragments. Advantageously, 23 antibodies are administered by subcutaneous injection in "i the fields of the lymphatic drainage to detect lymph node metastases. In relation to breast cancer identification, antibodies may be injected into the arm, chest or breast 27 itself.
Antibodies employed may be monoclonal or polyclonal 29 or Fab, (Fab) 2 portions thereof. Antibodies may be of mouse, human, hamster horse, goat, rat, rabbit or other 31 animal origin. Antibodies may have one or more heavy or light chains from different species. Where monoclonal 33 antibodies are employed, they may be of any isotype. The tumour specific antibody and irrelevant antibody may be of b~ i r 6 1 the same or different isotype. Advantageously, antibodies of the same isotype are preferred. Antibodies of the IgG 3 or IgM isotype are preferred. Polyclonal or monoclonal antibodies and fragments thereof may be prepared by methods well known to persons skilled in the art, such as those described in Goding (1986).
7 Isotopically or paramagnetically labelled antibody or antibody fragments specific for a tumour marker 9 (hereinafter referred to as "tumour specific antibody") o and non-tumour antibody labelled with a non radioactive 00 11 isotope or non paramagnetic element (hereinafter referred to as "cold labelled antibody") may be administered to a o 00 0e o 13 patient at the same time or substantially at the same time 0000 °oo (within an interval of a few minutes). Alternatively, the 0'Oo1 15 cold labelled antibody preparation may be administered approximately up to five hours before administration of 17 the tumour specific antibody.
0000 o The molar ratios of tumour specific antibody to cold oa 0 19 labelled antibody may vary considerably. Generally, the cold labelled antibody is in considerable molar excess, 00 0 o a 21 such as two to twenty fold excess.
Antibodies or fragments thereof may be labelled with o 23 Kadioactive or non-radioactive isotopes of an element S, according to methods well known to persons skilled in the art, and described for example in Goding (1986).
I Radioisotopes of iodine 131I, 1 25 I, 1231) indium 27 Xe.g. 11 lIn) and technecium 99 mTc) may be employed in the Sinvention, as may the non-radioactive isotopes of these 29 elements. The present invention is not limited to tho use of the aforementioned radioisotopes. Other radioisotopes 31 emitting a detectable signal may be employed. Preferably, the tumour specific antibouy and irrelevant antibody are 33 labelled with the same element, that is, the tumour specific antibody is labelled with a radioactive isotope 7 1 of a particular element, and the irrelevant antibody is labelled with a non-radioactive form of the same element.
3 Suitable paramagnetic labels which may be employed in the invention include atoms or ions that slightly increase a magnetic field, having an odd number of electrons and a partially filled inner shell, such as 7 found in transition elements, rare earth elements and those of the actinide series. Such magnetic labels may 9 include manganese copper (II) and cobalt (II).
Other suitable paramagnetic labels are described in G.N.
o, 11 La Mar et al, (1973) which is incorporated herein by 0 o0o reference o 0a a" o 13 In the case of metal ions, methods of attachments oo are similar to those previously discussed for 0o 0 15 radionucleotides. Methods for introducing paramagnetic labels into molecules are disclosed in Paik et al (1982).
17 Selective binding of a tumour-specific antibody to o tumour is generally allowed to proceed for a period of 0° 0 19 about one hour to fo-r days before tumour imaging is carried out. Advantageously, the time period is from 12 0 a 21 to 36 hours, and preferably from 16 to 24 hours.
Tumour imaging is carried out utilizing a device or 23 detector capable of detecting radionuclide emissions such o 0 o So 0 as a gamma scintillation counter. Where paramagnetic labelling is used, a NMR (Nuclear Magnetic Resonance) spectrometer may be used (Mansfield and Morris 1983).
27 The immunoscintigraphy method of the present invention is safe and easy to perform. The non-specific 29 uptake of radiolabelled antibody-by normal lymph nodes is reduced with the addition of the second cold labelled 31 antibody, resulting in greater differential uptake between malignant and normal lymph nodes with improved tumour 33 detection compared with previous methods.
In accordance with another aspect of the present 8 1 invention, there is provided a tumour imaging composition which comprises: 3 a tumour specific antibody or a fragment thereof labelled with a radioactive isotope of an element or a paramagnetic conjugate; and (ii) a non-tumour reactive antibody labelled with a 7 non-radioactive isotope of the same or other element, or a non-paramagnetic conjugate; 9 in association with a pharmaceutically acceptable carrier OA or excipient.
0 0q# cooa 11 Pharmaceutically acceptable carriers or excipients soao include sterile water, physiological saline, albumin and 0 0 0o0 0 13 other proteinaceous carriers, and carbohydrate based 0000 'Do 0 solutions such as mannitol or dextrose. Other examples of oe P 15 pharmaceutically acceptable carriers or excipients are 0 00 detailed in Remington's Pharmaceutical Sciences, 16th Ed.
17 Mach Publishing Co., edited by Osol et al., which is 0000 oo 0 hereby incorporated by reference.
SQ0 19 ABBREVIATIONS MoAb monoclonal antibody a 0 o 21 PBS phosphate buffered saline; 23 The present invention will now be described, by way o o of example only, with reference to the following non-limiting examples.
27 EXAMPLE 1: MATERIALS AND METHODS 29 Monoclonal Antibodies (MoAbs): The monoclonal antibody used was 17.1 (IgG2a). The 31 17.1 MoAb is a mouse IgG2a immunoglobulin raised by immunising inbred Biozzi mice with the MCF-7 breast cell 33 line (Thompson et al 1983). It reacts with most breast cell lines and breast carcinomas but has no reaction with
V-
9 1 other tissues of relevance to this study muscle, fat, blood vessel, erythrocytes). The Ly-2.1 MoAb was 3 reactive with the murine Ly-2.1 specificity but not with human breast cancer (Hogarth et al 1982). Both 17.1 and Ly-2.1 MoAbh were isolated from ascitic fluid by precipitation with 40% ammonium sulphate, dissolution in 7 PBS and dialysis with the same buffer. Prior to adsorption onto Protein A-Sepharose (Pharmacia Inc., 9 Piscataway, NJ), the column was washed extensively with go PBS (isotonic saline bufered with sodium phosphate and 0 0, 11 disodium phosphate to a pH of 7.3) and the immunoglobulin 0 0ooo eluted with 0.2M glycine-HCI (pH After 0 00 0 oo 13 neutralization, MoAbs were dialysed against PBS, aliquoted 000o 0 0o and stored at -700C.
0oo o oo 15 Antibody activity was determined by rosetting with 0 00 ,heep anti-mouse immunoglobulin (Parish, C.R. 1978) and in 17 the case of 17.1 also by immunoperoxidase methods on 0000 oo 0 snap-frozen sections of fresh breast cancer (Thompson 0 0Q o0OO 19 et al 1983). The prepared antibodies were retested for 0 activity after all procedures, filtered through a 0.22pm °o 21 Millex-GV filter (Millipore, Bedford, Ann Harbor, Michigan, USA) and batch tested for pyrogens and sterility 04 23 before and after radiolabelling (Pharmacology Department, Melbourne University and Sigma Pharmaceuticals, Clayton, Vic., Australia).
27 Iodination of Monoclonal Antibodies: The 17.1 antibody (400pg) was labelled with 2piCi of 29 131I (200pCi/ml, Amersham Inc., -UK) to a specific activity of lpCi of 131I per mg of 17.1 antibody using the 31 enzymobead reagent (Morrison) and the final radio-iodinated antibody was suspended in a 1% human serum 33 albumin solution (HSA; Commonwealth Serum Laboratories, Melbourne, Australia). The murine MoAb Ly2.1 (2mg) was 10 1 labelled with cold sodium iodide (non-radioactive Nal) by means of the chloramine T method (Greenwood). lodinated 3 antibody was separated from free iodine on a Sephadex column (PDI0, Pharmacia, Sweden) which had previously been equilibrated with 3ml of 25% human serum albumin and of sterile normal saline (Travenol Laboratories, NSW, 7 Australia). The radioactivity of the iodinated antibody was measured in both a gamma counter (LKB Wallac 1260, 9 Finland) and radioisotope calibrator (Capintec CRC-2Ni ie capintec, New York, USA), and the radiolabelled protein t 11 peak pooled. The sample was then centrifuged at 100,000 x a s so g for 60 minutes to remove aggregated proteins. Finally, o 13 the radiolabelled antibody was filtered through a 0.22im oooo oo° Millipore filter in a sterile laminar-flow hood, before So 1.5 clinical use.
The antibody 17.1 (200pg) was also labelled with 17 lmCi of 1311 (200pCi/ml, Amersham Inc, UK) to a specific o° 3 activity of lI.Ci of 131I per mg of 17.1 antibody using the 0 0* °o 0O 19 chloramine T (Greenwood 1963), iodobead (Markwell 1982) or enzymobead method (Morrison 1970) to compare the effect on 00 0 0 o 21 antibody activity by different iodination methods (see Example 2).
23 0 0 o Patient Section: Thirty-six patients with histologically or cytologically proven breast cancer were studied. All 27 patients except four (Stage IV, 2 patients; benign breast disease, 2 patients) had Stage I or II breast cancer using 29 the standared UICC classification (American Joint Committee on Staging). The clinical assessment of 31 axillary lumph node status was performed independently by two experienced breast surgeons. All patients with stage 33 I or II breast cancer had axillary disscection either as part of Patey mastectomy, or in addition to partial I-13 -I _PsYC __P^_UI 11 1 mastectomy. Two patients with a benigr. '.east lump (sclerosing adenosis) was studied: initial fine needle 3 aspiration cytology (FNAC) suggested the presence of malignant cells but subsequent excision biopsy failed to find any malignancy. Two patients had breast cancer proven by fine needle aspiration cytology but subsequently 7 were found to have Stage IV breast cancer with bone metastases; neither had axillary dissection but they both 9 had malignant axillary lymph nodes on clinical 0 examination. Of the patients studied, 11% (4/36) received o 11 radioiodinated 17.1 injected subcutaneously into the arms, o a s o 3% (1/36) had periareolar injection into the breast of 0 00 0o0 o 13 radioiodinated 17.1, 81% (29/36) received radioiodinated 0000 o0o° 17.1 (reactive with breast cancer) together with cold o°o 2 15 labelled Ly2.1 (non-reactive with breast cancer) 0 0 administered subcutaneously into both arms, and 8% (3/36) 17 received radioiodinated 17.1 together with unlabelled 0000 0 0 0o 0 Ly2.1 injected subcutaneously into both arms. One patient o 0 19 who received radioiodinated 17.1 alone in her arms had daily scans performed up to the fourth day after o o °o 21 injection. To inhibit thyroid uptake of radioactive iodine, each patient received potassium iodide (5ml of 23 16.54% w/v) and sodium perchlorate (400mg) orally, one O hour before the subcutaneous injection of radioiodipmt a monoclonal antibody. The potassium iodide was continued for 5 days after the injection of 1311.
27 Blood samples were obtained from all patients immediately before, and 6 weeks after injection of 29 radioiodinated MoAbs for- determining human anti-mouse antibody (HAMA) response.
31 Imaging: 33 Scintillation-camera images were recorded 16-18 hours after injection of the radiolabelled antibody.
antibody is labelled with a paramagnetic conjugate; after a period of time sufficient for selective binding of t-a labelled tumour Specific antibody to the tumour scanning the human or animal subject with a detector to locate the site or sites of uptake of labelled antibody or fragments thereof.
/2 74, 12 1 Anterior axillary and upper body scans were recorded with a Toshiba GC A402 gamma camera using a high-energy 3 collimator and computerised acquisition with an informatek Simis 4 computer (Informatek, Sydney, Australia). A window setting of 360 KeV with a 20% window was used.
Imaaes were obtained over a period of 600 seconds and then 7 digitally recorded into a matrix of 128 x 128 words.
Regions of interest on the images were defined by 9 manual drawing over the axillary lymph node regions on both sides using anatomical landmarks as well as adjacent 11 background and the heart. Definition of anatomical coo regions was performed by 2 independent investigators and I, 13 has been found to be remarkably reproducible. The o fraction of radiolabelled antibody localised in the o' 15 axillary nodes was estimated by measurement of nocal uptake with the gamma camera, and comparison with 17 uptake in the other regions of interest and the amount of o radiolabelled antibody injected Nodal uptake was r.o 19 adjusted for background activity camera response and attenuation through the anterior axillary fold using an %o 21 attentuation factor calculated using a known source placed in the axilla. The following formulae were used: 23 S0, Formula 1: F (NI/I) x 100 Formula 2:N2 bPn A 27 N n x 29 Pb -ux 31 Formula 3: N 2 (cpm) p Nl(pCi) x camera sensitivity 33 Where F Fraction of antibody localised in the nodes.
N nodal uptake (N 1 in pCi, N 2 in cpm) n total gammp camera counts (cpm) over lymph 37 node regions 1 actual injec 8d dose (pCi) i C 13 1 b background activity (cpm) Pn number of pixels in region of intest (lymph 3 node region) Pb number of pixels in background A =attenuation factor (1/1.37) e-Px= correlation factor for isotope decay 0.94 7 for 131I at 18 hrs Camera sensitivity was determined by counting the 9 amount of counts per minute (cpm) with a known amount o (ipCi) of 131I.
0 044 Scans were reported as positive, and therefore o o o 13 indicative of lymph node metastases, if the number of o countes per pixel in the axilla on the tumour side oa° 15 exceeded the normal side by a ratio greater than 1.5:1, after adjustment for background activity as indicated 17 above.
0o o Analysis of Excised Lymph Nodes and Breast Cancer tissue: o 'o 19 In most instances patients had surgery 36-40 hrs after the injection of radio-iodinated antibody. At 00 a o 21 surgery the nodes were removed in a tissue block and pinned onto a foam board to facilitate orientation. After 23 formalin fixation at room temperature overnight, each node 00
I
o was dissected free of fat and in some cases gamma camera images, radioactive count and the weights of individual nodes were obtained.
27 Each node was processed, stained and 6im sections were examined histologically tumour. To -assess 29 binding of the antibody to any tu,"-)ur-cells present in the lymph nodes, immunoperoxidase staining of snap-frozen 31 sections from some patients was performed using both the indirect two-stage and a one stage direct application of 33 rabbit anti-mouse-peroxidase conjugate (Thompson, C.H., Epenetos et al). In addition, fresh breast cancer tissue V^ yLtS LiL*Alr*Y J'..tnl.Vi LAS~ IP-- ii- 1" -14 14 1 was also obtained from some patients, snap-frozen and tested for its reactivity with 17.1 antibody by 3 immunoperoxidase staining. A non-reactive, negative control was processed for each lymph node section by incubating with an anti-human colon MoAb 30.6, as the primary antibody. The sections were then assessed by 7 light microscopy to estimate the percentage of carcinoma cells stained with the antibody and the result expressed 9 semi-quantitatively as 0-4 according to whether there was a, no staining up to 25% 26-50% 51-75% or 0 o O. 11 more than 75% of cells stained.
o °q o 13 EXAMPLE 2: t on Comparison of Radioiodination Methods: 0 0, s 15 To select the best radioiodination method, a 0 00 comparison was made of the yield of incorporation of the 17 17.1 antibody after radioiodination with Chloramine T, o o. iodobead and enzymobead methods (Table Using the S 19 rosetting assay and immunoperoxidase staining independently, the Chloramine T method resulted in a S. 21 greater loss of immunoreactivity than either the iodobead or enzymobead method as assessed by the titre of antibody 23 and percentage of rosette forming cells and by the S staining score on immunoperoxidase. Immunoreactivity after labelling was also tested by radioimmunoassay (RIA) for 17.1 against a breast cancer cell line. There was a 27 25-fold difference between the reaction of 131 I-labelled 17.1 on the breast carcinoma cell line (T47D-17.1+j 29 compared to melanoma cell line (COLO 239-17.1-), and labelled antibody competed well with unlabelled antibody 31 for antigen binding. The enzymobead method resulted in minimal loss of reactivity, while chloramine T method 33 resulted in greatest loss of reactivity. The enzymobead method was, therefore, used as the method of choice for labelling 17.1.
15 1 EXAMPLE 3 3 IMMUNOLYMPHOSCINTIGRAPHY 1311-17.1 (400pg) and cold Labelled Ly-2.1 (2mg): Arm Injection 7 Twenty-six patients (Table 2) received radioiodinated 17.1 antibody (reactive with breast cancer) 9 with a specific activity of ImCi 131I per mg of 17.1 (by II enzymobead method) together with cold labelled Ly2.1 11 (non-reactive with breast cancer) injected subcutaneously into both arms just above the ante-cubital fossae. The 13 Chlroamine T method was used to iodinate Ly2.1 antibody to o o maximally produce damaged moAb. Correct prediction of C,°o S 15 lymph node status was achieved in 92% (24/26) patients).
Fourteen of the 26 patients had histologically proven 17 axillary lymph node metastases and 13/14 patients 00oo0 Sooo were detected by the scan, while 12/26 patients did not o0oo 19 have lymph node metastases and a negative scan was Sobtained in 11/12 of patients.
00 0 o 0o 21 Several representative examples of patients in this study are described in more detail below.
23 Patient WT had right breast cancer and clinically 00 0 0 aC pe non-involved axillary lymph nodes. She underwent right Patey mastectomy and a single focus of miicrometastases was identified in 1 of the 12 axillary lymph nodes recovered S27 on histology. The preoperative immunolymphoscintigraphy showed an increased localisation of 1311-17.1 in the right 29 axillary lymph node region with a ratio betweeh axilla of interest and contralateral axilla of-1.5:1.
31 Patients KS clinically was considered to have axillary lymph node metastases but scanning showed equal 33 uptake of 1311-17.1 in both axillae with ratio of background substracted count density of 1:1 between .he 2 1 I signal from different radionuclides, thus making 33 this proceducure costly and cumbersome. In addition, the isotopes used in substraction (e.g.
i 16 1 axillae. Histology of the 14 lymph nodes recovered showed reactive hyperplasia only with no metastases. The 3 immunoperoxidase staining of regional lymph nodes with 17.1 antibody also did not identify any tumour deposits.
(ii) 1311-17.1 (1mg) and Cold Labelled Ly-2.1 (2mg): 7 Arm Injection Three patients (Table 2) received a higher dose of 9 specific MoAb (17.1 Img) while maintaining the same amount of 13 1I (2pCi) to a specific activity of 0.5mCi of 1 3 1 11 per r.g of antibody. The same amount cold iodine-labelled o Ly-2,1 MoAb was given. Correct prediction of lymph nodes S0oo 13 status was achieved in 3/3 patients including 1 patient who had a benign breast lump but the amount of radioactive 0 oo00 I o° 0 15 uptake in either axilla is less with a poorer quality 0o0 image.
0oo 17 Tables 3 and 4 summarise the results and statistical 0 analysis of preoperative clinical assessment and axillary 19 immunolymphoscintigraphy in 26 patients who received 0000 0o0 o 1311-17.1 (400pg) and cold labelled Ly-2.1 (2pg). This 0 00 Ao0 21 new method of immunoscintigraphy with the use of two antibodies is much more sensitive and specific (92%) 00 0 0°o 23 than preoperative clinical assessment (57% sensitivity, 58% specificity) but still not quite as accurate as axillary dissection and histological assessment which is o o the standard against which it is compared. Overall 27 accuracy of this new-method of immunoscintigraphy (92%) was undoubtedly superior to preoperative clinical 29 assessment Patients tolerated the immuno lymphoscintigraphy 31 procedures easily and there were no allergic reactions or sepsis locally or generalised. One patient who had 33 chronic asthma and was receiving steroids, had an exacerbation of her asthma which was easily controlled by i I, r 33 The present invention is based on the surprising finding that the background non-specific antibody binding i, i 17 1 increasing the dose of the steroid. Human anti-mouse antibody (HAMA) response was detected in the serum of only 3 1 patient at 4 weeks after injection.
Table 5 shows the amount of radioactivity uptake in metastatic lymph nodes, normal lymph nodes and axillary fat in 2 patients, one of whom (Patient 1) had 1311-17.1 7 (400pg) alone, the other (Patient 2) had 1311-17.1 (400pg) and cold labelled Ly-2.1 (2mg). Correction was made to 9 account for the physical half-life of 131 I and to obtain a relative count with regard to the injected amount. The 11 localization index refers to the ratio of radiolabelled Oo antibody present in the ratio counted. Only 2 axillary o 13 specimens were examined by the method, but the 00.* localization index of radioactivity in the involved nodes 0 00 o0 15 were higher than the normal nodes in the patient who 0 o° received 1311-17.1 and cold labelled Ly-2.1 than the o0°o 17 patient who had 1311-17.1 alone. The background counts Q 00 from the surrounding fat were low.
19 00ooo00 (iii) 1311-17-1 (400pg) and Unlabelled Ly2.1 (2mg): 0 0Q 0oo0o 21 Arm Injection Three patients (Table 2) were injected with 00 0 °o o 23 radioiodinated 17.1 (400pg) together with unlabelled Ly2.1 (2mg). Incorrect prediction of lymph node metastases were 25 obtained in all three patients.
00 t 0o o t 27-iv) 1311-17.1 (400pg): Arm Injection Four patients (Table 2) received radioiodinated 17.1 29 alone. One of the four patients had sclerosing adenosis, a benign condition, while the other three patients had 31 breast cancer. Correct prediction of lymph node metastases was obtained in 1/4 patients. In 2/4 patients, 33 the uptake of radioiodinated 17.1 was, in fact, higher in the normal axilla. Tne fourth patient, with advanced 18 1 breast cancer (Stage IV) and malignant left axillary lymph nodes on clinical examination, was also injected with 3 131 I-labelled 17.1 (400pg) into both arms subcutaneously.
Serial scans were obtained at 16hrs, 40hrs and 64hrs after injection and showed equal uptake in both axillae. The differential uptake between abnormal and normal axillae 7 did not change with time and appears to be optimal at 16hrs after injection.
9 In a further experiment, three patients received radioiodinated Ly2.1 alone. Two patients without axillary 11 lymph node metastases received 131 I-Ly2.1 with a specific o activity of 0.25 mCi/mg (total amount of antibody injected 0 00 g 13 2mg) and one patient with right axillary lymph node metastases received 1 31 I-Ly2.1 with a specific activity of 0 00 o 15 1 mCi/mg (total amount of Ly2.1 injected 400pg). In ooo 0° 0 each patient, the amount of radioactive uptake between the o"o 17 two axillae was the same, even in the presence of axillary o o lymph node metastases. It appears from this study that 19 the uptake of radioiodinated Ly2.1 is the same between o 00oo 00 0 node positive and node negative axillae suggesting that 0 00 0 a0 21 the blocking of non-specific uptake was independent of the size of the lymph nodes or the presence of lymph node 00 0 o o 23 metastases.
25 EXAMPLE 4 00 t 00 0 Correlation with Immunoperoxidase Staining: 27 To assess the reacti.v-ty of th breast cancer with 17.1 and the binding of 17..i to any tumour cells present 29-in the axillary lymph nodes, immunoperoxidase staining of snap-frozen sections using both the indirect two-stage 31 (primary breast cancer and lymph nodes) and a one-stage (lymph node) direct application of rabbit 33 anti-mouse-peroxidase conjugate was performed. Fresh sections were obtained from 10 patients, all of whom had 33 antibodies are employed, they may be of any isotype. The tumour specific antibody and irrelevant antibody may be of -19- 1 1311-17.1 and cold labelled Ly-2.1 and 6/10 patients had lymph node metastases. Five out of these six patients had 3 primary breast cancer and lymph node metastases which reacted strongly with 17.1 (staining score 3-4) These five patients had strongly positive scans. On the other hand, 1/6 patients had primary breast-cancer and lymph 7 node metastases which reacted only poorly (staining score 1) with 17.1 and the immunoscintigraphy failed to detect 9 the lymph node metastases in this case, suggesting that failure of the method to localise lymph node metastases 11 may be contributed, in part, by the poor reactivity of the S«17.1 MoAb with the particular tumour.
S 13 In these experiments, antibodies of the IgG subclass were used. Studies we have performed with antibodies of S o00 .0 0 15 different subclass, show that antibody subclass is of 000oo S00 little importance.
"o0 17 It has previously been established that 17.1 reacted with only 90% of fresh breast cancer sections examined 19 (unpublished observations) presumably due to the 0000 So phenomenon of tumour heterogeneity both within and between o 0 21 different tumour deposits. As shown in a patient whose breast cancer only reacted poorly with 17.1, the 00 0 c0 23 immunoscintigraphic scan failed to identify the lymph node metastases. This may account for false negative rate in example 3(i) of this study or but this has yet to be proven.
27 The mechanism of non-specific antibody uptake by normal lymph nodes and the mechanisms of action of the 29 second cold labelled antibody are not clear at.this time.
The improvement in results obtained with the addition of a 31 second antibody in excess may be due to the competition for non-specific binding to normal lymph nodes.
I-
II
20 1 3 7 9 11
OI
o 0 013 0 00 0000 0 00 00 0 00o 0 17 0 00 o0o 0 1 0 25 0 0 0 00 0 0 00 00 a 0 00 23 a t 27 29 31 33
REFERENCES:
American Joint Committee on Staging, Manual for Staging of Cancer, 2nd Edition, Philadelphia, Lippinsott (1983).
Greenwood, Innter, W.M. and Glover, Biochem.
J. 1063; 89: 114-123.
Goding, Monoclonal Antibodies: Principles and Practice, 2nd Edition, Academic Press (1986).
Hogarth, Henning, M.M. and McKenzie, I.F.C., Immunology; 46: 125-144 (1982).
La Mar, NMR of Paramagnetic Molecules, Principles and Applications, Academic Press, New York (1973).
Mansfield, and Morris, Advances in Magnetic Resonance (NMR) Imaging, C.L. Partain et al., eds.
w.B. Saunders Co. (1983).
Markwell, Annals Biochem.; 125: 472-432 (1982).
Morrison, M. and Boyse, Biochemistry; 9: 2995-3000.
Paik, J. Nucl. Med.; 23: -37 (1982).
Parish, G.R. and McKenzie, J. Immunol. Methods; 173-183 (1978).
Thompson, Jones, Whitehead, R.H. et al., J. Natl. Cancer, Inst.; 70: 409-419 (1983).
I
21 TABLE 1 Comparison of Three Methods of Iodination on the Activity of 17.1 JMoAb by Rosetting Assay and imnu.voperoxidasea T'echnique 9 Chloramine T Iodobead 11 Enzymobead Unlabelled Control Incorporation 82 78 81 Antibody Titre (%RFC)b 1/5,000 1/5,000 1/20,000 1/100,000 00 0 0 0*00 0000 a 06 0 0 0 000 0 0000 0 00a 00 06 00 0 0 00 0 0 a 00 15 Technique Staining Densityc (and of carcinoma stained) at different-MoAb dilutions 17 1/102 1/103 1/104 1/5x,0 4 1/7x10 4 1/105 Chioramine T 4 4 3 2 1 0 19 lodobead 4 4 3 2 J 0 Enzymobead 4 4 4 3 2 0 21 Unlabelled Control 4 4 4 3 2 1 0be 23 aAll tissue6 were obtained fresh, immediately snapfrozen, and stored at -70 0
C.
b RFC, rosette forming cells.
c Staining score was graded as no stain <25% 27 26.50% (20, 51-75% >75% of carcinoma cells stained.
29 33 line (Thompson et al 1983). It reacts with most breast cell lines and breast carcinomas but has no reaction with 22 1 TABLE 2 Method Number of 3 Patients 1. 1 3 1 1-1.7.1 (400pg) cold labelled Ly-2.1 (2mg): Arm Injection 26 2. 1311-17.1 (1mg) cold iodine labelled 7 Ly-2.J1 (2mg): Arm Injection 3 3. 13 1 1-17.1 (400pg) unlabelled Ly-2.1 9 (2mg); Arm Injection 3 4. 1311-17.1 (400pg): Arm Injection 4 11 0o, TABLE 3 0 000 13 Comparison of Immunolymphoscintigraphy Using 0900 S. 131 I-17.1 MoAb (400pg) and Cold Iodine Labelled 15 (Blocking) ly2.1 MoAb with Clinical and Pathological 000 Soo Assessment of the Axillae in 26 Patients with 00 0 o 17 Breast Cancer 0 0 Pathological Assessmenta 19 Node +ve Node -ve Scan Result b o0 21 +ve 13 1 -ve 1 11 0 0
I
0 23 Clinical Assessment +ve 8 -ve 6 7 a. Node +ve implies nodal metastases 27 Node -ve implies no nodal metastases b. Node +ve =.palpable axillary lymph nodes felt to 29 corLain tumour deposits.
Node -ve nodes not palpable, or if alpable, felt 31 not to contain tumour deposits.
33 albumin solution (HSA; Commonwealth Serum Laboratories, Melbourne, Australia). The murine MoAb Ly2.l (2mg) was -23 1 TABLE 4 Comparison of Irmunolymphoscintigraphy and Clinical 3 Examination in Predicting Axillary Lymph Node Metastases Parametera Imrunolympho Clinical Immunolymphoscintiscintigraphy Examinations graphy Clinical Examinaition 7 Sensitivity 93% 57% 100% Specificity 92% 58% 9 Accuracy 92% 58% 77% 11 Sensitivity
T
TP+FN
13 Specificity JtL
TN+FP
00 0Accu racy TP TN 06 Total No. of patients tested P900 0000 0TP True Positive; TN =True Negative; 0:00:0FP F~lse Positive; FN =False Negative 00 0
L
24 TABLE Concentration dose/gm Tissue) of Specific MoAb in Different Tissues 1 31 -IgG; Mean 13 1 1_,gG Cold IgG*; Metastatic LN 7 Normal LN Axillary Fat 6.4 x 1j-2 4.2 x 10-2 2.0 x 10-4 Mean 1.6 x 10-1 9.2 x 10-3 4.1 x 10-4 00 0 000 0009 0*00 00 0 0 0000 0 0 0 OP 0I 00 go 0000 0 44 0 0 0I 00 0 0 0 4( I 4
Claims (11)
1. A method for identifying and/or determining the location of a tumour which produces or is associated with a cytoplasmic, intra-cellular or cell surface marker substance, tha method comprising the steps of: injecting a human or animal subject with tL ,our specific antibody cr a fragment thereof specific for the marker and labelled with a radioactive isotope of an element or a paramagnetic conjugate; and an irrelevant (non-tumour reactive) antibody which has been labelled with a non-radioactive irotope where the tumour specific antibody is labelled with a radioactive isotope, or a non-paramagnetic conjugate where the tumour specific antibody is labelled with a paramagnetic conjugate; after a period of time sufficient for selective binding of the labelled tumour specific antibody to the tumour scanning the human or animal subject with a detector to locate the site or sites of uptake of labelled antibody or fragments thereof.
2. A method according to claim 1, wherein the tumour specific antibody is labelled with a radioactive isotor- of iodine, indium or technecium.
3. A method accordig to claim 1, wherein the paramagnetic conjugate is selected from manganese (II), copper (II) or cobalt (II).
4. A method according to any of claims 1 to 3, wherein the specific antibody and irrelevant antibody are of the same isotype. A method according to claim 4, wherein the humour specific antibody and irrelevant antibody both coumprise IgG, IgM or fragments thereof. 901219,PASDAT.062,29547-89.rsp,25 j_ i 26 0 0 0 0 000, 0 0 00 o o 0 0 00 00 G o o o 0 0 0 0 06 o00 00 0 00 oo a 0040 0 0 0 0 oo a 0 0 W 0 0 0 0 W 0 0
6. A method according to claim 1, wherein the tumour specific antibody and irrelevant antibody are both monoclonal antibodies.
7. A method according to any one of claims 1 to 6, wherein a human or animal subject is injected subcutaneously with the tumour specific antibody and irrelevant Fntibody.
8. A method according to any one of claims 1 to 7, wherein the tumour specific antibody and irrelevant antibody are administered at the same or substantially the same time, or the irrelevant antibody is administered up to five hours before administration of the tumour specific antibody.
9. An imaging composition which comprises: a tumour specific antibody or a fragment thereof labelled with a radioactive isotope of an element or a paramagnetic conjugate; and (ii) a non-tumour reactive antibody labelled with a non-radioactive isotope of the same or other element where the tumour specific antibody is labelled with a radioactive isotope, or a non-paramagnetic conjugate where the tumour specific antibody is labelled with a paramagnetic conjugate; in association with a pharmaceutically acceptable carrier or excipient. Il '2- r i i i" -i i J 27 An imaging compositicin according to claim 9, wherein the tumour specific antibody is labelled with a radioactive isotope of iodine, indium or technecium.
11. An imaging composition according to claim 9 or wherein the specific antibody and irrelevant antibody are of the same isotype.
12. An imaging composition according to any one of 00 claims 9 to 11, wherein the tumour specific antibody and ,irrelevant antibody are both monoclonal antibodies, a0 0 S13. An imaging composition according to claim 9, wherein the tumour specific antibody is 17.1 and the non- 0 0o tumour reactive antibody is Ly2.1 as hereinbefore o00 a 0 0 0 V o defined. o o
14. Methods for identifying and/or determining the 000*0 location of a tumour according to any one of claims 1 to 00 00 8; and imaging compositions according to any one of 0 claims 9 to 13, substantially as hereinbefore described. o0 a0 00 000 a 6 DATED this 19th day of December, 1990 THE UNIVERSITY OF MELBOURNE by its Patent Attorneys DAVIES COLLISON 901219,PASDAT.062.2954789.rsp,27
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| AU4778585A (en) * | 1984-08-31 | 1986-03-24 | Hybritech Incorporated | Monoclonal antibodies against metal chelates |
| AU6267186A (en) * | 1985-09-20 | 1987-03-26 | Regents Of The University Of California, The | Dual isotope color functional imaging utilizing radiolabelledmonoclonal antibodies |
| AU7865687A (en) * | 1986-09-19 | 1988-04-21 | Immunotech Partners | Affinity enhancement system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| AU4778585A (en) * | 1984-08-31 | 1986-03-24 | Hybritech Incorporated | Monoclonal antibodies against metal chelates |
| AU6267186A (en) * | 1985-09-20 | 1987-03-26 | Regents Of The University Of California, The | Dual isotope color functional imaging utilizing radiolabelledmonoclonal antibodies |
| AU7865687A (en) * | 1986-09-19 | 1988-04-21 | Immunotech Partners | Affinity enhancement system |
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