Classifications

• • 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
• • 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
• • 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
  • A61K51/1093—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 conjugates with carriers being antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2123/00—Preparations for testing in vivo

Definitions

• the present invention relates to a technetium-antibody conjugate.
• the present invention provides a conjugate of technetium with a radical having an antigen binding site uiherein the technetium thereof is radioactive.
• the preferred technetium isotope is 99m Tc.
• the present invention provides a technetium-antibody or antibody fragment conjugate which is preferential ly absorbed by a tumour cell as compared to a non-tumour cell.
• the conjugation is via a sulphide linkage.
• the present invention also provides a compound of formula Ab-Y-S-NTc(Hal) 3 where Hal is chlorine, bromine or iodine and including mixed halides, and Y is a conjugating chain and Ab is an antibody radical or a radical having an antigen binding site.
• Y is of the formula
• X Z " ' -[NH-C-(CH) n ] z wherein Z is H, alkyl, aryl, carboxy,halide hydroxy or amino, n is 1-10, X is NH, O or S and z is 0 or 1.
• Alkyl groups preferably 1 - 6 carbon atoms, aryl groups preferably 5 - 16 carbon atoms.
• the present invention also provides compounds of formula Ab-S-NTc(Hal) 3 Ab-NH-Y-S-NTc(Hal) 3 wherein Ab, Ab-NH or Ab-S represents an antibody radical or a radical having an antigen binding site and Y and Hal have the meaning given above.
• the present invention also provides the intermediate compounds Ab-SH Ab-NH-Y-SH wherein Ab, Ab-NH or Ab-S and Y have the meaning given above.
• Compounds in accordance with this invention may be produced by taking one of said intermediate compounds and reacting with TcN(Hal) 4 wherein Hal has the meaning given above.
• the intermediate compounds may be formed by a) reducing an antibody to form free sulphydryl groups.
• Such reduction may be effected in a number of ways but it is presently preferred to use dithiothreitol (DTT), b) reacting an antibody succinimidyl pyridyldithiopropionate (SPDP) or an analogue thereto appropriate to the compound desired to obtain an antibody conjugate containing a -S-S-group, reducing the conjugate to form a -SH group, c) using S- acetylmercaptosuccinic anhydride (SAMSA) or SH introducing compounds to produce a side chain on an antibody containing a -S-linkage and reducing to form a -SH group, It is preferred that said radical is an antibody.
• the antibody may be a monoclonal antibody.
• Antibodies useful in the present invention included those showing specificity for breast, brain, melanoma, lung, pancreas and colon tumours.
• the antibody may be an intact immunogobulin or a fragment of an immunogobulin maintaining a sufficiency of an antigen binding site such that it is preferentially absorbed by a tumour cell as compared to a non tumour cell.
• F(ab') 2 and F(ab') fragments in addition to whole antibodies, it is also possible to utilize F(ab') 2 and F(ab') fragments.
• Still further antibody polymers such as antibody pentamers IgM and derivatives of these such as immunogobulin monomers may be used.
• IgG 2a , I g G 2b , IgG 1 and IgG 3 are also useable.
• the compounds of this invention may be combined with pharmaceutically acceptable carriers.
• the mode of administration of the compounds of this invention will be as selected.
• the compounds of this invention may be administered intravenously, intraperitonealy, intrapluraly, intrapericardialy, intracerebospinal fluid and subcutaneously.
• the technetium-antibody conjugates of the present invention may be formed into pharmalogical compositions with appropriate pharmaceutically acceptable diluents.
• the technetium-antibody conjugates of the present invention are useful for in vivo detection of tumours such as by immunoscintigraphy.
• Radioactive isotopes of technetium coupled to MoAb have been used by us in the search for specific methods of diagnosing sma l l tumours. In this l ight we have successfully located tumours in both mouse and man, and antibodies have been administered either intravenously or subcutaneously, or by other routes. It is clear that radiolabelled MoAb can indeed localise in tumours in vivo and with the use of computer assisted tomography, with subtraction for non-specific effects, this method can then be utilized for the specific detection of tumours - both primary and secondary. However, there are problems of specific activity, specificity and high blood background which need attention before this technique can be accepted as a usefu l diagnostic tool.
• the isotope has an optimal gamma energy (140keV) for detection with currently available scintigraphic instrumentation and produces very little radiation exposure to patients undergoing scanning procedures.
• tumour cell lines Two tumour cell lines were used: one, the E3 clonal variant of the thymoma ITT(1) 75 NS(1) which was obtained by three successive rounds of fluorescent activated cell sorting of ITT(1)75NS cells stained with monoclonal Ly-2 antibodies and selected for the most fluorescent 1 % of cells.
• the murine cell line E3 was maintained in vitro in DME supplemented with 10 % heat inactivated newborn calf serum (Flow Laboratories, Sydney, Australia), 2mM glutamine (Commonwealth Serum Laboratories, Melbourne, Australia), 100 IU panicillin/ml and 100 mg streptomycin/ml (Glaxo Laboratories, Melbourne, Australia).
• E3 cells were washed twice in DME (without additives) and twice in DME containing 0.5 % BSA and used in the in vitro binding assays.
• the E3 cell line was maintained in vivo by the passaging of cells from ascites fluid produced in BCF 1 mice. Ascites were washed in DME and PBS, solid tumour grew after the s.c. injection of 10 6 -10 7 cells.
• the second cell line used was a human colonic carcinoma, COLO 205 (2), maintained in culture with RPMI containing the same additives; adherent cells were harvested with 0.125% trypsin (Commonwealth Serum Laboratories, Australia) washed with RPMI and injected s.c.
• MoAb Two MoAb were used: (i) anti-Ly-2.1 (IgG2a), an antibody raised against the murine alloantigen Ly-2.1 (3); and (ii) 250-30.6 (IgG2b), an antibody to human colonic secretory epithelium (4).
• the MoAb were isolated from ascitic fluid by precipitation with 40% ammonium sulphate, followed by dissolution in 0.01 M Tris buffer pH 8.0 and extensive dialysis against the same buffer and further purif ied by af f inity chromatography using protein -A Sepharose (Pharmacia Inc., Piscataway, NJ, U.S.A.)and purity was confirmed by gel electrophoresis and antibody activity assayed by a rosetting test (5).
• MoAb were label led with 99m Tc using two methods - the new method described herein, and a method using stannous chloride.
• the fractions (1 ml) containing the protein peak were added to the dried 99m TCcl 4 - salt residue and the mixture brought to pH 3.0 with 0.2M hydrochloric acid; after 2 minutes at room temperature, 0.1 M sodium phosphate was added and the pH adjusted to 7 by the careful addition of sodium hydroxide. Purification of the labelled MoAb was then achieved by gel chromatography with a Sephadex G-25 co lumn ( PD-10, Pharmacia).
• MoAb 100 microg, 1 mg/ml were labelled using the chloramine-T method (6): 2.5mCi of carrier-free Na 125 I (Amersham International Ltd., Amersham, England) and 3 microl of chloramine-T (1 mg/ml) were mixed with protein for 2 minutes at room temperature and the reaction then terminated by the addition of 3 microl of sodium metabisulf its (2.4 mg/ml).
• Iodinated MoAb was separated from free iodine by gel filtration using a PD-10 column.
• Serological Analysis A binding assay was developed to determine the stability and specificity of the 99m TcN-MoAb complexes.
• MoAb complexes were tested in one of two ways - either a) using one MoAb and two cell lines; or b) using two different MoAb and one cell line - both MoAb being labelled identically, one being reactive with the cell line, the other non-reactive.
• Polyvinyl chloride 96 well plastic plates (Pynatech Laboratories, Inc., Alexandria, Va) were washed with 1% bovine serum albumin (BSA) in PBS. In this assay either the number of cells or the quantity of MoAB could be kept constant while the other was varied.
• BSA bovine serum albumin
• Biodistribution Nude mice bearing COLO 205 xenografts or BCF 1 mice bearing the E3 thymoma were used.
• the first study compared the distribution of two 99m TcN-MoAb in BCF 1 mice; groups of 4 mice were sacrificed at 20 hrs, 30 hrs and 35 hrs after the injection of labelled MoAb.
• the second study compared the binding of a 99m TcN-MoAb complex to two different tumours - the E3 thymoma and COLO 205 xenografts.
• mice bearing the E3 thymoma were given i.v injections of either approximately 115 micro Ci of 99m TcN-labelled anti-Ly-2.1 (specific MoAb) and 200 micro Ci (4microCi/micro g) of 125 I labelled anti colon (non-specific MoAb) or 115 micro Ci of 99m Tc-labelled anti colon MoAb.
• Twenty-seven hours after injection mice were anaesthetised by intraperitoneal injection of 4% chloral hydrate (0.01 ml per g body weight).
• Vertical views of the mice were taken using a Toshiba GC 402A gamma camera and a low energy parallel hole collimator. A setting of 50 keV with an 80% window and 140 keV with a 20% window was used to image the 125 I and 99m Tc photons respectively. Data were stored in digital form by an MDS Modumed computer.
• MoAb were partially reduced with DTT to generate free sulfhydryl sites and mixed with the 99m Tc Cl 4 -, leading to the formation of 99m TcN-MoAb complexes.
• the 99m TcN-MoAb complexes were then tested in different serological assays to determine whether the label ling procedure damaged or altered the binding or specificity of the MoAb, and whether the complexes formed were stable.
• Fig.2 shows the specific binding of anti-Ly-2.1 labelled with 99m TcNCl 4 - on RF/J and C57BL/6 thymocytes; Amount of radioactivity bound as a function of antibody in the reaction mixture.
• RF/J reactive cells
• C57BL/6 non-reactive cel ls
• specific ratio cpm RF/J / cpm C57BL/6
• Fig.1 shows the binding of 99m Tc labelled anti-Ly-2.1 with SnCl 2 reduction on RF/J and C57BL/6 thymocytes; amount of radioactivity incorporated as a function of ce l l number.
• Fig.3 shows the specific binding of 99m TcNCl 4 - laballed anti-Ly-2.1 and anti-colon Mo-Ab on ITT(1) 75NS E3 target cells.
• 99m Tc/ml of 99 Tc eluted (8) As the number of labelled binding sites on the antibody molecule is determined by the chemical quantity of technetium present, the effect of labelling with increased quantities of Tc may be studied by the addition of 99m TcCl 4 - carrier to the 99m TcO 4 - used for labelling. The addition of 2 micro g 99 Tc to the reaction mixture was thus equivalent to increasing the 99m Tc activity used by a factor of 200. This approach was adopted to avoid the radiation hazards associated with the handling of high levels of activity and to overcome "dead-time" problems which would arise in the gamma counting of very high activities.
• Fig.5 shows the binding of two ati-Ly-2.1 conjugates - one containing added Tc carrier, the other carrier free on
• Biodistribution The in vivo localization and biodistribution of 99m Tc-MoAb complexes was examined by injecting mice with 99m -Tc-MoAb and determining the relative amounts of radiolabel accumulated in the tumour or the tissue. These results were used to calculate the localization ratio derived as follows: tissue (cpm/g) / blood (cpm/g).
• the non-specific antibody 250-30.6 the localization ratio of the liver, spleen and kidney were observed to be higher than that of the blood and at 30.5 hours the liver localization ratio was 5 times greater than that of blood - the reason for this high ratio is unknown, but may be due to the different reactivity of the MoAb.
• tumours (0.23-1.2g) could be visualized with the use of a small animal scanner as early as 2 hrs after injection of the specific 99m TcN-MoAb (results not shown) the visualization became well defined with time.
• the mouse in B. had an E3 tumour (1.0cm in diameter) which was easily seen as a distinct single entity on the right hind leg.
• the tumour was dissected and found to have a localization ratio (tumour to blood approximately 2.0). Radioactivity in this image is also concentrated in the central region of the mouse, indicative of significant distribution of antibody to large vascularized organs such as liver, lung and heart; a phenomenon that tends to obscure visualization of small tumours.
• the second image B The second image B.
• TcNCl 4 - complexes The chemical stability and activity of TcNCl 4 - complexes was determined in several serological assays which involved MoAb reactive and non-reactive cells; either one MoAb, two different target cells or conversely, two MoAb and one call target. In all studies specific binding of radiolabelled MoAb to target cells was demonstrated, the complexes were not non specifical ly "sticky" nor unstable with the release of 99m Tc to bind to other non reactive target cells. In vitro studies have shown that 99m TcNCl 4 - may be used to produce chemically stable MoAb complexes that retain their activity for at least 24 hours. Furthermore these complexes may be prepared at a clinically useful specific activity without any changes in the in vitro properties. For example, it was possible to increase the amount of 99m Tc bound to b 200 fold without affecting MoAb activity (text fig.5).
• tumours (0.8 - 1.1 cm in diameter) could be easily visualised (A.) but the ultimate sensitivity of this technique lay in the detection of small tumours (0.3 - 0.6cm in diameter) that were located near vascular organs, such tumours being detected without the requirement of a blood pool subtraction
• TcNCl 4 - monoclonal antibody can be simply produced and have high activity for specifically localizing tumours both in vitro and in vivo.
• the coupling method we consider the coupling method to be superior to other methods of coupling 99m Tc to antibody and the immunoscintigraphic findings to be superior to that obtained with radiolabelled iodine.
• mice RF/J, CBA, AKR, C57BL/10(B10), BALB/c, C57BL/6(B6), and (C57BL/6xBALB/c) F1 (B6CF1) mice were brdd in our colony.
• Tumor Cell Lines Human tumor cell lines (CEM and Bordin - an EBV induced B cell line) were cultured in RPMI 1640 medium with L-glutamine. BW 5147 and several clonal variants (E3,D1) of the murine thymoma ITT(1)75NS (20) were cultured in DME with L-glutamine.
• the clonal variant ITT(1)75NS.E3 (E3) was maintained by serial passage in ascitic fluid in (B6CF1) mice.
• B6CF1 mice For imaging experiments 10 6 -10 7 cells injected subcutaneously into B6CF1 mice and reached a size of 0.5-1.0 cm in diameter prior to experimentation.
• Monoclonal Antibodies The details of MoAb are shown (Table 1).
• IgM antibodies were isolated from ascitic fluid by dialysis against water at 4°C, after which the precipitate was collected and resuspended in phosphate buffered saline (PBS, pH 7.3): IgG antibodies were prepared by precipitation with 40% ammonium sulfate [NH 4 (SO 4 ) 2 ], followed by dissolution of the preparation in 0.01 M Tris buffer (pH 8.0); after dialysis against the same buffer, the IgG fraction was further purified by either: (i) adsorption onto Protein-A-Sepharose, washing with PBS and eluting with either 0.2 M glycine-HCl (pH 2.8) or citrate buffers (pH 5.0, 4.0, 3.0) and neutralization with saturated Tris, after which antibodies were dialyzed against PBS; or (ii) ion-exchange chromatography on DEAE-Sephacel and with elution using a linear gradient of 0.5 M NaCl In 0.01 M Tri
• MoAb were labeled with 99m TcNCl 4 - by one of two procedures: (i) 100-200mug of MoAb (1 mg/ml) was directly added to the dried 99m TcNCl 4 - salt residue, or (ii) a modified version of the method previously described (19) where 20 mul of dithiothreitol (DTT, 115 mg/ml) was added to 200 mug MoAb (1 mg/ml) and the solution allowed to stand for 30 minutes at room temperature; it was then transferred to Biogel P6 to remove unreacted DTT and the column eluted with 0.1 M sodium acetate (pH 4.0).
• DTT dithiothreitol
• the protein fraction (1.5 mis) was added to the dry TcNCl 4 - residue, reacted for 2 minutes at room temperature prior to adjusting the pH to 7 with sodium hydroxide.
• the 99m TcN- labeled MoAb was purified by passage over Sephadex G-25 (PD-10) and sterilised using a 0.22 mum membrane filter.
• Serological Analysis In vitro cel l binding studies were performed on cultured tumor cell lines or mouse thymocytes (19). 99m TcN- MoAb complexes were tested in one of two ways: (i) using one MoAb and two different target cells; or (ii) using two different MoAbs and one target cell line. The ability of the MoAb to bind to target cells was assessed after each step.
• tumor cells or thymocytes were incubated for 30 minutes on ice with one of the following: (i) untreated MoAb; (ii) DTT treated MoAb; or (iii) 99m TcN- labeled MoAb.
• the cells were then washed 3 times with PBS (0.5% BSA), resuspended in PBS and then treated with iodinated sheep anti-mouse immunoglobulin ( 125 I-SAM) for 30 minutes on ice.
• the cells were then washed 3 times with PBS (0.5% BSA) to remove unbound 125 I-SAM and the amount of 125 I-SAM bound determined.
• mice bearing E3 tumors were used in two studies. The first compared two identically labeled different MoAb and one tumor; the second compared a specific MoAb in mice bearing several tumors.
• B6CF1 mice bearing the E3 thymoma 0.5 - 1.0cm in diameter
• mice bearing the E3 thymoma were given intravenous injections of approximately 115muCi (12muCi/mug) of 99m TcN- labeled anti-Ly-2.1 (specific MoAb) or anti-Ly-1.1 (nonspecific MoAb).
• Each animal was given an intraperitoneal injection of 4% chloral hydrate (0.01 ml/g body weight) imaged 4-28 hours after injection.
• mice Vertical views of the mice were taken using a Toshiba GC 42A gamma camera and a low energy parallel hole collimator using a setting of 140 keV with a 20% window to image the 99m Tc photons. Data were stored in digital farm by a MDS modumed computer.
• RESULTS Radiolabeling of MoAb with 99m TcNCl 4 - After radiolabeling, unbound reaction products were removed by passage of the final reaction mixture through a gel permeation column of Sephadex G-25 (PD-10). The yield of 99m TcN passing through the column was then a measure of the success of radiolabeling and typical yields were 80-90%. (A typical example of the elution profile is shown in Figure 6).
• 99m TcN-anti-Ly-2.1 achieved almost identical binding to both cell types ( Figure 7a), with a specific ratio (cpm bound RF/J / cpm bound B10) of approximately 1.2.
• the conclusion is that the 99m TcN-MoAb complexes produced in this way were either unstable or "sticky" and on exposure to target cells the 99m TcN bound nonspecifically.
• 99m _TcNCl 4 - complexed to sulfhydryl groups after partial reduction As the former label ling method gave law specificity (previously due to nan-specific labelling) an alternative method of labeling was designed, utilizing the known ability of 99m TcNCl 4 - to form a stable covalent linkage to sulfur atoms. MoAb were partially reduced with D T T to generate free sulfhydryl sites and mixed with the 99m TcNCl 4 -, leading to the formation of 99m TcN-MoAb. These complexes were shown by sodium dodecyl sulfate polyaerylamide gel l1ectrophoresis (SDS-PAGE) to consist of intact IgG. The binding assay demonstrated 99m TcN-MoAb complexes produced in this way to be specific and to yield workable specificity ratios. The E3 (Ly-3+) cell line bound
• the partial reduction method was then used to radiolabel a panel of eleven different MoAb, including some of the same specificity but of different isotypes. All MoAb were testsd in the binding assay, and bound specifically to reactive target cells (Table 2). Four different Ly-2.1 MoAb were tested; there was a 10-15 fold difference between the binding of 99m TcN-labeled IgG 2a and IgM anti-Ly-2.1 (monomer) MoAb on reactive target cells (CBA, RF/J) compared to that found with nonreactive target cells (BALB/c, C57BL/6), whereas the IgG 1 and IgG 3 Ly-2.1 MoAb produced specificity ratios of 30-50 and 50-70 respectively. Other MoAb were also highly selective e.g.
• the immunoperoxidase method was also used to assess the MoAb activity of two IgM MoAb, before and after labeling with 99m TcNCl 4 -: (a) 3E1.2, which reacts strongly with membrane and cytoplasm of breast carcinoma and with the luminal membrane of normal breast and (b) 5C-1 , which reacts with colonic carcinoma; the labeling procedure used did not significantly alter the binding ability of the radiolabeled MoAbs (Table 3).
• Immunoreactivity of 99m TcNCl 4 - labeled MoAb It was necessary to show that the partial reduction procedure used to label MoAb with 99m TcNCl 4 - did not significantly compromise the binding ability of the MoAb to bind reactive target cells and this was demonstrated in three ways.
• E3 and 01 are high and low Ly-2 + variants of the ITT(1 )75NS cell line, and BW 5147 being Ly-2- was used as a control.
• the amount of binding was in proportion to the antigen density and 99m TcN-anti-Ly-2.1 bound the E3 cell line 8 times more antibody than the D1 cell line and incorporated up to 100 times more radiolabel than did the nonreactivs BW 5147 cell line (Figure 13).
• Imaging The four different Ly-2.1 MoAb (IgG 1 , IgG 2a , IgG 3 and IgM) were used in imaging experiments using B6CF1 mice bearing E3 tumor grafts to determine which subclass best localized the tumor in vivo.
• mice with E3 tumor (0.82 cm in diameter) located on one thigh were given intravenous injections of 99m TcN-anti-Ly-2.1 (IgG 2a ) or 99m TcN-anti-Ly- 1.1 (IgG 2a ), the control antibody.
• Scintigrams images obtained 28 hours after injection demonstrated the specific localization of the 99m TcN-anti-Ly-2.1. Radioactivity was concentrated in the central region of the mouse, indicative of the significant antibody distribution to vascularized organs such as the liver, lung and heart but the tumor was easily defined.
• Iilhen 99mTcN-anti-Ly-1.1 was used as a nonreactive isotype control, the definition of the tumor was poor relative to the images obtained with specific MoAb and only blood pool activity in the tumor was observed, with no specific localization.
• B6CF1 mice hosting three E3 tumors were scanned 28 hours after the intravenous administration of 99mTcN-anti-Ly-2.1 (IgG2a), and all three tumors could be visualized.
• the high blood pool activity hindered visualization of the tumor close to the vascular organs such as the heart and liver.
• the IgM Ly-2.1 MoAb (monomer) was used to specifically localized E3 tumors in B6CF1 mice, and mice with two E3 tumors (0.62 cm and 0.55 cm in diameter) were scanned 4 and 28 hours after an intravenous injection. Both tumors could be visualized 4 hours after injection, and the tumors became progressively better defined with time. From the scans obtained it was apparent that the IgG, Ly-2.1 MoAb resulted in superior images compared to the images obtained with the IgM MoAb and also those obtained with the IgG1 and IgG3 MoAb (data not shown).
• the labeling procedure adopted did not damage the binding specificity of the antibody molecule and did not alter the antibody antigen binding capacity (Figure 12) as can occur with the conjugation of metallic radionuclides via bifunctional chelates (6). Up to 60% of the radiolabeled preparation was able to bind specifically to target cell ( Figure 11). Furthermore, the degree of antibody binding wa3 dependent on the antigen density of the target cells, hence the Ly-2HIGH E3 cell line bound 8 times more 99m TcN-anti-Ly-2.1 than the Ly-2LOW D1 cell line (one has approximately B times the antigen density of the other) and 10 times more than the nonreactive BW5147 (Ly-2.1-) cell line ( Figure 13). Finally, high specific activities were achieved which allowed the specific localization of labeled MoAb in the appropriately reactive murine tumors.
• Second 99m TcN-MoAb could specifically detect more than one tumor and this study showed that several tumors in the one mouse could be specifically localized.
• the method is useful to detect murine tumors and results indicate value in patients with cancer.
• Perkin AC, Pimm MV and Birch MK The preparation and characterization of 1 11 In-labeled 791T/36 monoclonal antibody for tumor immunoscintigraphy. Eur J Nucl Med 10:296-301 , 1985. 9. Rairweather DS, Bradwell AR, Dykes PW, et al: Improved tumor localization using indium-111 labeled antibodies. Br Med J 287:167-170, 1983. 10. Rainsbury RM, Ott RJ, Uestwood JH, et al: Localization of metastatic breast carcinoma by a monoclonal antibody chalats labeled with Indium-111. Lancet :934-938, 1983. 11.
• Figure 11 Percentage binding of 99m TcNCl 4 --labaled anti-Ly-2.1 ( ⁇ ) to E3 target cells; the control anti-Ly-1.1 antibody did not bind >5% at any dilution (not shown). The amount of radioactivity incorporated as a function of cell number is shown.

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