MXPA98005565A - Antibodies with positive reduced load - Google Patents

Abstract

Antibodies that have been modified by chemical conjugation with reactive agents with free amino groups. Among the chemical agents used in connection with the invention are the heterobifunctional agents and biotin. Modified antibodies are also useful in the diagnosis and therapy of cancer and other mammalian diseases. The diagnosis used includes immunoscintigraphy. The modified antibodies can also be conjugated with biologically active labels or molecules for use in diagnostics and therapies. The modified antibodies can also be formulated in pharmaceutical compositions for these purposes.

Description

ANTIBODIES WITH REDUCED POSITIVE LOAD Background of the Invention Field of the Invention The present invention relates, in general, to modified antibodies. More specifically, the invention relates to chemically modified antibodies having an increased binding specificity, improved pharmacokinetics, and localization capabilities. These modified antibodies are particularly useful in the diagnosis and treatment of cancer and other diseases in mammals.

DESCRIPTION OF THE PRIOR ART The use of antibodies, particularly monoclonal antibodies ("MABs") has the potential to be an extremely valuable approach in the diagnosis and treatment of cancer.An important property of MABs is their specificity. for simple antigens. MAB 's specific for antigens of tumor cells have been produced. It has also been shown that MAB's can be efficiently coupled to attachments such as radionucleotides. These MAB 's radio-labeled are useful for providing data or clinical information, as for example the image of a tumor from immunosynatography, also known as camera image or radioimmunoimage. In immunosynatography, MAB's are allowed to bind to a specific tissue or tumor types that have antigens recognized by MABs. Then, the radionucleotides are visualized by using the appropriate technology, such as through the use of a germanium chamber. It is the unique specificity of MAB's that allows * its application in the immunosynatography of tumors and others types of tissues. However, the use of MAB 's in immunosy - tography has been limited due to the high levels of posterior imaging and the low binding capacity of MAB' s with their antigens. Experimental studies suggest that The biodistribution of radiolabelled MABs depends on many factors, including the specificity and time of antibody release. For an effective diagnosis of a tumor by immunosynatography, the antibody that binds to an antigen that is dense and homogeneous on the surface of the tumor cell. Effective diagnosis by means of immunosyntatography also requires that the selected antibody is effectively bound to the tumor antigen. However, frequently MAB's that bind to the appropriate antigen frequently does not require a high binding affinity. In addition, even the use ß of these MABs that bind with a relatively high affinity to other MAB 's can produce a high level of non-specific binding, resulting in high levels of subsequent imaging when used in immunosynactography. Thus, there is a need for a method that improves the effectiveness of MAB binding in order to improve immunosynatography as a diagnostic tool. Additionally, the cytotoxic effect of MAB's can increase markedly by coupling radionucleotides, drugs or toxins. The unique specificity of MABs increases the hope of developing immunotherapy. In immunotherapy, biologically active agents are supplied, using MABs, for a cell type Particularly unwanted cells, such as tumor cells, for example, affect the unwanted cell types without affecting other cells of the subject. However, immunotherapies require antibodies with an extremely high specificity in order to avoid affecting them healthy tissue. Thus, the method to increase the specificity of MABs will be highly beneficial in the achievement of effective and safe immunotherapies. Many MAB 's remain in circulation for several days after their introduction into the subject. These do not is desirable at least for two reasons. One of these reasons is that the MAB's circulating produces high levels of later images in the scintografía. A second reason is that circulating MABs coupled to radionucleotides or other potentially cytotoxic agents can produce undesired side effects in the subject, after a prolonged exposure. Thus, there is a need for a method to reduce the time of release of MABs. Of course, a very sharp reduction could result in the MAB's being eliminated before any effective use can be made of MAB 's. Thus, there is a particular need for a method of reducing the release time of MABs without substantially affecting the uptake or absorption of MABs in the tumor or other target tissue. One factor that is critical in determining both the specificity and the time of release of the antibody is the form of the antibody. According to the use herein, "intact" antibody molecule will refer to an unmodified antibody molecule comprising two heavy chains and two light chains, The entire, intact antibody molecule is considered to be one of the reactant of the equation Chemistry of Figure 1. As can be seen in Figure 1, the intact molecule is divided into dominant Fc and Fab. The bivalent F (ab ') 2 form of the Fab fragment can be produced by digestion of the dominant Fc with a protease.

* The two heavy chains (designated "H" in Figure 1) cooperate with each other by one or more disulfide bridges. In intact molecules these disulfide bridges are normally protected from the reducing agents. However, it has been found that removing the dominant Fc allows easy reduction of the disulfide bridges. Thus, F (ab ') 2, the monovalent form, can be produced from F (ab') 2 by the action of a mild reducing agent. The author Perham, P. in Fragmentation of monoclonal IgGl, IgG2 and IgG2b, from BALB / c mice. J. Immunol 131: 2895 (1983), the presentation of which is incorporated herein by reference, describes the method for the production of F (ab ') and F (ab') 2. The schematic representation of the changes that are created occur with this method show by the chemical equation of Figure 1. It has been found that Fc is responsible for many of the non-specific bonds of antibody molecules. It is also believed that the molecular weight of the fragments is below the threshold for glomerular filtration, thus allows the rapid removal of fragments. Therefore, one approach to increasing the release time of the antibodies for use in radioimage is to break the intact antibody into several fragments, such as F (ab ') 2 and its divalent form, F (ab') 2. As expected, these fragments are release into the body so quickly that it reduces its * utility. Moreover, these fragments can result in reduced uptake or absorption by the tumor or other target tissues relative to the intact antibody. Thus, although the use of these fragments in immunosynatography may provide better release and target tissue greater than the average posterior image with intact MABs, it has been found that the absolute concentration of MABs in the antigen target tissue content to which binds • > The MAB 's is up to three times or more with MAB' s intact than with any of the fragments. In addition, both types of fragments are removed from the bloodstream very quickly. According to this, the effective time for diagnosis or therapeutic techniques using these fragments is very short. 15 The heterobifunctional reagents are reagents that have two groups capable of participating in the different reactions. For example succinimidyl 3- (2-pyridyldithio) propionate (SPDP) is heteobifunctional since its N-hydroxysuccinimido ester group reacts with amino groups and structure 2 -pyridyl disulfide reacts with aliphatic thiols. Orlandi et al. In contrast to the reactivity of a monoclonal anti-tumor antibody after the introduction of 2-pyridyl disulfide groups, Hybridoma 5: 1-8 (1986) reported that a increase in the in vivo link of MAB 's that arises against the Human ovarian carcinoma can be obtained after chemical conjugation with a heterobifunctional reagent, SPDP. The conjugated MABs used by Orlandi et al., Has an average of 11 PDP groups per molecule. Orlandi et 5 al. found that modified MAB 's increases its in vitro binding activity to the point where molecules not detected by the modified MAB' s can be detected. These researchers did not report studies about the use of conjugated MABs in vivo. Additionally, these researchers believed that molecules that have a very low number of antigenic sites were detected by conjugated MABs. According to these, the modified MAB's of PDP has a really reduced target cell specificity in relation to its unmodified counterparts. Thus, in spite of the advantages mentioned above, there is still a need for modified antibody fragments which have a higher specific activity for tumor antigens, allowing the absolute concentration of the antibody to accumulate in the tumor, and also that it has a relatively rapid release time in the blood volume, and that it is not so fast that it reduces the therapeutic and diagnostic effectiveness.

Brief description of the Figures Figure 1 shows a schematic representation of the changes that are believed to occur in a method for producing fragments of F (ab ') and F (ab') 2. Figure 2 shows the retention of all the antibody of the different preparations of Lym-1 MAB's radio labeled in mice, without fur, athymic. Figure 3 shows the biodistribution as the% dose / gram injected Lym-1 of MAB's and Lym-1 modified in mice without palaje that carry a human lymphoma seven days after the injection. Figure 4 shows the biodistribution as average Lym-1 tumor / organ of MABs and modified Lym-1 in uncoated mice, which carry a human lymphoma, seven days after injection. Figure 5 shows the biodistribution as% dose / gram injected of F (ab ') 2 of Lym-1 MABs and modified Lym-1 in mice without fur, which carry a human lymphoma after 5 days of injection. Figure 6 shows the biodistribution as a average tumor / organ of F (ab ') 2 of Lym-1 MAB's and Lym-1 modified in mice, without fur, carrying a human lymphoma, after five days after injection. Figure 7 shows the image obtained 7 days after the injection of intact Lym-1 labeled with 1-25 131.

Figure 8 shows the image obtained 7 days after the injection of modified Lym-1, labeled with 1-131. Figure 9 shows an image obtained 7 5 after injection of modified Lym-1 labeled with 1-131. Figure 10 shows the image obtained 5 days after injection with modified Lym-1 labeled β with 1 -131. Figure 11 shows retention in the whole body of different preparations of radiolabeled monoclonal antibodies B72.3 in nude mice without fur. Figure 12 shows the biodistribution as% dose / gram injected of MABs B72.3 and B72.3 modified in 15 mice, without fur, carrying a carcinoma of colon LS174T, four days after injection. Figure 13 shows biodistribution as a tumor / organ average of MABs B72.3 and B72.3 modified in mice, without fur, carrying a colon carcinoma LS174T, four days after injection. Figure 14 shows the image that is obtained a few days after the injection of modified B72.3, labeled with 1-131.

Figure 15 shows the image obtained on the fourth day of injection of modified B72.3, labeled with 1-131. Figure 16 shows the retention throughout the body 5 of the different preparations of. TNT-1 MAB's radiolabelled in mice, without fur, athymic. Figures 17A-D show a series of bar graphs. Figures 17A and 17C represent the percentage of intact, injected TNT-1 and biotinylated TNT-1 located in a tumor and in various tissues. Figures 17N and 17D represent the averages of labeled antibodies located in a tumor and in various organs. Figure 18 shows a line graph depicting the whole body release of intact TNT-1, modified TNT-1 and F (ab ') 2 fragments in Balb / c mice. Figure 19 shows a linear plot showing that ß * represents the release throughout the body of intact Lym-1, 1 modified and F (ab ') 2 fragments in Balb / c mice.

Summary of the Invention One aspect of the present invention relates to an antibody conjugated to a chemical reagent in at least a plurality of free amino groups disposed in the antibody to produce a modified antibody. He The antibody has a net positive charge compared to the intact antibody. Also, the antibody has an average release in vivo between the average release of F (ab ') 2 fragments and intact antibodies of the same type. In the antibodies in this aspect of the invention, the chemical reagent is not a heterobifunctional agent. The antibodies also include a reagent portion attached thereto. The antibody can be a monoclonal antibody or a polyclonal ß antibody. The chemical reagent can be biotin a chelate math, such as N2S2 or N2S4, another chelator, such as EDTA, DPTA or TETA or a dye, such as FITC. The chemical part is often labeled as a radionucleotide. The radionucleotide can be a Technicon or halogen radionucleotide, such as 1251 or 1311. In certain embodiments, labels are detected using magnetic resonance imaging. The chemical part can be a biologically active molecule, such as a toxin, a drug or a chelate. Appropriate drugs include methotrexate, 5-fluoro-uracil, cis-platinum and adriamycin. An appropriate toxin is chain A ricin. Another aspect of the present invention is the pharmaceutical composition for immunosynography. The composition includes a labeled antibody conjugated with a chemical reagent in free amino groups arranged in the labeled antibody, such that it has a charge positive, net, reduced compared to the intact antibody, # and the pharmaceutically acceptable excipient, carrier or base for immunosynography. Yet another aspect of the present invention is a method for preparing a modified labeled antibody that has an increased antigen binding specificity, a reduced non-specific binding, or a reduced in vivo release time. The method includes the following steps: obtaining an intact antibody having binding specificity for an antigen to be detected, the antibody The native has a plurality of free amino groups disposed therein, reacting at least one of the free amino groups with a chemical agent to produce a modified antibody, such as for example the modified antibody having an isoelectric point lower than the isoelectric point of the intact antibody, and label the modified antibody with a label that can be detected. The method produces a modified labeled antibody. The label in this method can be detected by immunosynatography, such as a gamma camera. Still another aspect of the invention is a method for locating the antigen in mammals. This method includes obtaining a labeled modified antibody that has a binding specificity for the antigen to be located. The modified antibody labeled has less free amino groups and a reduced isoelectric point in comparison with the unmodified antibody of the same type, and has a detectable label incorporated therein. The labeled modified antibody is administered to mammals. The method allows the antigen and the modified antibody to bind in vivo. The labeled modified antibody bound or bound to the antigen is detected, whereby the antigen is localized. The antibody can be an intact antibody chemically modified at the free amino groups. This intact antibody can be chemically conjugated with a heterobifunctional reagent or chemically conjugated with biotin. A further aspect of the invention relates to the method for treating a disease condition in mammals. This method includes obtaining an intact antibody, specific for diseased tissues in mammals. The intact antibody has a plurality of free amino groups therein. The method also includes modifying at least one of the free amino groups by conjugation with a chemical reagent other than the heterobifunctional reagent to produce a modified antibody. The modified antibody has a reduced isoelectric point compared to the intact antibody. The biologically active molecule binds to the first binding site arranged in the modified antibody different from the chemical reagent. Then, the antibody is administered to the mammal, so the disease condition is treated.

DETAILED DESCRIPTION OF THE INVENTION We have discovered that chemical modification of antibodies, including MAB's, human antibodies, genetically engineered antibodies, chimeric antibodies, synthesized antibodies and polyclonal antibodies, by conjugation with a reagent that modifies the free amino groups, can increase the binding specificity of the antigen, reducing the non-specific binding and reducing the release time in vivo. Antibodies that are modified in this way have a positive, net, reduced charge, compared to the intact antibody. Examples of the reagents we use to modify the free amino groups according to the present invention include heterobifunctional reagents such as SPDP, or biotinylated reagents. However, those skilled in the art will recognize that a wide variety of chemical reagents can be used to modify free amino groups and thereby reduce the average isoelectric point of the antibody. Thus, for example, methyl chelates, such as N2S2 and N2S4, other chelators such as EDTA, DPTA and TETA, and a number of temps such as FITC can be used to achieve effective results according to the present invention. In Nucí .Med. Biol. , 18: 179-185 (1991) describes a N2S4 bond with antibodies for purposes other than those described according to the present invention. The article is incorporated as a reference. The chemical modifications to the free amino groups surprisingly lead to an improved accumulation of the modified antibodies in the target cells containing antigens to which the antibodies will bind. It is believed that heterobifunctional reagents different than SPDP, including sulfosuccinimidyl 2- (p-azido salicylamido) ethyl 1-1, 3 '-dithiopropionate (SASD), sulfosuccinimidyl 2- (m-azido-o-nitrobenzamido) -ethyl-1,3'-dithiopropionate ( SAND), sulfosuccinimidyl (4-azidophenyldithio) propionate (sulfo-SADP) and 2-aminothiolane * HCl (reagent of Traut) provide similar results when conjugated with antibodies according to the present invention. The modified antibodies of the present invention can be linked, advantageously, elsewhere chemistry to provide a benefit in diagnosis and treatment. For example, any of a variety of well-known labels, such as radionucleotides or an enzyme, can be linked. The therapeutic part can also be linked, such as the antineoplastic compound or toxin.

Both heterobifunctional agents and biotin have previously been used with bonds to bind labels and other parts to the antibodies. Biotin itself can function as a label in certain circumstances. However, neither the biotin nor the heterobifunctional agents have been used with the aim of modifying the antibodies to achieve an improved binding specificity, reducing the non-specific binding and reducing the release times in vivo. Thus, unlike the present invention, the above antibodies had no modifying agent, such as a heterobifunctional agent or biotin, attached to the first place therein and a linked label or a chemical part in the second place thereof. In the present invention, the second binding or binding site will generally not have a binding modifier agent of the same type as the modifying agent linked to the first binding site. Thus, the improved accumulation of the modified antibodies is due to the modified specific binding capacity. We have found that by conjugating, on average, only one PDP group per antibody molecule, a dramatic increase in the specificity of the molecule occurs for its target cells, relative to the unmodified antibody. Similar results were obtained using biotinylated antibodies.

Wp We have also found that the chemical modification of the free amino groups in IgG by conjugation with a heterobifunctional reagent or biotin, advantageously, also improves the release from the normal tissues. Although a link is not desired by a particular explanation of this effect, it is conceivable that these modifications lead to fragmentation of the antibodies in a form having a molecular weight below the glomerular filtration threshold, thus allowing the rapid removal of the fragments. It is still possible that fragmentation of the antibody occurs in the monovalent form of the antibody. Whatever the exact shape of the resulting fragments, the removal of these fragments is, advantageously, not so fast as to overshadow the effectiveness of the diagnosis and treatment of modified antibodies. As presented above, the modified antibodies useful in the practice of the invention are chemically modified at the free amino groups and are additionally labeled with a detectable label. Significantly, while both the antibody itself or the chemical reagent used to modify the free amino groups can be labeled, we have also shown that the labeling of the modified antibody in a different place A free antibody can be provided to the free amino groups useful in the practice of the invention. In addition, when the label is chemically conjugated with an antibody, either before or after the chemical modification of the antibodies to the free amino groups, this label can be linked to the antibodies in a different place from the free amino group and in a specific site. different from the reagent that modifies the amino group when this reagent is a heterobifunctional reagent. ß As it is presented specifically here, the tyrosine residues present in the antibody protein can be modified by radioiodination. However, detectable labels that can bind to the tyrosine residues of the antibody are not limited to iodine. Other labels that can bind to the tyrosine residues in the antibody include halogen radionucleotides, such as isotopes of F, Cl, Br, I and others. The binding of these halogen radionucleotides is described in ilbur, Bioconj. Chem., 3; 433-470 (1992), whose presentation is mentioned in the invention as a reference. Technicio radionucleotides bound to other residues of antibody molecules. Further, Other labels and methods for labeling antibody proteins are useful in the practice of the invention, as will be apparent to one skilled in the art. Those of us skilled in the art will appreciate that the functional groups in the amino acid side chains of the antibody protein can serve as places for union of labels. The selection of labels, binding sites and methods to conjugate the label and the antibody. They will be appreciated by those skilled in the art. The important consideration with respect to the operability of the invention is that the modified antibody has a bound tag. Generally, we have found that chemically modified antibodies have reduced β-isoelectric points (pls) relative to unmodified antibodies that present a specificity towards an improved objective. More especially, our results demonstrated that the chemical modification of the free amino groups in the antibodies can confer this specificity towards the improved target. These chemical modifications can include modifications by agents such as, but not limited to, the heterobifunctional agents and biotin, referred to above. In FACT, Any chemical modification of the free amino groups present in the antibody that will effectively reduce the pl of the antibody will provide the specificity towards the improved target. While we do not wish to be limited to any particular theory to explain the origin of these improvements, we postulate that the non-specific antibody binds due, in part, to non-specific electrostatic interactions.

These are reasonable in view of the observation that MAb 's are positively charged with a physiological pH, while the cells of mammals are negatively charged (Eichmann et al., J. Exp. Med. 131_207) 1970); Silvia Filho 5 et al. J- Leu ocite Biol. 41: 143 (1987)). Thus, altering the character of the positive charge of the intact antibody effectively decreases the non-specific binding thanks to the ß interactions between the negatively charged tissues and the positively charged antibody proteins. When minimizing these non-specific interactions, the specificity of the antibody is attributed to the antigen binding domains of the antibody is mainly responsible in determining the specificity of the binding. Thus, any antibody having a plurality of amino acid parts modified as the pl of the antibody is reduced relative to the unmodified antibody which will exhibit an improved target specificity by virtue of having reduced non-specific interactions with non-cognate antigens. However, we also discovered that a second The characteristics of the antibodies modified according to the invention make them particularly useful for localization of antigens in vivo. Two factors that can improve the average signal, in relation to sound, which can be represent as an "average tumor / organ", in the F. Procedures for antigen-based images of the antibody are: (1) increase the tumor's location, and (2) reduce the levels of labeled, specially bound antibodies. We can now find that chemically modified MAb's that have reduced isoelectric points relative to intact MAb 's, unmodified, can advantageously increase link specificity * while reducing the non-specific binding and reducing the release time throughout the body in relation to the unmodified antibody. In addition, we have discovered that the increase in antibody specificity and localization capabilities can be achieved using chemically modified antibodies that have a label on them. detectable. This label can, for example, be a radionucleotide. More especially, we have found that antibodies modified to contain parts of biotin have a substantially improved capacity for the bound antigen. As will be indicated below, the Biotinylated labeled antibodies have been used in a method to localize tumor cells in vivo. In the practice of the method of the invention, it is essential for the chemically modified antibody to be directly labeled. This contrasts with the methods they employ indirectly labeled antibodies like the one l describes in Khawli et al. in Antibody, Immunoconjugates and Radiopharmaceuticals, 6:13 (1993), the disclosure of which is incorporated herein by reference. Thus, reagents useful in procedures for the improved localization of tumors can be produced by obtaining an antibody having a binding specificity for the antigen of the desired target, chemically modifying the free amino groups in an antibody using a reagent as a reagent Heterobifunctional or biotin and then the antibody is labeled with a detectable label as a radionucleotide. In practice, the order of steps for chemical modification and radiolabelling is optional. In addition, a step can be eliminated to radiolabel antibodies substantially purified if the antibodies employed in the method are MAb 's and if the hybridoma produced by these MAb' s is propagated in the growth medium containing the labeled precursors that are incorporated into the MAb 's products of the hybridoma. So, for example, the MAb 's Radiolabelling and biotinylation useful in connection with the invention can be produced by propagating the cell line that produces MAb 's in the growth medium containing radiolabeled amino acids, by collecting the radiolabelled MAb' s, and the biotinylated MAb 's and radio labels. Alternative methods for labeling antibodies, either before or after the biotinylation step, will be apparent to those skilled in the art. In the method described here, while is related to the present of Khawli et al., In Antibody, Immunoconjugates and Radiopharmaceuticals, supra, advantageously involves fewer steps to achieve images of the Hr antigens of tumor cells, in vivo, and unexpectedly provides better results than those that only use radiolabelled antibodies lacking biotin groups. Thus, we describe in the present that biotinylated antibodies have an improved target location when compared to non-biotinylated antibodies. Since an antibody useful in connection with the invention can be By detecting by virtue of the label carrying antibody, the presence of labeled biotin groups in the antibody provide obvious advantages with respect to the target and detection. Thus, the essential characteristics of the antibodies useful in connection with the invention and those of the The antibodies are: (1) having aminolateral moieties chemically modified such that the pL of the modified antibody is reduced relative to unmodified antibody, and (2) they house a label that can be detected by the detection mechanisms.

Advantageously, the modified antibodies of the present invention have a surprisingly improved diagnostic and therapeutic effectiveness in relation to antibody fragments, such as for example F (ab ') or F) ab') 2. The following examples show the exemplary method for introducing, on average, a PDP group into a monoclonal antibody. EXAMPLE 1 Modification of Lym-1 with SPDP 10 Lym-1 (IgG2a), in monoclonal antibody against B-cell lymphoma is obtained as in Epstein, A.L. et al., Two new monoclonal antibodies, Lym-1 and Lym-2, Reaction of human B-lymphocytes and derived tumors, with potential for immunodiagnosis and immunoreactivity, Cancer re., 47: 830-840 (1987), the presentation of which is incorporated herein by reference. MAb's of Lym-1 are made functional using SPDP, a heterobifunctional reagent that reacts with the free amino groups of the antibodies as in Carlson, J. et al. Thiolation of protein and Reversible protein-protein conjugation: N-succinimidyl 3- (2-pyridyldithio) propionate, A new heterobifunctional reagent, Biochem. J. 173: 723-737 (1978), the disclosure of which is incorporated herein by reference. To a 5 ml test tube containing 1 ml of Lym-1 (10 mg / ml) in PBS, pH 7.2, add 20 μl of 3 mg SPDP in 1 ml of # ethanol and 40 μl of N, N-dimethylformamide. This mixture is incubated for 15 minutes at room temperature with continuous mixing using an orbital shaker apparatus which is placed at normal speed. After incubation, the functionalized Lym-1 5 solution was purified by passing it through a PD-10 column equilibrated with PBS. The degree of functionality of Lym-1 with SPDP was determined at? F an average of one PDP group per molecule by measuring the release of pyridine-2-thione at 343 nm after the introduction of an aliquot into the Lym-1 solution with molar excess of 7 mg dithioerythritol in phosphate buffered saline (PBS), pH 7.2, as in Grassetti, DR and Murray, J.F., Determination of sulfhydryl groups with 2,2'- or 4,4'-dithiodipyridine, Arch. Biochem. Biophys. 15 119: 41-49 (1967), the description of which is incorporated herein by reference. The modified antibody of Example 1 was analyzed by rapid protein liquid chromatography (FPLC) in order to show the antibodies remaining substantially intact. The analysis is shown in Example 2. EXAMPLE 2 Analysis of modified Lym-1 by liquid, fast, protein chromatography (FPLC) The analysis of the modified antibody, of Example 1, is accomplished by chromatography, rapid, liquid protein (FPLC) equipped with a fixed-wavelength UV spectrometer which is placed at 280 nm. A size exclusion chromatography was carried out on a seperose 12 column (Pharmacia) with PBS, pH 7.2 as the solvent system, eluting the flow average of 1 ml / min. The modified Lym-1 appears with a retention time of 690 seconds, equal to the retention time of the intact Lym-1, without 10 labeling. Thus, Example 2 shows that the modified SPDP antibodies behave virtually unchanged as the unmodified antibodies in FPLC. These data show that the modification itself does not lead to the breaking of the intact molecules in vitro. In order to further study the modified MAb 's for an in vivo evaluation, a radiolabeling of modified MAb' s was carried out. Radiolabelling is shown in Example 3. EXAMPLE 3 Direct radioiodination of modified Lym-1 A batch of modified Lym-1 of intact PDP and Lym-1 was iodinated with 1251, and another batch labeled with 1311 using the Chlamine-T method modified de Mills, SL et al., 1251 monoclonal antibody labeling for * In vivo procedures, Hybridone 5: 265-275 (1986), the disclosure of which is incorporated herein by reference. Slowly, to a 5 ml test tube containing 100 μg of a monoclonal antibody in 100 μl PBS, add the appropriate 5 isotope of iodine, 1251 or 1311 depending on the batch, and 10 μl of 43 nM of an aqueous solution of chloramine T. The reaction is allowed to stand after 3 minutes with 20 μl of 120 nM of a sodium metasulfite solution. The radio-labeled antibodies were purified using a column Saphadex G-25. This column consists of a plastic serological pipette (8mm x 200mm) capped at one end with a cotton swab (Vo = 4.51, Vl = 12ml). Each reaction mixture is loaded onto a column and eluted with PBS, pH 7.2. The individual tubes contains 1 ml aliquots are counted and the Radiolabelled antibodies are recovered in a tube 6 with a production of 85-90%. These radiolabelled antibodies are stored in a refrigerator and are administered to the mice within 4 hours after labeling. The radiolabelled MAb 's of Example 3 is submitted to a thin layer instantaneous chromatography (ITLC) in order to determine the purity of the MAb 's labeling. This analysis is shown in Example 4.

EXAMPLE 4 25 Analysis of modified Lym-1, radiolabelled by thin layer flash chromatography (ITLC) The modified Lym-1 radiolabeled with 1311 and modified lym-1 radiolabeled with 1 51 by the chloramine T method of Example 3 was analyzed using an ITLC analytical system consisting of glass fiber impregnated with gel of silicon. The bands (2 x 20 cm) were activated by heating them at 110-1 / 2 ° C for 15 minutes before using them, - they were stained or spattered with 1 μl of the sample; dried in air and eluted with MeOH / H20 (80:20) for approximately 12 cm; again it was dried and cut into halves and a count was performed to determine the protein binding and the radioactivity without protein binding. Both forms of Lym-1 antibodies had an Rl value of p and showed a radiochemical purity of > 99% The analysis of intact Lym-1, labeled in the same manner as in Example 3 revealed the same purity. Thus, Example 4 shows that radiolabelled antibodies with high purity can be obtained. The immunoactivities of these radiolabelled MAb 's were evaluated by their ability to bind Raji cells. This analysis is shown in Example 5.

EXAMPLE 5 Analysis of the modified radiolabelled Lym-1 by an immunoreactivity assay The in vitro immunoactivity of radiolabeled modified Lym-1 and intact Lym-1 was evaluated by conventional in vivo assay of 106 tube / Raji cells by means of the Epteins method. TO THE. et al, supra. Slowly, Raji cells were resuspended in 100 μl of 1% bovine serum albumin in PJBS, and pipetted into test tubes with a pipette. One thousand μl of labeled Lym-1 was added to each test tube (100,100 cpm / tube) and incubated for 30 minutes at room temperature with a mixture continue using an orbital shaker. After incubation, the cells were washed three times with 1% bovine serum in PBS by rotating the tubes at 100 rpm for 5 minutes, decanting the upper layer and resuspending the cells in 200 μl of PBS. After that completed the washings, the Lym-1 bond was detected by measuring the radioactivity of the link in the cells, using a gamma counter. The results showed that the activity of the modified Lym-1 binding was 87%, while intact Lym-1, which served as a control, had an activity in the 80% link. Thus, Example 5 shows that the modified Lym-1 was more immunoreactive than the unmodified Lym-1. In order to obtain a preliminary assessment of the stability of the activity of the modified antibodies in Live ß, modified MAb's were analyzed to determine their stability in serum, as shown in Example 6.

EXAMPLE 6 5 Analysis of radiolabeled modified Lym-1 by serum stability Monoclonal antibodies of modified Lym-1 and intact Lym-1 which are directly labeled with 1251 are added to each of the triplicate sets of serum from fresh mouse with a final concentration of 100 μg / ml. The tubes were incubated at 37-1 / 2 ° C in a humidified incubator maintained at 5% C02 in air. From time to time between day 0 and day 8, the activity of the bound protein is determined by adding 900 μl of trichloroacetic acid to the 100% (TCA) to 100 μl of aliquots. After five minutes of incubation at room temperature, the protein precipitates are pelleted by centrifugation, and 500 μl of the supernatant is removed from each tube and counted for radioactivity in a gamma counter. The data were expressed as the average percentage of the precipitate counts minus those of the control tubes. The results show that each time after incubation, the modified Lym-1 of 1251 was stable as the intact Lym-1 labeled 1251 which serves as a control strain.

Subsequent results showed that > 92% activity present in the modified Lym-1 followed by the 8 days in incubation at 37-1 / 2 ° C was TCA that can be precipitated. Thus, Example 6 shows that the activity stability of the modified antibodies was maintained in the serum for at least 8 days. In order to evaluate if the modified MAb 's remain intact after the incubation in serum, the HPLC analysis of the modified Lym-1 was carried out, as shown in Example 7.

EXAMPLE 7 Analysis of HPLC-modified Lym-1 HPLC analysis was carried out in a Waters system equipped with size exclusion columns (SW 300) with a 0.1M neural phosphate buffer as an elution solvent and an average flow of 1 ml / min. The elusion was detected with a radioisotope detector. The modified Lym-1 labeled product of the mixture of Example 6 revealed a higher peak of a low molecular weight species with an elution time of 750 seconds, plus a small amount at 690 seconds.

The intact lym-1 gave a single peak with a retention time of 690 seconds. Thus, Example 7 shows that the samples of modified Lym-1 incubated in the serum had an apparent molecular weight in the HPLC analysis lower than that of the intact Lym-1.

In contrast, Example 2 showed that Lym-1 modified without ~ "V) j * ^ conceal had a retention time identical to intact Lym-1 Thus, modified Lym-1 showed an apparent molecular weight loss in FPLC analysis after incubation in serum. Apparent loss of the molecular weight of the modified Lym-1 after incubation in serum, electrophoresis was carried out with polyacrylamide gel of the samples as shown in the Example # EXAMPLE 8 Analysis of radiolabeled modified Lym-1 by SDS-Poloacrylamide gel electrophoresis (SDS_PAGE) The same aliquots were incubated in a mixture of the serum of Example 6 and serially verified by a non-reductive SDS-PAGE. For this study, the samples were placed in 10% acrylamide gel, carefully dried and exposed in the usual way to the photographic film as in Laemmli, UK, key of the 20 structural proteins during the head set of bacteriophage T4 , Nature 227: 680-685 (1970), the description of which is incorporated herein by reference. This analysis revealed that intact Lym-1 1251 was evident at Mf200,000, while modified Lym-1 1251 was observed in a different band 25 corresponding to a lower molecular weight of * approximately Mf 116,000. Thus, the present example shows that the incubation of the modified antibodies in serum results in a molecular weight apparently modified in acrylamide gel, checking the results of the HPLC analysis.

EXAMPLE 9 Test for the Desiodination of Lym-1 Serum Labeling The same samples of Example 6 were examined in * an 8-day study to verify if there was loss of radioactivity of the radiolabelled Lym-1; this loss can be interpreted as evidence of serum desiodination. The data showed, virtually, that there is no loss in this period, confirming that a very stable iodine bond is obtained in these immunoconjugates. Thus, Example 7-9 showed that the modified antibodies, in addition to virtually retaining the total activity after incubation in the serum, exhibit a breakdown of the apparent molecular weight molecules of 116,000. As specified above, it is possible that this loss of molecular weight is due to the breaking of the antibodies in their monovalent forms. In any case, it is believed that the apparent loss of molecular weight is due to the breaking of the modified antibodies into fragments thereof.

After discovering the unexpected previous change in the apparent molecular weight loss of the modified antibodies when incubated in serum, we evaluated the stability of the modified MAb 's in vivo. We conducted 5 in vivo tests in order to determine the time of release throughout the body. One of these tests is shown in Example 10.

# EXAMPLE 10 10 Total release in the body The experiments were carried out, in which three groups of mice, without fur, athymic (n = 5) were given peritoneal injections of (a) an intact antibody, (b) fragments of F (ab ') 2 or (c) of an antibody modified Lym-1 labeled with 1-131 using the chloramine T method. The activity was measured at the injection and, serially, with a dosimeter. This study showed that the total release in the body of radioactivity varied depending on the preparation of the antibody. The results are shown in Figure 2. Figure 2 shows that modified Lym-1 was released throughout the body faster, with a mean biological life (t%) of 20 hours, more than Lym-1 (t% = 5 days) intact. However, the release of F (ab ') 2 fragments was twice as fast, with an average biological life of 10 hours, than the modified Lym-1. The data showed that the modified Lym-1 was released at an intermediate average between the rapidly released F (ab ') 2 fragments and the intact antibody released slowly. Thus, it can be seen from the data of Example 10 that the modified antibodies are released from the body more rapidly than the relatively higher intact antibodies, but are not yet as fast as the F (ab ') 2 fragments. The ideal agent for immunotherapy would persist in the bloodstream for a period long enough to produce the desired toxic effect, although it is not yet so long as to cause toxic side effects, which are not desired. The data of Example 10 suggest that modified antibodies will exhibit potentially ideal persistence times when used in immunotherapy. As discussed above, an agent for immunotherapy can also have a very high specificity in relation to the cells that are the target. Thus, we evaluated the specificity of the modified MAb 's in relation to both MAb' s and fragments F) ab ') 2 in the following Examples. Example 11 shows the methods used in all biodistribution studies to continuation.

EXAMPLE 11 Biodistribution studies To two groups of mice, without fur, with 6 weeks of life, Raji cells (107) were injected subcutaneously into a region of the thigh. The tumors grew for three weeks until they reached more than 1 cm in diameter. The studies were carried out, as described ^ S «previously, using each group of mice. In the first group (n = 6), each mouse was injected with i.p. with an inoculation containing 0.2 ml of 10 μg of modified Lym-1 labeled with 1-131 in 12 μCi / μg (120 μCi / mouse) and 10 μg of Intact Lym-1 labeled with 1-125 at 2.5 μCi / μg (25μCi / mouse). In the second group (n = 4), the mice received 0.2 ml inoculation containing 10 μg of modified Lym-1 labeled with 1-131 at 12 μCi / μg (120 μCi / mouse), and 10 μg of F (ab ') 2 labeled with 1-125 at 2.5 μCi7μg (25 μCi / mouse). In all the experiments, the mice were sacrificed by cervical dislocation at times preselected, after the injection, and several organs, blood and tumor were removed, and weighed on an analytical balance. Then, samples were counted in a gamma counter, to determine activity 1251 and 1311. Count 1251 was adjusted for an intersection from channel 1311 by subtracting 17% of the counts of channel 1311, the formula was determined experimentally using a gamma counter Compugamma 1282 (LKB). Data was also corrected for decay or reduction of isotope 1311 radiation according to the days on which the 5 animals were slaughtered. For each mouse, the date was expressed as cpm per gram of tumor / cpm per organ gram,% dose / gram and% dose / organ. From these data, MAb 's average and the standard deviation for each group were calculated.

Example 12 compares the biodistribution of the 10 modified MAb 's with the intact MAb' s using the methods of Example 11. EXAMPLE 12 Biodistribution Study of Modified Lym - 1 versus Invisible Lym - 1 15 For this study, the antibody was compared Lym-1 intact with modified Lym-1 antibody in the methods of Example 11. Intact Lym-1 produces a blood activity of 0.64% ID / g at 7 days after injection, as recorded in Table 1. At the end of the same interval of time, the tumor had an activity of 3.92% ID / g. As reported in Table I, compared to intact Lym-1, modified Lym-1 is released from the blood faster and produces a blood activity of 0.14% ID / g at 7 days. At the end of this time interval, the tumor produces ^^ r 'r ^' »7.7% which tends to be significantly greater than the corresponding activities of intact Lym-1. The results of the reactivity of the antibody of Example 12 in different organs is reported in Table 1 and is shown graphically in Figure 3 (% dose / gram) and Figure 4 (tumor / organ). TABLE I BIODISTRIBUTION OF A MONOCLONAL ANTIBODY LYM-1 INTACT AND MODIFIED IN RAJI MICE WITH NO HAIR THE TUMOR (N = 6) 7 DAYS AFTER INJECTION Lym-1 Modified Organ tumor cpm / g% dose / g% dose / organ organ cpm / g Blood 140.07 (81.30) * 0.14 (0.20) Skin 93.98 (43.40) 0.09 (0.04) Muscle 364.53 (232.97) 0.03 (0.03) Bone 126.96 (55.86) 0.06 (0.02) Heart 137.34 (67.96) 0.07 (0.04) 0.01 (0.00) Lungs 28.31 (10.34) 0.28 (0.10) 0.06 (0.02) Liver 96.80 (49.03) 0.09 (0.05) 0.15 (0.08) Spleen 12.02 (5.62) 0.79 (0.53) 0.03 (0.01) Pancreas 286.43 (159.92) 0.04 (0.02) 0.00 ( 0.00) Stomach 71.21 (30.72) 0.11 (0.03) 0.02 (0.01) Intestines 133.31 (80.82) 0.07 (0.04) Kidneys 17.63 (7.63) 0.45 (0.12) 0.14 (0.03) Tumor 7.70 (3.95) 2.98 (1.71) Intact Lym-1 (Control) Blood 30.72 (17.74) 0.64 (1.26) Skin 8.83 (3.05) 0.41 (0.20) Muscle 44.39 (26.16) 0.15 (0.21) Bone 19.94 (6.08) 0.21 (0.18) Heart 28.79 (13.76) 0.19 (0.22) 0.02 (0.02) Lungs 16.98 (8.22) 0.36 (0.46) 0.07 (0.01) Liver 11.84 (5.95) 0.37 (0.25) 0.59 (0.46) Spleen 3.93 (3.74) 1.52 (1.14) 0.06 (0.03) Pancreas 29.35 (12.88) 0.16 (0.17) 0.02 (0.02) Stomach 11.00 (4.55) 0.32 (0.11) 0.07 (0.03) Intestines 18.06 (8.79) 0.23 (0.13) Kidneys 22.44 (10.61) 0.20 (0.17) 0.06 (0.05) Tumor 3.92 (3.11) 1.02 (0.27) * Mean (standard deviation).

It can be seen from Figure 3 that the modified antibodies produce a greater signal in the tumor than the intact antibodies. Additionally, the modified antibodies react with less force than the intact MAb for each organ tested, except for the kidneys. It is not unexpected that the greatest signal was found in the kidneys, since you expect the antibodies to be released through this * 5 * "organ." Because the average faster acceleration of the modified MAbs relative to the intact MAbs found in Example 10, a greater amount of modified MAbs in the kidney was expected. Figure 4, this shows the same data as Figure 3 but in a different way, it can be seen that the modified MAbs produce an average tumor / organ significantly higher than the intact MAbs in each organ tested, except for the kidneys. Thus, it is expected that the modified antibodies would produce a significantly lower support when used in immunography. Moreover, it was also expected that the modified antibodies would be more effective when used in immunotherapies due to both higher affinity with the tumor and the lower affinity in non-target tissues. When used in immunotherapies, modified antibodies would be expected to have greater toxicity to tumors and less toxicity to tissues that were not studied. The use of immunotherapy of The modified antibodies of the present invention are explained below in greater detail. We then compared the distribution of the modified MAbs with F (ab ') 2 fragments of other unmodified antibodies. Example 13 illustrates these experiments. 25 EIJTEMPLO 13 Biodistribution study of Modified Lym-1 versus Fragments F (ab 'of Lym-1) For these studies, fragments of F (ab 2 with Lym-1 MAbs modified.) The experiments were carried out as in Example 11. The results are reported in Table II and are shown graphically in Figures 4 and 5. TABLE II BIODISTRIBUTION OF A MONOCLONAL ANTIBODY LYM-1 INTACT AND MODIFIED IN RAJI MICE WITHOUT HAIR, WHICH CARRIES THE TUMOR (N = 4) 5 DAYS AFTER THE INJECTION Lym-1 Modified Organ tumor cpm / g% dose / g% dose / organ organ cpm / g Blood 39.59 (14.84) * 0.09 (0.03) Skin 13.69 (3.15) 0.24 (0.06) Muscle 73.32 (16.22) 0.04 (0.01) Bone 26.79 (7.18) 0.12 (0.04) Heart 44.42 (11.34) 0.08 (0.04) 0.01 (0.01) Lungs 15.78 (3.88) 0.21 (0.07) 0.04 (0.01) Liver 12.19 (4.25) 0.29 (0.12) 0.27 (0.10) Spleen 2.68 (1.01) 1.34 (0.55) 0.07 (0.03) Pancreas 42.17 (11.23) 0.08 (0.03) 0.01 (0.00) Stomach 14.16 (4.43) 0.24 (0.07) 0.05 (0.02) Intestines 28.36 (9.96) 0.12 (0.05) r ^. Kidneys 12.53 (3.15) 0.27 (0.09) 0.09 (0.03) Tumor --- 3.18 (0.89) 3.16 (1.09) Fragments F (ab '(Control) Blood 29.27 (13.17) 0.05 (0.02) Skin 10.54 (2.78) 0.12 (0.02) Muscle 55.50 (14.49) 0.02 (0.01) Bone 23.73 (7.89) 0.06 (0.02) Heart 35.19 (11.01) 0.04 (0.02) 0.00 (0.00) Lungs 12.57 (3.69) 0.10 (0.03) 0.02 (0.00) Liver 10.43 (4.20) 0.13 (0.05) 0.12 (0.04) Sow 2.59 (1.03) 0.54 (0.21) 0.03 (0.01) Pancreas 31.55 (9.82) 0.04 (0.02) 0.00 (0.00) Stomach 8.08 (3.31) 0.17 (0.06 0.03 (0.01) Intestines 22.27 (7.93) 0.06 (0.02) Kidneys 10.05 (2.71) 0.13 (0.04) 0.04 (0.01) Tumor 1.23 (0.24) 1.42 (0.48) * Mean (standard deviation) Table II shows that modified Lym-1 is released more slowly from the blood than fragments of F (ab ') 2. The modified Lym-1 produces an activity in the blood of 0.09% ID / g greater than the fragments (0.05%) in 5 days after injection.Figure 5 shows that the activity of the modified Lym-1 tumor was approximately two and a half times greater than the corresponding activity of the fragments ß of F (ab ') 2. The activity of the modified Lym-1 was greater than the F (ab ') 2 fragments for all the different organs tested, including the kidney. This is consistent with the theory that antibodies accumulated in the kidney are released more rapidly. In addition, Figure 6 shows that the modified turaor-organ average for Lym-1 is greater than for F (ab ') 2 fragments for all tested organs. Thus, the experiments of Examples 12 and 13 are confirmed of the modified antibodies of the present invention have a higher activity in the tumo to be treated, that the MAbs or fragments F (ab ') 2 intact. Additionally, the tumor data in relation to the organ from those experiments show that the modified antibodies have a greater specificity for the tumor, than the F (ab ') 2 fragments or intact MAbs. Thus, we tested the ability of the modified MAbs of the present invention to produce improved immunoscintigraphic results. An example of this test is shown in Example 14. 20 EXAMPLE 14 Lym-1 Image Studies Images of unhuttered mice, which carry the tumor using a pin hole collimator and a spectrum range camera, will be imaged. (Raytheon) The analysis of the image of these animals provide an estimate of the distribution of antibodies in relation to the tumor and the whole body after the injection. Seven days after the injection, the mice were anesthetized with 2 mg of ketamine HCl and 0.4 mg of xylazine administered as a 5 inoculation of 0.2 mL s.c. Immobilized mouse imaging was then taken at a later position with the camera present to record 10,000 counts. Backup subtraction is not carried out. The photographic images were obtained using a Polaroid Film Type 330 Pack. HE defined two areas in each image: (a) region 1, the entire body; (b) region 2, tumor. Figures 6 to 8 show exemplary scintografías (also known as scintogramas) produced by these experiments. Immunosynography image with intact Lym-1 was performed 7 days after the injection and was not satisfactory, as can be seen in Figure 6. Figure 6 shows that although the tumor was visualized, the rest of the animal was also visualized. Figures 7 and 8 show the images of two different tumor bearing animals injected with modified Lym-1 labeling, taken at the same time subsequent to injection. It can be seen that both Figures 7 and 8 show a modified Lym-1 concentration, labeled in the tumor at levels much higher than the concentration in the tumor produced by intact Lym-1, which is appreciates in Figure 6. More important, the average labeling ß in the tumor, in relation to the backing of the whole body of the mouse produced by the modified Lym-1, many times greater than that of the intact Lym-1. Thus, Figures 7 and 8 show a clear definition of the tumor, with little or almost no radioactivity in the surroundings or in the posterior part. Furthermore, satisfactory visualization of the tumor could be obtained 5 days after the injection when the modified Lym-1 is used. Figure 10 shows an image taken at 5 days from the same animal shown in the 10 Figures 9 at 7 days. As can be seen, the 5 day image of Figure 10 is significantly superior to the image produced by intact Lym-1 at 7 days (Figure 7). The similar result for all the animals evaluated. This study suggests that the use of fragments of modified antibodies has a higher specific activity for tumor antigens, allowing a more absolute concentration of the body that accumulates in the tumor. This is confirmed by said results, which show the absolute concentration of the modified Lym-1 fragments is approximately 2 times the concentration of intact Lym-1, 7 days after the injection and approximately two and a half times of the F fragments ( ab ') 2 after five days. The much faster release of fragments Lym-1 modified also significantly produce the time required to achieve high tumor averages ß in relation to the space that surrounds it and the posterior space and thus results in a better image in less time than with the intact antibodies. In order to demonstrate the general usefulness of the modification of the present invention to improve the specificity and activity of the antibodies, we modify additional MAbs. The tests of these different iK modified MAbs. are shown in Examples 15-18. EXAMPLE 15 10 Average Antibody Release B72.3 B72.3 (IgG-, monoclonal antibody against colon carcinoma is obtained, as in Colcher, D. et al., A Spectrum of Reactivity of Monoclonal Antibodies with Cells from Tumor in the Glands Mammary Human, Proc. Nati Acad. Sci. 78: 3199-3203 (1981), the disclosure of which is incorporated herein by reference. The functionality of B72.3 MAb was measured with an average of one PDP group per molecule according to the method of Example 1. The modified B72.3 MAb was radiolabelled by the method of Example 3. The times of total release in the body were measured as in Example 10. Figure 11 shows the results of these experiments and the total release in the body. The modified antibodies showed a reduction in half the time in the body from about 6 days of the intact MAb towards about 2.5 days of the modified antibodies. The release of half the time of the F (ab ') 2 fragments was, like the Lym-1 fragments, faster than the modified antibodies, with an average time of about 12 hours. Thus, the results indicate that the modified B72.3 behaves similarly to the modified Lym-1 since there is an average time of intermediate release between that of the F (ab ') 2 fragments and the intact antibodies.

EXAMPLE 16 Biodistribution of B72.3 The labeled biodistribution studies were performed in two groups of five mice, each carrying, in a mouse without fur, human LS174T colon carcinoma.

One group was injected with B72.3 labeled 1-125 intact, while the other was injected with a B72.3 labeled 1-131 modified. The experiment also compared the biodistribution in the tumor, blood and various organs. The methods employed were as those used in Examples 11 to 13.

The data are reported in Table III, and are shown graphically in Figures 11 and 12.

TABLE III BIODISTRIBUTION OF AN ANTIBODY B72.3 MONOCLONAL LYM-1 25 INTACT ß IN THE MOUSE, WITHOUT PEEL, WHICH CARRIES HUMAN COLON LS174T CARCINOMA (N = 5) 4 DAYS AFTER INJECTION B72.3 Modified Organ tumor cpm / g dose / g% dose / organ organ cpm / g Blood 6.16 (2.32) * 1.10 (0.45) Skin 20.81 (3.92) 0.31 (0.12) Muscle 61.58 (16.16) 0.11 (0.05) * Bone 65.25 (15.04) 0.10 (0.04) Heart 31.41 (18.44) 0.24 (0.11) 0.03 (0.02) ) Lungs 11.89 (3.25) 0.54 (0.18) 0.14 (0.04) Liver 21.61 (10.36) 0.33 (0.14) 0.43 (0.17) Spleen 37.89 (15.00) 0.18 (0.09) 0.02 (0.01) Pancreas 60.23 (23.73) 0.12 (0.06) 0.02 (0.01) Stomach 37.74 (9.20) 0.17 (0.05) 0.04 (0.01) Intestines 68.31 (28.27) 0.10 (0.04) Kidneys 24.56 (10.07) 0.29 (0.13) 0.09 (0.04) Tumor 6.02 (1.33) 6.45 (1.53) B72.3 Intact (Control) Blood 3.43 (1.13) 1.34 (0.60) Skin 10.44 (1.80) 0.41 (0.15) Muscle 31.78 (8.86) 0.14 (0.05) Bone 33.36 (9.84) 0.14 (0.06) * Heart 16.57 (8.28) 0.30 (0.14) 0.04 (0.02) Lungs 6.42 (1.59) 0.68 (0.28) 0.18 (0.08) Liver 11.85 (4.78) 0.39 (0.16) 0.52 (0.24) Sow 18.94 (5.61) 0.24 (0.12) 0.02 (0.01) Pancreas 29.80 (9.42) 0.15 (0.05) 0.02 (0.01) Stomach 18.88 (3.83) 0.22 (0.04) 0.05 (0.01) Intestines 35.61 (13.48) 0.13 (0.06) Kidneys 15.20 (6.40) 0.33 (0.18) 0.11 (0.06) ) Tumor 4.04 (0.84) 4.28 (0.78) * Mean (standard deviation). As can be seen in Table III, the intact B72.3 antibody produces a blood activity at 1.34% ID / ga on the 4 days after injection, and an activity of 4.04% on the tumor, as shown in the Table III. Compared with intact B72.3, modified B72.3 produces a lower blood entity (1.1% ID / g) and greater tumor activity ((6.02% ID / g) after 4 days. Figure 13, all 10 activities in the various organs was greater than for the modified B72.3, except for the kidney, as expected for a more rapid release of the antibody.Thus, as shown in Figure 14, the average tumor in relation to the organ for the modified B72.3, it was significantly greater than the corresponding 15 average for the intact B72.3. ß organ-tumor was still improved for the kidney due to increased activity of the modified antibody at the tumor site. EXAMPLE 17 Image of B72.3 in Tumor Carrier Mice 5 The analysis of the mouse image carrying the LS174T tumor injected with a modified B72.3 provides a tumor distribution / all of the antibody after injection. Figure 14 shows an immunosynography after the first day after injection. The image shows a clear definition of the tumor with a little relativity in its surroundings. Figure 15 shows immunosynography after 4 days of injection. After 4 days, the tumor was clearly visible with little radioactivity remaining in the animal's blood volume. The results were similar for all the animals. Thus, it was found that the modified B72.3 is very useful to obtain high quality immunosyntaggregates with a short time period of injection of the tumor reagent with B72.3. 20 TNT-1 is an IgG2a monoclonal antibody that is used for necrotic tumors as a target for its selective linkages with human cancers. We modified these antibodies with an average of one PDP group per molecule as in Example 1, and we analyzed the retention time in all the body as indicated in Example 18.

EXAMPLE 18 Use of the Monoclonal Antibody TNT-1 We obtained the TNT-1 as Epstein, A.L. and collaborators, Novel Method for the Detection of Necrotic Lesions in Human Cancer, Cancer Res. 48: 5842-5848 (1988), which is presented in the invention as a reference. The TNT-1 MAb was radiolabelled by the method of Example 3. The total release time in the body was measured as in Example 10. Figure 16 shows the results of these total release experiments in the body. The modified TNT-1 MAb shows a reduction in the average time of release throughout the body in relation to the inactivated TNT-1, and a relative increase in the F (ab ') 2 fragments of the TNT-1. Thus, the modified TNT-1 behaved similarly to the other modified antibodies. We, therefore, hope that the utility of the modified TNT-1 MAb is equivalent to the other modified antibodies tested. Example 19 describes a useful method for preparing biotinylated antibodies. EXAMPLE 19 Preparation of Biotinylated Antibodies The MAb of TNT-1 and Lym-1 was separately conjugated with 6- (biotinamido) hexanoate by reacting it with its sulfo ester N-hydroxysuccinimide (NHS-LC-biotin). Typically, it prepare the standard solution of 2 mg NHS-LC-biotin in 1 mL of One solution comes out at 9%. Add 5 mL of a test tube containing 1 mL of antibody (10 mg / mL) in a bicarbonate buffer solution, with a pH of 8.5, to 1 mL to a standard solution of NHS-LC-biotin (average molar 50: 1 NHS-LC-biotin / MAb). The reagent mixture was incubated for 2.5 hours at room temperature with continuous agitation at low speed. After incubation, chromatography of the coupled antibody was performed on a PD-10 column (Pharmacia) equilibrated with PBS, pH 7.2. The purity of the coupled antibody preparations was tested with an FPLC using a supersized column 12. The results of these procedures indicated that the biotinylated antibody is obtained with at least 99% purity. The average of the biotin groups coupled to each antibody molecule was determined spectrophotometrically according to the method described by Green in Biochem J. 94: 23c-24c (1965). Briefly, the biotinylated antibody was digested enzymatically with 1% protease at a temperature of 37 C for four hours. To a 5 mL solution containing 800 L of 100 M HABA in 0.1 M PBS, at a pH of 7.2, 70 L of to 17 L of streptavidin solution are added. The estraptivine-HABA solution was titrated with increased volumes of the biotinylated antibody solution digested, and the change in absorption was determined at 500 mm.

^^ For this treatment, the concentration of biotin in the protease treated antibody was calculated using the standard curvature of the biotin solution. The results indicate that an average of 3 to 4 parts of biotin was incorporated into each antibody molecule. The conjugated biotin antibody was radiolabelled with 125 using the chloramine T method described essentially in Example 3. yf Example 20 describes the methods used for radioiodination of antibodies and antibody fragments. EXAMPLE 20 Direct Radioiodination of Antibodies All the antibodies (intact, modified and F (ab ') 2 fragments) were iodinated with 125 or 131 using the The modified chloramine T method is essentially described in Example 3. Typically, 0.5-1.0 mCi of iodine-125 or iodine 131 and 10 L of a 43 mM aqueous solution of chloramine T was added to a test tube containing 50 to 100 g of a monoclonal antibody in 100 L PBS. The reaction was mixed after three minutes with 20 L of a 120 M solution of sodium metabisulfide. The radiolabelled antibodies were purified using a Sephadex G-25 column. This column consists of a plastic serological pipieta (8 x 200 mm), covered at the end with a cotton swab (V0 = 4.5 mL, Vt = 12 mL). Each The reaction mixture was loaded onto a column and diluted with PBS, pH 7.2. Individual tubes containing 1 mL of aliquots were counted in a scintillation counter. Typically, the radiolabelled antibodies were recovered with a production of 85 to 90%. All antibodies were radiolabelled with the chloramine T method and analyzed using the analytical, instantaneous, thin layer chromatography (ITLC) system on gel impregnated glass fibers. The bands (2 x 20 cm) were activated with heat at a temperature of 110 C for 15 minutes before use. HE stained or splashed with 1 L of the sample, air-dried and eluted with methanol / H20 (80:20) for approximately 12 centimeters, again dried, cut into halves and counted to determine the radioactivity of the protein and non-protein bonds. The results of this The procedure indicated that more than 99% of the antibodies had protein bonds, therefore, it is confirmed that biotinylated, radiolabelled functional antibodies can be obtained with high purity. Radiolabelled biotinylated antibodies are prepared according to the procedure described above and stored at a temperature of 4 C, and were administered to mice within four hours after labeling. The following two examples present the results of the analytical procedures used to prove that Radiolabelled biotinylated antibodies retain the ability to bind the antigens that are the subject of study and were stable in the presence of serum components. Example 21 describes the method used to demonstrate that antibodies that have been modified by biotinylation and radiolabelling retain both the ability to bind antigens and the structural integrity. EXAMPLE 21 Evaluation of Immunoreactivity Immunoreactivities were evaluated within radiolabeled Lym-1 preparations with a live cell assay using the Raji according to the method described in Epstein et al., Cancer Res. 47: 830 (1987). The Raji cells (106 / test tube) were suspended in 100 L of 1% bovine serum albumin (BSA) in PBS. The labeled Lym-1 (100 1) was added to each test tube (approximately 10 Ci / g, 100,000 cpm / tube) in triplicate and incubated for one hour at room temperature with mixtures continuous using an orbital shaker. After incubation, the cells were washed three times with 1% BSA in PBS by centrifuging the tubes at 1000 revolutions per minute for 5 minutes, decanting the supernatant layer, and resuspending the cells to 200 L PBS. After that completed the washings, Lym-1 bonds were detected measuring the radioactivity associated with the groups of cells using a gamma counter. The results of this procedure were substantially identical to those presented in Example 6. More specifically, 75% of an intact radioactive tagged 5 and 75% of target cells with biotinylated Lym-1 radiolabelling. This indicates that the antigen binding activity of the biotinylated Lym-1 re-labeled was comparable to that of the intact immuno # radiolabelling that serves as a standard control. The immunoreactivity of the modified TNT-1 MAb was evaluated using the fixed cell radioimmunoassay described by Gaffar et al in J. Immunoassay, 2:11 (1991). Briefly, the radiolabelled TNT-1 and the biotinylated TNT-1 radiolabel were incubated for 30 minutes with Raji cells previously fixed with 20% paraformaldehyde PBS, at room temperature, and then treated with acetone at F., - a temperature of -20 C. Then the cells were washed in 1% BSA in PBS and counted in a gamma counter. The results of this procedure indicated that approximately 60% of Both antibody preparations bind to fixed cells. This indicates that the antigen binding activity of the biotinylated TNT-1 re-labeled was comparable to that of the intact radiolabeled TNT-1 which serves as the standard control.

Example 22 describes the methods used to prove that antibiotics of antibiotics are not usually signaled to the degradation and presence of serum. EXAMPLE 22 5 Modified Antibody Serum Stability MAb labeled 125 was added directly to a triplicate set of tubes containing fresh serum mouse at a final concentration of 100 g / mL. All samples were incubated at 37 C in a humidified incubator and maintained a 5% C02 in air. On several occasions between 0 and 8 days, the radiolabelled protein bond was determined by adding 900 L of 100% trichloroacetic acid (TCA) to 100 L of aliquots of each sample, incubating the room temperature for five minutes and recovering the precipitates of protein by centrifugation. The aliquots (500 1) of the upper layer were removed from each tube and a radioactivity count was performed using a gamma counter. These results indicated that the radiolabelled biotinylated antibody is stable in vi tro at all points. Plus Specifically, at least 97% of the radiolabel remains associated with the protein after 8 days of incubation. This confirmed that the biotin parts present in the MAb do not have opposite effects on the stability of the radiolabelled associated proteins.

The same aliquots of each incubated serum mixture were verified serially by non-reducing SDS-PAGE and autoradiography. For this study, the electrophoresis samples were made in 5% -10% polyacrylamide gel and visualized by exposure to an X-ray film. The molecular weights of the samples were determined by comparing molecular weight standards. The results of this procedure indicate that the intact MAb f. radiolabelling and radiolabelled biotinylated MAb have substantially similar molecular weights. More specifically, the major bands for the radiolabelled Lym-1 and biotinylated radiolabelled Lym-1 have Mrs about 200,000. Similarly, the main bands for the radiolabelled TNT-1 and biotinylated TNT-1 radiolabels have Mrs about 150,000. An isoelectric focus was performed on polyacrylamide gels in a BioRad Model 111 Mini IEF cell. The samples were made electrophoresis by means of a pH gradient built with a mixture of BioLyte ampholytes (BioRad) at anhydrite concentrations 3/10 to 1.2% and anfolite 5/8 to 0.8% according to the protocols provided in BioRad. The IEF standards (BioRad) included in each time for the calibration of pl. The IEF gels were stained with Coomassie Blue R-250 and dried overnight. The results of this procedure confirm that the Biotinylation of MAb substantially alters the electrical charge properties of macromolecules. While the radiolabelled Lym-1 has a pl value of 7 to 8, the radiolabelled biotinylated Lym-1 has a pl of 5 to 6. Similarly, while the radiolabelled TNT-1 MAb has a pl value of 5.5 to 6.5 , the biotinylated TNT-1 MAb of radiolabelling has a pl of 4.5 to 5.0. Thus, as expected, the modification of the free amino groups in the MAb protein effectively eliminates or neutralizes some of the positive charges on the proteins as evidenced by the lower basic characters of the biotinylated antibodies. Example 23 describes the method used to demonstrate that the MAb has parts of amino acids chemically modified to result in a macromolecule having a reduced pl relative to the native antibody, which advantageously has: (1) an increased target specificity, (2) a specific non-reduced link and (3) a reduced release time. EXAMPLE 23 Biodistribution Studies Two groups of unleaded mice six weeks old were injected with Raji lymphoma cells, LS-174T colon carcinoma cells, or cervical carcinoma cells ME-180 according to the methods described in Khawli and collaborators, in Antibodies, Immunoconj ugados, and Radiopharmaceuticals 6:13 (1993). The tumors grew by 10 to 21 days until they were approximately 1 centimeter in diameter. 5 (a) Studies of the tagged pair. In the first group of mice (n = 6), each mouse was injected intravenously with 0.2 mL of inoculation containing 120 Ci / 10 g of MAb 131 -labeled modified and 25 Ci / 10 g of intact MAb 12S -labelled . In the second group (n = 4), the mice received an inoculation of 0.2 mL containing 120 Ci / 10 g of MAb 131 - modified labeling and 25 Ci / 10 g of F (ab ') 2 of labeled MAb of 12s. In all experiments, the mice were sacrificed by servical dislocation at pre-selected time after injection and several were removed organs of blood and tumor and weighed. The samples were then counted in a gamma counter to quantify activities 131 and 125. The counts 125 were adjusted in transverse from channel 131 by subtracting 17% of the counts from channel 131, a formula that is determined experimentally for the 1282 CompuGama counter. The data was also corrected for the decay of radiation from isotope 131 according to the time in which the animals were sacrificed. For each mouse, the data were expressed by cpm per gram tumor / cpm per gram of organ and% of the dose above grams. For these data, the standard deviation was calculated for each group. The same par-labeled biodistribution studies were carried out using Lym-1 in the Raji lymphoma model and in B72.3 in the human colon carcinoma model LS-174T. 5 The results of these procedures are presented in Figure 17. As indicated in panel 17A, both the TNT-1 / biotin-modified MAb and intact TNT-1 MAb were located in the tumor tissue on the day after the injection. , the biotinylated antibody showed higher location that is measured as the percentage of dose / gram injected from the tumor. The non-specific binding of 2 MAbs labeled in a variety of tissues was also evident. It was very important, the amount of biotinylated TNT-1 that is associated in a non-specific way with these tissues was uniformly less than the amount of intact TNT-1 represented these non-specific bonds. Thus, the level of specific binding was raised and the level of non-specific binding was reduced for the biatinylated antibodies related to the unmodified antibody. The advantage of The modified antibody is represented quantitatively in Figure 17B, where the average of antibodies located in the tumor relative to the localized antibody is not especially present for each tissue. As illustrated, the biotinylated antibody advantage as an image of "W reagent was particularly evident in the muscles and in the pancreas. Similar traces were observed for the measurements taken after three days of the injection. Figure 17C 5 shows that the biotinylated TNT-1 antibody was located in the tumor tissue more efficiently than its non-biotinylated antibody. At the same time, the amount of non-specific links was advantageously lower for the species biotinylated. Figure 17b confirms quantitatively that the The average tumor / organ, which reflects the average signal in relation to the sound in several tissues, was higher in muscles and pancreas. Notably, Figures 17C and 17D include the results obtained using F (ab ') 2 fragments of TNT-1 having averages of rapid whole body release.

Although the tumor / organ average was higher for the antibody fragments compared to the biotinylated TNT-1 in several tissue types, the fraction of the injected biotinylated TNT-1 that localized the tumor was advantageously higher. These results illustrate that biotinylated MAb localized for the tumor tissue in a form that was superior to the intact unmodified MAb or to the MAb F (ab ') 2 fragments. (b) Liberation in the Whole Body. Experiments were carried out where different groups of Balb / c mice (n = 45) were given intravenous injections of radiolabeled antibodies. Activity throughout the body fl was measured after the injection and at selected times with a dose calibrator. Figures 18 and 19 present the results of the studies for the average of the whole body release of Lym-1 and TNT-1 antibodies and their derivatives. The results presented in Figure 18 indicate that the MAb of TNT-1 modified by its SPDP or biotin has reduced release times relative to the intact antibody. The TNT-1 fragment of F (ab ') 2, which has the fastest release average of the antibodies tested, is used as a positive control in this procedure. Thus, the biotinylated TNT-1 MAb advantageously exhibits a faster release rate than the non-biotinylated antibody. Similarly, the results presented in Figure 19 indicate that Lympho-1 MAb modified by SPDP or biotin has a reduced release time relative to the intact antibody. This illustrates that agents that chemically modify the parts of free amino groups to reduce the pl of the antibodies advantageously reduce the binding of the specificity of the antibody binding and the average release throughout the body. We believe that modifications of any antibody using the methods of the present invention provide a useful reagent to improve the tumor image. With the aim of producing an image of any desired tissue used in the methods of the present In the invention, antibodies must first be obtained for this type of tissue. Polyclonal antibodies can be obtained in a conventional manner known to those skilled in the art. Alternatively, monoclonal antibodies can be prepared with the aim of obtaining an increased specificity provided in these antibodies, which is also a method known to those skilled in the art. Then, the antibodies are chemically modified into free amino groups by conjugating them with a heterobifunctional agent, biotin or other The agent that leads to the modified antibody product having an isoelectric point lower than the isoelectric point of the unmodified antibody. After conjugation, an appropriate label is applied to the modified antibodies. Although the previous examples use images of In a label comprising a radionucleotide that emits gamma radiation, many other labeled types of imaging systems are contemplated within the scope of the present invention. For example, radiopaque materials, such as barium, celsium or iodine, can create images using conventional X-rays. Paramagnetic or supermagnetic particles can be used as labels, using MRl imaging technology to produce an image of a location from an antibody location. Additionally, you can use technology as a label. These labels # Alternatives can be conjugated with modified antibodies using conventional methods. The labeled antibodies can be included in pharmaceutical preparations for the introduction of labels in subjects including pharmaceutically acceptable excipients, carriers or bases. Suitable carrier carriers or bases include saline solutions, phosphate buffered saline solutions, glycerol, calcium carbonate, and the like. These compositions are then introduced by any of many means, such as for example a local injection, an intravenous injection, oral administration in cases where a signal with reduced force is required or when the image of the tissue in the oral cavity is to be taken. However, preferably, the The administration is carried out by means of a systemic injection in order to maximize the exposure of the target tissue to the antibody. We believe that the chemical modification of the free amino groups disposed of the antibodies by the addition of A heterobifunctional agent, biotin or other agent to result in a modified antibody having an isoelectric point lower than the intact, unmodified, corresponding antibody according to the present invention will produce significantly improved results when these modified antibodies are incorporated into an agent immunotherapeutic These therapeutic agents generally they comprise an antibody specific for a tumor or other diseased tissue combined with one or more biologically active molecules. The active and biologically appropriate molecules which function in these agents are toxic, eg toxic diphtheria (resin) of chain A or any of the variety of toxic plants known to those skilled in the art; radionucleotides; as for example radioactive isotopes of yttrium, iodine, phosphorus and others commonly radiotherapeutic agents commonly used; drugs, such as methotrexate, 5-fluoro-uracil, or adriamycin; chelates, which include EDTA and EGTA; cis-platinium and other toxic organometallic agents and any other therapeutic agent. 15 Therefore, the promise of effective immunotherapy is not yet fully achieved. We believe that the activity and increased specificity of the modified antibodies of the present invention will produce immunotherapeutic agents having an activity and specificity The deficiencies of the above immunotherapeutic agents are sufficient for their specific objectives for solutions. Thus, the diseased tissue that is the target of study can be killed without significantly affecting the healthy tissue of the subject, when the subject is injected with an agent suitable immunotherapeutic.

In the use of these immunotherapeutic agents, a specific antibody must first be obtained for a particular type of unwanted tissue. If the desired antibody is not available, antibodies can arise in a suitable organism that is injected into the body with antigens and obtain serum from a mammal, as is known to those skilled in the art. Alternatively and preferably, the monoclonal antibodies may arise or may be obtained in the manner known to those skilled in the art. Then, the antibodies are chemically conjugated with an agent such as, for example, a heterobifunctional agent, biotin or another agent capable of modifying the free amino groups present in the antibody molecules. Following conjugation, the resulting modified antibodies are also modified by conjugation with a biologically active agent, such as for example a therapeutic agent or a detectable label as described above. The antibodies are combined in pharmaceutical compositions containing a pharmaceutically acceptable carrier, excipient or bases. These excipient carriers or pharmaceutically acceptable bases include normal saline solutions for systemic injections, glycerol, and calcium carbonate. The compositions are then ready for introduction into the patient, such as a mammal.

The antibodies are then introduced into the subject by means of the administration that is known. For example, the compositions may be introduced by means of systemic injection, local injection into the affected tissue, they may be applied topically or externally to the affected tissue and may also be terminated orally in the case where a relatively weak signal is required or when Tissue therapy when tissue therapy is to be carried out in the oral cavity. The dose of the active agent directly containing an antibody will depend on the sensitivity of the tissue to be studied to the toxin, the amount of tissues affected, the route of administration, the affinity of the antibody, the means of release and other factors. However, representative doses will generally be between 1 g / kg of the total body mass up to 1 mg / kg. In the measurement of the applications this dose will preferably be from 5 to 200 g / kg. The following Example illustrates the effectiveness in immunotherapy against tumors in Raj i mice. Although PDP-modified antibodies are used in the Example, antibodies that have free amino groups modified by other agents such as biodine, which produce a modified antibody having a polynucleotide, are expected to lower than the corresponding intact antibody also works. EXAMPLE 24 Treatment of Raj i Tumors in Mice PDP modified Lym-1 is obtained as in Example 1. Then, the modified antibody is treated to introduce, on average, a chain to resin per antibody molecule. The intact Lym-1 and the F (ab ') 2 fragments are combined in a similar with the toxin. 10 Twenty-five mice were divided into five groups. Group I received an intraperitoneal injection at 10 g / kg of the total weight of modified Lympho-PDP-recine in buffered saline solution (PBS) once a week for 8 weeks. Group II received injections of an equivalent amount of intact Lym-1 resin. Group III received equivalent amounts of F (ab ') 2-recine fragments of Lym-1. Group IV received an equivalent amount of non-conjugate recin. Group V received PBS only. After 8 weeks, immunosyntation was performed on all 20 surviving Iso isolates using Example 14. Group I mice showed reduced visualization of the tumor compared to any other group. The surviving mice of Group II and Group III showed some improvement, although less dramatic than that of Group I mice.

The mice in Group IV were really sick or died. Thus, Example 24 shows a particular tumor treatment using modified antibodies of the present invention. Example 19 shows the superior results that were obtained when modified PDP antibodies of the present invention were used. It is believed that the substitution of the use of other antibodies specific for other tumors or diseased tissues in mice or other mammals, such as, for example, human, produces similar effective results in the treatment for specific tumors or diseased tissues. Moreover, it is believed that the substitution of other known toxins also produces similar effective results. Example 20 shows the use of a similar effective therapy against the pancreatic cancer in humans. Although modified PDP antibodies are used in the example, antibodies having free amino groups modified by other agents, for example biotin, which produced a modified agent which has a lower than the corresponding intact antibody, will also work very well. EXAMPLE 25 Treatment of Pancreatic Cancer in Humans A monoclonal antibody is obtained that is specific to an antigen found in tumors pancreatic. This antibody is modified by the conjugation with, on average, a PDP group per antibody molecule, as in Example 1. Menoxtrase is then conjugated to these modified antibodies as described for the Resin in Example 19. 5 Two groups of ten patients with pancreatic cancer are treated. . The first group receives intravenous injections of the PDP-MAb test in PBS at 20 g / kg of total body weight on a weekly basis, combined with traditional therapy. The second, receives injections of PBS in combination with the traditional therapy as a control. After 10 weeks, the immunosynography of the surviving patients is carried out. In immunosynatography, the average size of the tumor images of the first group of patients was reduced relatively in relation to the control group. Thus, the previous example illustrates the usefulness of modified antibodies in immunotherapy in humans. As described above, in a preferred form of the invention, the modified antibodies are formulated in pharmaceutical compositions. Thus, modified PDP antibodies that are conjugated with the drug for immunotherapy can be incorporated into injectable compositions having an effective amount of cytotoxicity of the conjugated toxic antibody conjugated to the present invention. The following is an example of the composition effective cytotoxicity against B-cell lymphomas in humans . EXAMPLE 26 Pharmaceutical Composition 5 Effective Against Lymphoma Cell B in Humans 10 mg / mL of Lym-1 Modified Radiolabel from Example 18. Balance Saline solution buffered with phosphate, balanced (0.9%) In addition, the modified radiolabeled MAb can be formulated in effective compositions to visualize their specific antigens in immunosocgraphy. The following is an example of this compound. EXAMPLE 27 An Effective Pharmaceutical Composition in the Immunoscitography of the Carcinoma Colon 10 mg / mL of B72.3 Modified radiolabelling of Example 15 Balanced with a buffered saline solution of 20 Phosphate (0.9%). Although Example 23 illustrates a method for locating biotinylated radiolabelled antibodies, those skilled in the art will appreciate which methods also can be used to locate alternative methods. radiolabeled antibodies. For example, the distribution of Radiated biotinylated MAb antibodies can also be localized by means of the immunoscintographic image exactly as defined in Example 14. Thus, for example, the radiolabeled biotinylated MAb having a binding specificity for a tumor antigen would also be useful in the of diagnosis. The following example describes how these imaging procedures are carried out. Example 28 describes a method in which radiolabelled biotinylated MAb can be used to perform images of tumor cells in vivo. EXAMPLE 28 Images of Melanoma Tumor Cells First, a human patient who has been diagnosed with metastatic melanoma is identified. The analysis immunohistological indicates that the patient's melanoma expresses a surface antigen stained with an antimelanoma MAb. First the antimelanoma MAb is chemically modified by biotinylation of the free amino groups according to the method of Example 19 and radioiodinated with 131 according to with the method of Example 20. Then, the biotinylated, radiolabelled, substantially purified antimelanoma MAb is combined with a pharmaceutically acetable excipient and administered to the patient. After three days, the modified MAb injected has substantially located the cells expressing the malanoma antigens. The MAb The localization is then visualized by the immunocytography image using a collimator with a needle hole and a spectrum camera with range 91 obtained from Raytheon. The photographic record indicates a small area of radioactivity in the patient's skin, therefore it identifies a secondary fear. It will be appreciated that certain chemical and mechanical variations may be suggested to those skilled in the art. The examples and detailed description above will be understood more clearly by way of illustration, and the spirit and scope of this invention is limited only to the appended claims.

Claims (31)

1. CLAIMS 1. A study composition, comprising, an antibody conjugated with a dye, EDTA, DPTA, TETA or biotin in at least a plurality of free amino groups arranged in 5 the antibody to produce a modified antibody, this antibody has a reduced net positive charge compared to the intact antibody, the antibody has an average release in vivo between the release rates of the F (ab ') 2 fragments and the intact antibodies the same type; 10 and a radioneuclotide, toxin, drug or chelate bound to the modified antibody.
2. 2. The compound of Claim 1, wherein the antibody is a monoclonal antibody.
3. 3. The compound of Claim 1, wherein the antibody is a conjugate with biotin. jj ^ L
4. 4. The compound of Claim 1, wherein the antibody is conjugated to EDTA, DPTA or TETA.
5. 5. The compound of Claim 1, wherein the halogen or tenoion radioneucléotide binds to the modified antibody.
6. 6. The compound of Claim 5, wherein 125 or 131 binds to the modified antibody.
7. 7. The compound of Claim 1, wherein the antibody is conjugated with methotrexan, 5-fluoro-uracil, cis-platinum or adriamycin.
8. 8. The compound of Claim 1, wherein the antibody is conjugated to racin A chain.
9. 9. The pharmaceutical composition for immunosynatography, comprising: an antibody according to any of Claims 1 to 6; and a pharmaceutically excipient, carrier or base for immunosynography.
10. 10. The compound of Claim 9, for use in the localization of an antigen in mammals.
11. 11. The compound of Claim 10, for use in a gamma camera for immunosynatography.
12. The composition according to any of Claims 1 to 8, for use in the treatment of diseases in mammals.
13. The method for preparing the modified antibody according to claim 1, wherein the antibody has an increased antigen binding specificity, a reduced non-specific binding and a reduced in vivo release time, which comprises the following steps : get this antibody, when the antibody it is intact; react at least one of the free amino groups, with the dyes, a chelator or the biotin to produce 5 the mamified antibody, such that the modified antibody has an isoelectric point lower than the isoelectric point of the intact antibody; and labeling the modified antibody with a radioenucleotide, toxin, drug or chelate to produce a labeled modified antibody.
14. 14. The method according to claim 13, wherein the label is a radionucleotide selected from a group consisting of a halogen radionucleotide and technetium.
15. 15. The method according to claim 14, wherein the label is selected from a group consisting of 125 131
16. 16. A method for locating an antigen in mammals, comprising the steps of: obtaining a labeled modified antibody that has a binding specificity for an antigen to be located, the labeled modified antibody has lower free amino groups and a reduced isolelectric point compared to the unmodified antibody of the same type, the labeled modified antibody has a detectable label incorporated therein; administering the labeled modified antibody to a mammal, thereby allowing the labeled antigen and modified antibody to bind in vivo; and detecting the modified antibody labeled 5 bound to the antigen, whereby the antigen is localized.
17. 17. The method according to Claim 16, wherein the labeled modified antibody is labeled with a radionucleotide.
18. 18. The method according to claim 17, wherein the radionucleotide is selected from a group consisting of a halogen radionucleotide and techcinium.
19. 19. The method according to Claim 16, wherein the detection step comprises immunosynography.
20. 20. The method according to Claim 19, 15 wherein the immunosy- tography comprises the use of a gamma camera.
21. The method according to claim 16, wherein the labeled modified antibody comprises an intact chemically modified antibody in amino groups 20 free.
22. 22. The method according to claim 21, wherein the intact antibody is chemically conjugated to a heterobifunctional regent.
23. 23. The method according to Claim 21, wherein the modified antibody is chemically conjugated with biotin.
24. The method according to claim 16, wherein the chemical reagent is selected from a group consisting of methylkelates, other chelates and dyes.
25. 25. The method according to claim 24, wherein the chemical reagent is selected from a group consisting of N2S2, N2Se, EDTA, DPTA, TETA and FITC.
26. 26. The method for treating mammalian diseases comprising: obtaining an intact antibody specific for diseased tissue in mammals, this intact antibody is arranged in a plurality of free amino; 15 modify at least one of the free amino group by conjugating them with a chemical reagent to produce a modified antibody, the modified antibody has a reduced isoelectric point compared to the intact antibody, with the proviso that the chemical agent is not an agent 20 heterobifunctional; joining the biologically active molecule to the binding site arranged in the modified antibody different from the place of the modification by the chemical reagent; and administering this antibody to a mammal, by 25 what sick disease or tissue is treated.
27. 27. The method according to Claim 26, where the chemical reagent is biotin.
28. 28. The method according to Claim 26, wherein the chemical reagent is selected from a group that 5 consists of methylchelates, other chelates and dyes.
29. 29. The method according to Claim 28, wherein the chemical reagent is selected from a group consisting of N2Sz, N2Se, EDTA, DPTA, TETA and FITC.
30. 30. The method according to Claim 29, 10 wherein the biologically active molecule is selected from a group consisting of a toxic plant, such as a drug or a chelate.
31. 31. The method according to claim 30, wherein the drug is selected from a group consisting of 15 methotrexate, 5-fluoro-uracil, cis-platinum and adriamycin.