The method of treatment, antibodies, hybridoma


(57) Abstract:

The invention relates to medicine, in particular to Oncology and immunology, and for the treatment of b-cell lymphoma. The proposed new immunologically active chimeric antibody anti - CD 20, including radioactive labeled, as well as hybridoma, secreting specific in relation to SD 20-antigen antibody. The method of treatment is the introduction of drugs specified antibodies, including phased in three stages using different therapeutically effective amounts of these antibodies. The invention increases the specificity of the effects on malignant cells. 8 C. and 7 C.p. f-crystals, 27 ill., 3 table.

The copyright notice 37 C. F. R. 1.74 (d) / (e)

Part stated in this patent document contains material which is subject to copyright protection. The author has no objection to the reproduction by anyone of this patent document or material stated in it, in the form in which it appears in the files of the patent or registration protocols Patent and Trademark office, but in all other cases, reserves all copyrights.

Applications relating to the subject matter of this application


U. S. Serial N (submitted concurrently with this application). These related to the subject of the patent documents incorporated herein by reference.


A. the Scope of the invention

B. Background of the invention

C. Summary of the invention

D. Brief description of the drawings

E. Detailed description of the preferred options

F. Examples


A. Receiving anti-CD20 monoclonal (mouse) antibodies ("288")

B. Obtaining conjugate 2B8-MX-DPTA

i. MX - DTPA

ii. Getting 2B8

iii. The conjugation 2B8 with MX-DTPA

iv inclusion MX-DTPA

v Immunoreactivity 2B8-MX-DTPA

vi Obtaining labeled with indium-[III] 2B8-MX-DTPA ("12B8")

vii Obtaining labeled with yttrium-[90]-2B8-MX-DTPA ("Y2B8")

C. animal Studies (not the man)

i the Distribution of the radioactive-labeled 2B8-MX-DTPA

ii Localization 12B8 in tumor

iii Study bearsdley and tumor localization with the use of 2B8-MX-DTPA D And the technical analysis 12B8 (tomography) and Y2B8 (therapy)

a. Clinical trial phase I/II:

The study of therapy with the use of a single dose.

Clinical trial phase I/II:

the study of therapy with the use of multiple doses


A. Design of expressing the chimeric anti-CD20 immunoglobulin vector DNA

B. Creating producing chimeric anti-CD20 CHOB and SP 2/0-transfected

C. determination of the immunological activity of chimeric anti-CD20 antibodies

i Analysis with human C1q

ii Complement - dependent cell lysis

iii Effector analysis-dependent cellular cytotoxicity antibody


A. Study in primates

B. Clinical analysis C2B8

i Clinical trial phase I/II C2B8: a study of therapy with the use of a single dose

ii Clinical trial phase I/II C2B8: a study of therapy with the use of multiple doses


A. Obtaining Y2B8

B. Obtaining C2B8

C. Results




H. FORMULA IMAGE is information, described prior to the date of submission of this document, and nothing contained herein should not be construed as a waiver, expressed or definitely in the form of a hint that these links are "previous work" or that these applicants are not eligible to date back a number of such descriptions by previous inventions or priority, based on previously submitted applications.

This invention relates to the treatment of B-cell lymphoma using chimeric and radioactively labeled antibodies to the antigen Bp 35 ("CD20") the surface of B-cells.

B. Background of the invention

The immune system of the spine (e.g., primates, which includes humans, apes, monkeys, and so on) consists of a number of types of organs and cells that develop to: accurately and specifically to learn alien microorganisms ("antigen") that inhabit the vertebrate host; specifically bind to such alien microorganisms, eliminirovat/destroy these alien organisms.

Lymphocytes, among others, are crucial for the immune system. Lymphocytes produced in the thymus (thymus gland), spleen and bone marrow (adult) and approximately 30% of the total of calously of lymphocytes: T-cells and B-cells. T-cells are responsible for cell-mediated immunity, while B-cells responsible for producing antibodies (humoral immunity). However, T-cells and B-cells can be regarded as dependent in a typical immune response, because T-cells are activated by the binding of receptor T-cells with antigen fragments associated with glycoproteins major histocompatibility complex ("MHC") on the surface bearing the antigen cells; this activation induces the release of biological mediators (mediators), "interleukins"), which, in their accumulation, stimulate the differentiation of B-cells and antibodies (antibodies against this antigen.

Each B-cell is inside the host expresses a different antibody on its surface: one B-cell will Express the antibodies specific against one antigen, while the other B-cell to Express the antibody, specific for a different antigen. Thus, B cells are very diverse, and this diversity is critical for the immune system. In humans, each B cell can produce a huge number of molecules of antibody (approximately 107- 108). In most typical cases, this about is to sometimes proliferate specific B-cells are not attenuated; this proliferation can lead to cancer, called "B-cell lymphoma".

T-cells and B-cells contain cell surface proteins that can be used as "markers" for differentiation and identification. One of such markers of human B-cells is conservative (evolutionarily stable) differencirovany antigen Bp 35 B-lymphocytes of a man called "C20". CD20 is expressed during early development of precursor B-cells and remains in place until the differentiation of plasma cells. In particular, the CD20 molecule can regulate the stage of the activation process, which is necessary for initiation of the cell cycle and differentiation, and is usually expressed at very high levels in neoplastic ("tumor") B-cells. CD20, by definition, is present in normal B-cells, and "malignant" cells, i.e., those B-cells, not obstructed proliferation which can lead to B-cell lymphoma. Thus, the surface antigen CD20 could potentially be a candidate for "targeting" on the B-cell limpou.

In a compressed form such targeting can be summarized as follows: antibodies specific for surface antigen CD20 B-kletok, and malignant B-cells, anti-CD20 antibodies associated with surface antigen CD20, can lead to the destruction and depletion of neoplastic B-cells. Additionally with anti-CD20 antibodies can be conjugated to a chemical agent or a radioactive label, potentially destroying the tumor, so that the agent specifically "delivered", for example, neoplastic (tumor) B-cells. Regardless of the approach, the primary goal is the destruction of the tumor: a special approach can be determined by certain anti-CD20 antibodies, which are used. Thus, suitable approaches for targeting antigen C20 may differ greatly from one another.

For example, it was reported on the attempts of such targeting surface antigen CD20. It was reported that a murine monoclonal antibody IF5 (anti-CD20 antibody) was administered continuous intravenous infusion in patients with B-cell lymphoma. It was reported that extremely high levels (>2 grams) IF5 required for depletion of circulating tumor cells, and the results were described as "temporary". Press et al., Monoclonal Antibody IF5 (Anti-CD20) Serotherapy of Human B-Cell Lymphoma". Blood 69/2: 584 - 591 (1987).

A potential problem with this approach is what the STI human effector, i.e., they are not able to mediashout dependent complement lysis or to lyse human target cells through dependent antibodies cellular toxicity or indirectly with receptor phagocytosis. In addition, non-human monoclonal antibodies can be easily distinguished by the human host as a foreign protein; therefore, repeated injections of such alien antibodies can lead to the induction of immune responses, leading to a dangerous hypersensitivity reactions. In the case of murine antibodies is often referred to as the human response to anti - mouse antibodies, or "NAMA". Additionally, these "alien" antibodies can be attacked by the immune system of the host in such a way that they actually are neutralized before they reach their target customers.

Lymphocyte and simfonie cells are always sensitive to radiotherapy for several reasons: local emission of ionizing radiation radioactively labeled antibodies can kill cells with a target antigen or without it (for example, CD20 or without CD20) near the antibodies bound to the antigen, penetrating radiation may resolve the problem of limited access to antibodies in bulk or poorly vascularizing tumors; is that can destroy cellular DNA to such an extent, when the cellular repair mechanisms are not able to ensure the survival of the cells; therefore, if the target target cells are tumor radioactive label kills tumor cells. Radioactively labeled antibodies, by definition, include the use of radioactive substances that may require safety precautions for the patient (i.e. possible bone marrow transplantation), and for a person providing health care (i.e., the need to observe a high degree of caution when working with radioactivity).

Therefore, the approach to improve the ability of murine monoclonal antibodies to be effective in the treatment of B-cell disorders was to konjugierte radioactive label or toxin to these antibodies so that a label or toxin is localized at the site of the tumor. For example, the above-mentioned antibody IF5 "marked" iodine - 131 ("1311) and evaluated the biodistribution of the label in two patients. Cm. J. F. Eary "Imaging and Treatment of B-Cell Lymphoma" J. Nuc.Med. 31/8: 1257 - 1268 (1990); see also, al. "Treatment of Refractory Non-Hodgkin's Lymphoma with Radiolabeled MB-1 (Rufi-CD37) Antibocly" J. Clin.Onc.7/8 1027 - 1038 (1989) (showing that one patient who received1311 - labeled IF - 5, gave partial about the 991) (notified that 3 of the 8 patients receiving multiple injections, developed HAMA response); Appelbaum, F. R. "Radiolabeled Monoclonal

Antibodies in the Treatment of Non-Hodgkin's Lymphoma "Hem.Onc.Clinics of N. A. 5/5: 1013-1025 (1991) (review article): Press, O. W. et al. "Radiolabeled-Antibody Therapy of B-Cell Lymphoma with Antologous Bone Marrow Support" New England Journal of Medicine 329/17: 1219-1223 (1993) (labeled with iodine-131 anti-CD20 antibodies 175 and B1) and Kaminiski, M. G. "Radioimmunotherapy of B-Cell Lymphoma with [1311]Anti-B1 (Aati-CD20) Antibody", NEYM 329/7 (1993) (labeled with iodine - 131 anti-CD20 antibodies B1, hereafter referred to as "Kaminski")

Toxins (i.e., chemotherapeutic agents, such as doxorubicin or mitomycin C) was also manuginobili with antibodies. Cm. for example, PCT published application) WO 92/07466 (published may 14, 1992)

"Chimeric antibodies, i.e. antibodies which contain parts of two or more different species (e.g., mouse and human) have been developed as an alternative to the "conjugated" antibody. For example, Lin, A. Y. "Production of a Mouse - Human Chimerice Monoclonal Antibody to CD20 with strong Fc-dependent biologic activity". J. Immun. 139/10: 3521-3526 (1987), describes chimeric antibodies mouse/human anti-CD20 antigen. Cm. also PCT Publication 0/88/04936. However, there is no information about the validity, effectiveness or practicality of the use of such chimeric antibodies for treatment of B-cell disorders in which antibodies from cell-mediated cytotoxicity ("ADCC"), and so on) may not necessarily predict the ability of in vivo chimeric antibodies destroy or Deplete target cells, expressing the specific antigen. See, for example, Robinson, R. D. et al. "Chimeric mouse-human anti-cavcinoma antibodies that mediate different anti-tumor cell biological actirities" Hum. Antibol. Hybridomas 2: 84 - 93 (1991) (chimeric antibodies mouse-man, having redetection ADCC activity). Thus, the potential therapeutic efficacy of a chimeric antibody can be evaluated correctly only in vivo.

What is needed, and that would be a great success in this area is a therapeutic approaches targeting the CD20 antigen for treatment of B-cell lymphoma in primates, including (but not only) in man.

C. Summary of the invention

Here we describe therapeutic techniques for the treatment of B-cell disorders and, in particular, B-cell lymphoma. These protocols are based on the introduction of the immunologically active chimeric anti-CD20 antibodies for the depletion of peripheral B-blood cells, including B-cell associated with lymphoma; introducing a radioactively labeled anti-CD20 antibodies to target localized and associated with peripheral B-cells of the tumor; the introduction of a chimeric anti-CD20 antibodies and radioactively labelled anti-CD20 antibodies in a cooperative therapeutic strategies.

D. Brief about animago in obtaining immunologically active chimeric anti-CD20 antibodies ("TCAE 8");

Fig. 2A - 2E represent the sequence of the nucleic acid vector of Fig. 1;

Fig. 3A - 3F represent the sequence of the nucleic acid vector 1, additionally containing a murine variable region light and heavy chains (anti-CD20 in TCAE 8")

Fig. 4 represents a sequence of nucleic acid and amino acid sequence (including CDR and framework regions) murine variable region light chain derived from murine anti-CD20 monoclonal antibody 2B8;

Fig. 5 represents a sequence of nucleic acid and amino acid sequence (including CDR and framework regions) murine variable regions of the heavy chain derived from murine anti-CD20 monoclonal antibody 2B8;

Fig. 6 represents the results of flow cytometry, indicating the binding of fluorescently labeled human CIq with chimeric anti-CD20 antibody, and the quality controls used labeled CIq; labeled CIq and murine anti-CD20 monoclonal antibody 2B8; and labeled CIq and human IgG1,::

Fig. 7 presents the results associated with complement lysis comparing chimeric anti-CD20 antibody and mouse anti-CD20 monoclonal antibody 2B8;vectorname cells in vivo, comparing chimeric anti-CD20 antibody and 2B8;

Fig. 9A, 9B and 9C represent the results of the depletion of B - lymphocytes in the peripheral blood of a Primate (non-human) after infusion of 0.4 mg/kg (A); 1.6 mg/kg (B) and 6.4 mg/kg (C) immunologically active chimeric anti-CD20 antibody;

Fig. 10 shows the results, inter alia, the depletion of B - lymphocytes of peripheral blood of a Primate (non-human) after infusion of 0.01 mg/kg immunologically active chimeric anti-CD20 antibody;

Fig. 11 shows the results of destroying tumor cells actions UV in mouse xenograft models using B-cell lymphoblastic tumor;

Fig. 12 shows the results of destroying tumor cells actions C2B8 in mouse xenograft models using B-cell lymphoblastic tumor;

Fig. 13 shows the results of destroying tumor cells action combination UV and C2B8 in mouse xenograft models using B-cell lymphoblastic tumor;

Fig. 14A and 14B show the results of clinical analysis phase 1/11 C2B8, indicating a depletion of B-cell populations over time in patients, indicating a partial remission of the disease (14A) and a small remission Sabbah light and two heavy chains; these chains form a General "Y" shape, in which both light and heavy chains form the shoulders of Y and heavy chains form the base of the Y. the Light and heavy chain is divided into the domains of structural and functional homology. The variable domains of the light ("U2") and heavy ("Un") chains determine the recognition and specificity. The domains of the constant region of the light ("CL") and heavy ("CH") circuits responsible for important biological properties, for example, the Association of the antibody chains, secretion, transplacental mobility, linking Fcreceptor binding of complement and so on a Series of events leading to gene expression of immunoglobulin in producing antibodies cells is complex. Gene sequence of the variable region domain localized in individual line gene segments, called "Un", "D" and "YH" or "ULand YL". These gene segments are connected by rurangirwa DNA with the formation of complete neighbourhoods U, expressed, respectively, in the heavy and light chains. Rearrangeable United U segments (UL-YLand UH-D-YHthen encode the complete variable regions or antigen-binding domains of light and heavy chains, respectively.

l. (69 Blood 584, 1987, Supra); reported therapeutic responses, unfortunately, were temporary. In addition, 25% of the surveyed patients had developed a reaction against mouse antibodies (HAMA) in response to serotherapy. Press et. al., assume that these antibodies conjugated to toxins or radioactive isotopes may be more prolonged clinical benefit than unconjugated antibodies.

Due to the debilitating effect of B-cell lymphoma and a real need to ensure effective treatment approaches for this disease, we have taken a variety of approaches with the use of a specific antibody, 2B8, as a common link between these approaches. One such approach leverages the ability of systems mammals easy and effective recovery of B-cells in the peripheral blood, using this approach we are trying to get rid of B-cells or to Deplete B cells in the peripheral blood and lymphatic tissue as a means of eliminating and also B/ cell lymphoma. We do this by applying, inter alia immunologically active, chimeric anti-CD20 antibodies. In another approach, we are trying to destroy tumor cells, directing them in a radioactive label.

In applying section is theine 35000 daltons cell surface usually referred to as conservative (evolutionarily stable) differencirovany antigen Bp 35 B-lymphocytes person, usually called CD 20. In the application here, the term "chimeric" is used relative to anti-CD20 antibodies comprises antibodies that are most preferably produced by recombinant DNA technology and which contain the components as persons (including immunological "close" species such as chimpanzees), and human constant region of the chimeric antibody is most preferably essentially identical to the constant region of a natural human antibodies; the variable region of the chimeric antibody is most preferably manufactured from a source that is not a person and has the desired antigenic specificity against the CD20 antigen on the cell surface. Source other than human, may be any vertebrate animal, which can be used to generate antibodies to CD20 surface antigen of a person, or a material containing the surface antigen CD20 human cells. Such a source can be (but not limited to, rodents (e.g., rabbit, rat, mouse, and so on) and primates (not) (for example, the lower old world monkeys (martysh is here) is derived from a murine source. In the application here, the phrase "immunologically active" when applied to a chimeric anti-CD20 antibody refers to chimeric antibodies that bind human C1q, mediashout complement - dependent lysis ("CDC") lines B - lymphoid cells and are lysed target cells of a person due to dependent antibodies cellular cytotoxicity ("ADCC"). In the application here, the phrase "indirect labeling", "approach" indirect labeling" means that the antibody is covalently attached chelating agent and at least one radionuclide is built into this gelatinous agent. Preferred chelating agents and radionuclides described in Srivagtara, S. C. and. Mease, R. C. "Progress in Research on Ligands, Nuclides and Technigues for Labeling Monoclonal Antibodies", Nucl. Med. Bio. 18/6: 589-603/1991) ("Srivagtava") is incorporated herein by reference. Particularly preferred chelating agent is 1-isothiocyanatobenzene-3-metallitron - triaminopentauksusnoy acid (MX-DTPA"); a particularly preferred radionuclide for indirect labeling are indium [111] and the yttrium - [90] . In the application here, the phrase "direct labeling" and "approach direct labeling" means that the radionuclide is directly covalently attached to the antibody (usually through the remainder of the amino acids). Preferred radionuclides description is output via tyrosine residues. Especially preferred is the approach of indirect labeling.

Described herein therapeutic approaches based on the ability of the immune system of primates to quickly restore or rejuvenate B-cells in the peripheral blood. In addition, since the primary immune response of primates is T-cells, in the case when the immune system has the disadvantage of B-cells in peripheral blood and the need for "emergency" precautions (i.e., isolation of the patient, and so on) is not required. Due to these and other nuances of the immune systems of primates, our therapeutic approach to B-cell disorders allows you to delete the B-cells in peripheral blood using immunologically active chimeric anti-CD20 antibodies.

Because violations of B-cells in the peripheral blood, by definition, may indicate the need for blood processing, the route of administration of immunologically active chimeric anti-CD20 antibodies and radioactively labeled anti-CD20 antibody is preferably parenterally, in the application here, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration. From neither is the body and radioactively labelled anti-CD20 antibodies are usually obtained by standard methods in pharmaceutically acceptable buffer, for example, sterile saline, sterile buffered water, propylene glycol, or combinations thereof, etc., Methods of obtaining parenteral input agents are described in Pharmaceutical Carriers and Formulations, Martin, Remington's Pharmaceutical Sciences, 15 th Ed (Mack. Pub. Co. Easton, PA. 1975), incorporated herein by reference.

The specific therapeutically effective amount of immunologically active chimeric anti-CD20 antibodies applicable to obtain a unique therapeutic effect in any patient, can be determined by standard methods, well known to the specialists of ordinary skill in this field.

Effective dose (i.e., therapeutically effective amount) of immunologically active chimeric anti-CD20 antibody are in the range of from about 0.001 to 30 mg/kg body weight, more preferably from about 0.01 to 25 mg/kg body weight and most preferably from about 0.4 to 20.0 mg/kg of body weight. Other dose possible; factors influencing dose, include (but are not limited to) the seriousness, severity of disease, previous treatment approaches; General health of the patient, other participants diseases, etc. by a Qualified specialist will easily appreciate the particular patient and the CLASS="ptx2">

Introduction immunologically active chimeric anti-CD20 antibodies in these dose ranges can be carried out in a single injection or series of injections. In the case of chimeric antibodies preferably, this introduction was carried out in the form of a series of introductions, this is the preferred approach foreseen by the methodology of the treatment of this disease. Not wishing to be bound to any particular theory, note that since immunologically active anti-CD20 antibody immunologically active and bind to CD20, during the initial introduction of the immunologically active chimeric anti-CD20 antibody to the individual begins the depletion of B-cells in the peripheral blood, and we observed almost complete removal within approximately 24 hours after infusion. Therefore, the subsequent introduction (introduction immunologically active chimeric anti-CD20 antibodies (or radioactively labeled anti-CD20 antibody) to the patient allegedly: (a) removes the remaining B-cells in the peripheral blood; (b) begins to remove B cells from lymph nodes; (c) begins to remove B cells from other tissue sources, for example, from bone marrow, tumor, etc., In the result of repeated introductions immunologically active chimeric anti-CD20 antibodies, the number of event is. is the first "event" can be viewed as a directed mainly to the depletion of B-cells in the peripheral blood of the patient; subsequent events may be considered as aimed mainly at simultaneous or sequential removal of the remaining B-cells from the system by removing B-cells from lymph nodes or other tissues.

In fact, while single doses provide benefits and can be effectively used for the treatment/ management of the disease, the preferred treatment Protocol is carried out in several stages, most preferably between 0.4 and 20 mg/kg body weight immunologically active chimeric anti-CD20 antibody is administered to the patient once a week for a period of approximately 2 to 10 weeks, most preferably approximately 4 weeks.

In the case of the use of radioactively labeled anti-CD20 antibodies, preferably antibodies were not chimerical; it predicted a significantly long half-life of the chimeric antibodies compared with murine antibodies (i.e., longer half-life from blood radionuclide present in the patient for extended periods). However, radioactively labeled chimerical, associated with the murine antibody. This script allows you to reduce the toxicity to the bone marrow to acceptable levels with the preservation of therapeutic effect.

Many radionuclides are applicable for this invention, and specialists in this field can easily determine which radionucleid most suitable in many circumstances. For example, iodine [131] is a well-known radionuclide used for targeted immunotherapy. However, the clinical applicability of iodine [131] can be limited by several factors, including: eight-day physical half-life; dihalogenoalkane itinerancy antibodies in the blood and at the site of the tumor; characteristic radiation (for example, a large component of radiation), which may be suboptimal for the localized deposition of dose in the tumor. With the advent of excellent chelating agents attach metal chelating groups to proteins raised the possibility of using other radionuclides, such as indium [111] and the yttrium - [90]. The yttrium - [90] provides several advantages for use in radioimmunotherapeutic applications: the half-life of 64 hours of yttrium - [90] quite the high energy without accompanying gamma rays when it decays, with a range in tissue 200 - 1000 cell diameters. In addition, the minimum number of penetrating radiation makes possible the introduction of a labeled with yttrium - [90] antibodies to outpatients. In addition, the internalization of labeled antibodies is not required to kill cells, and local emission of ionizing radiation should be lethal to neighboring tumor cells that do not contain the target antigen.

One of the non-therapeutic limits for yttrium-90 is based on the absence of significant radiation, which makes it difficult to obtain images. To avoid this problem, you can use diagnostic "giving the image a radionuclide such as indium [111], to determine the location and relative size of the tumor before the introduction of therapeutic doses labeled with yttrium - [90] anti-CD20 antibodies. India [111] particularly preferred as a diagnostic radionuclide, because: you can enter without detectable toxicity of approximately 1 to 10 MCI; data tomography usually predict subsequent distribution labeled with yttrium - [90] antibodies. The majority of studies to obtain the images are labeled with 5 MCI India [111] antibodies, because this dose is safe and has a magnification obtained when 3 - 6 days after administration of the antibody. See, for example, Murray J. L. 26 Y. Nuc. Med. 3326 (1985) and Carraguillo, J. A. 26 Y. Nuc. Med. 67 (1985)

Effective dose is the only treatment (i.e., therapeutically effective amount) labeled with yttrium - [90] anti - CD20 antibody are in the range between approximately 5 and 75 MCI, more preferably between approximately 10 and 40 MCI. Effective one-time non-destructive bone marrow dose labeled with iodine [131] the anti - CD20 antibody are in the range between about 5-70 MCI, more preferably between about 5 and 40 MCI. Effective Deplete bone marrow dose for a single injection (i.e., which may require transplantation of autologous bone marrow) labeled with iodine [131] the anti - CD20 antibody are in the range between approximately 30 and 600 MCI, more preferably between about 50 and less than 500 MCI. In connection with chimeric anti-CD20 antibodies, due to the longer half-life in blood compared with murine antibodies, an effective single dose, do not destroy the bone marrow, labeled with iodine [131] chimeric anti - CD20 antibody is in the range between approximately 5 and 40 MCI, more preferably less than 30 MkI. Criteria for imaging, for example, for India [III] is usually less princie be carried out using a single treatment or multiple treatments. Due to the presence of the radionuclide component should preferably be taken before processing peripheral stem cells ("PSC") or bone marrow ("VM") for patients experiencing potentially fatal toxicity to bone marrow, resulting from the irradiation. VM and (or) the PSC taken using standard methods, then purified and freeze when possible reinfused. Additionally, most preferably holding before processing diagnostic dosimetry using diagnostic labeled antibodies (e.g., using India [III] on the patient. The objective of this review is to provide assurance that therapeutically labeled antibody (e.g., labeled with yttrium - [90] would not "concentrate" in any normal organ or tissue.

Described chimeric mouse/human antibody. See, for example, Morrison. S. L. et. al. PNAS 11: 6851-6854 (November 1984) European Patent Publication N 172404, Boulianne, C. L. 312:642 (December 1984); Neubeiger M. S. 314:268 (March 1985); European Patent Publication N=125023, Tan J. Immunol.135:8564 (November 1985); Sun, L. K. 5/1:517 (1986); Sanagan et. al, J. Immunol. 137: 1066-1074 (1986). Cm.Muron.Nature 312:597 (December 1984), Diekson, Genetic Ehgineering News 5/3 (Marck 1985), Marx, Science 229: 455 (August 1985) and Morrison Science 229:1202-1207 (September 1985). Robinson, in PCT Publation N WO88/04936 describe chimeric antibody with a human who REGO antibodies produced from murine monoclonal antibody 2H7 (gamma 2, Kappa). Although the author notes that the described chimeric antibody is an "excellent candidate" for the treatment of B-cell disorders, this statement can be regarded as no more than a proposal, and specialists in this field should determine whether this assumption is correct for that specific antibodies, especially because in this link there are no data supporting the application of therapeutic efficacy and, more importantly, there are no data from the use of higher order mammals, such as primates or people.

The methodology for obtaining chimeric antibodies are available for professionals in this field. For example, light and heavy chains can Express separately using, for example, the light chain immunoglobulin and heavy chain immunoglobulin in a separate playdoh. They can be cleaned and assembled in vitro in full antibodies, methods of performing such assemblies have been described. Cm. for example.Scharff M. Harveg Lectures 69: 125 (1974). The parameters of the reaction in vitro for the formation of IgG antibodies isolated from light and heavy chains were also described. Cm. for example Beychok, S. Cells of Immunoglobulin Synthesis, Academic Press, New York p.69,1979. Co-expression of light and heavy chains in the same kitchel also possible. Such co-expression can be performed using either the same plasmid or plasmids in the same host cell.

Another approach, which is our preferred approach for the learning chimeric non - human/human anti - CD20 antibodies, based on the use expressing vector, which contains, ab initio DNA encoding the constant region of the heavy and light chain of human origin. This vector allows the introduction of DNA that encodes a nonhuman variable region, so that you can get a lot of non - human anti-CD20 antibodies, which can be subjected to screening, analyzed for various characteristics such as the type of binding specificity, binding the epitope regions, etc.,); after that, the vector may be included cDNA encoding the variable regions of light and heavy chains of the preferred or the desirable anti-CD20 antibodies. We call these types of tandem vectors expressing chimeric antibodies vectors ("TCAE"). The most preferred TCAE vector that was used to generate immunological aktivnih chimeric anti - CD20 antibodies for therapeutic treatment of lymphomas is ivalsa TCAE 5.2. The difference is that in TCAE 5.2 start site of translation initiation dominant selectivity marker (neomycinphosphotransferase, "NEO") is a consensus Kozak sequence, whereas for TCAE 8 this area is corrupted consensus Kozak sequence. Details regarding the actions of the start site of translation initiation dominant breeding marker vector TCAE (also called "ANEX vectors") on the expression of the protein are discussed in detail in concurrently filed application submitted with this application.

TCAE 8 contains four transcriptional cassettes, in tandem order, i.e., the light chain of human immunoglobulin with no variable regions; human heavy chain immunoglobulin with no variable regions, DHER; NEO. Each transcription cassette contains its own eukaryotic promoter and a region polyadenylation (reference is made to Fig. 1, which is a schematic illustration of vector TCAE 8). Specifically:

1) the CMV promoter/enhancer ahead of the heavy chain of immunoglobulin is ukorochenniy version of the promoter/enhancer ahead of the light chain, from Nhe 1 sa is globulin obtained through amplification of cDNA using polymerase chain reaction (PCR), in TCAE 8 it was the constant region of the Kappa light of human immunoglobulin (numeriska Kabat, amino acids 108-214, allotype Km3 (see Rabat E. A. "Seguences of proteins of immunological interest" NIH Publication, Fifth Ed. N 91-3242, 1991) and the constant region of the heavy chain gamma 1 human immunoglobulin (Kabat numbering amino acids 114 - 478, allotype Gmba, Gmlz). Light chain was isolated from normal human blood (IDEC Pharmaccuticals Corporation, La Jolla, CA) RNA from it was used to synthesize cDNA, which is then amplified using PCR techniques (primers were obtained against the consensus of Kabat). A heavy chain was isolated (using method PCR) from cDNA derived from RNA, which in turn was obtained from cells transfected with the human vector (see 3 Prot. Eng 531, 1990; vector pN162). Two amino acids were changed in a dedicated human IgGI, to come to the consensus amino acid sequences of Kabat, namely: amino acid 225 changed from valine to alanine (G TT was replaced by GGA), and the amino acid 287, methionine was replaced by lysine (ATS replaced AAG);

3) Tape the light and heavy chains of human immunoglobulin contain synthetic signal sequence for secretion of these chains of immunoglobulin;

4) Cartridge light is not possible embedding of the variable regions of light and heavy chains of immunoglobulin, who hold the adapter frame read and do not change the amino acid, normally found in the chains of immunoglobulins;

5) Cassette DHER contained its own eukaryotic promoter (mouse main promoter of the beta-globulin, "the COUNCIL") and the district polyadenylation (district polyadenylation bovine growth hormone, "BGH"); and

6) the NEO Cassette contained its own eukaryotic promoter (VETA) and district polyadenylation (district early polyadenylation Sr50, "SV").

In the case of vector TCAE 8 and the NEO cassette district Kozak was partially damaged consensus Kozak sequence (which is included in the direction 3' to 5;the website Cla I):

< / BR>
(Vector TCAE 5.2 there is a change between districts Cla I and ATG, namely: CCACC).

The full sequence TCAE 8 (including specific components of the four transcription cassettes) are presented in Fig. 2 (SEQ ID NO. 1).

As will be clear to experts in this field, the vector TCAE can significantly reduce the time of receipt of the immunologically active chimeric anti - CD20 antibodies. The generation and allocation of non - human variable regions of light and heavy chains, followed by the inclusion in the transcriptional Cass is blast the heavy chain of human immunoglobulin allow you to get immunologically active chimeric anti - CD20 antibodies.

We received the most preferred non - human variable region with specificity against the CD20 antigen using a murine source and hybridoma technology. Using the method of polymerase chain reaction ("PCR")

variable regions of murine light and heavy chains were cloned directly into the vector TCAE 8. This is the most preferred way to include non - human variable regions in the vector TCAE. This preference fundamentally predicted by the efficiency of the PCR reaction and accuracy insertions (embedding). However, suitable and other equivalent procedure to perform this task. For example, using the TCAE 8 (or by an equivalent vector) we can obtain the sequence of the variable region of non-human anti-CD20 antibodies followed by oligonucleotide synthesis parts of this sequence or, if necessary, throughout this sequence; after that part or all of the synthetic sequence can be embedded in suitable locations within the vector. Specialists in this field are able to perform this task.

Our most preferred immunologically active chimeric anti - CD20 antibodies were polucheniya antibodies to CD20; these antibodies (which will be discussed in detail infra called 2B8.

The full sequence of the variable regions derived from 2B8 in TCAE (anti-C 20 in TCAE 8") are presented in Fig. 3 (SEQ ID NO. 2).

Line of host cells used for expression of the protein, most preferably is a line of mammalian cells, the experts in this field can determine the preferred line of host cells that are most appropriate to Express them in the target gene product. Cited as an example of a cell line of the host include, but are not limited to) line DG44 and DUB11 (line ovary cells Chinese hamster, DH7R minus), HELA (human cervical carcinoma), CVI (line kidneys of monkeys) COS (a derivative of CVI with T antigen SV40), R 1610 (fibroblasts Chinese hamster), BAL BC/ZTZ (mouse fibroblasts), NAC (kidney line hamster), SP 2/0 (mouse myeloma), P3 x 63 - Ag 3,653 (mouse myeloma), BFA - 1cIBPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney). Line to the host cells are typically available from commercial services, ADS or from published literature.

The preferred cell line DG44 host is ("CHO") or SP2/0. Cm. "Effect of gamma vays and the ture

276 : 269 (1978), respectively. Most preferably, the cell line DG44 host is a. Transfection of this plasmid into the host cell can be accomplished by any means available to specialists in this field. These methods include, but are not limited to) the content (including electrophoresis and electroporation), merge cells coated with DNA, microinjection, and infection of intact virus. Cm. Ridgway, A. A. G. "The Mammalian Expverssion Vectors".

Chapter 24.2.pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, MA 1988). Most preferably the introduction of the plasmid into the host via electroporation.

G. Examples

The following examples are not intended to limit the present invention. These examples are intended to show: dose imaging with the use of radioactively labelled anti - CD20 antibodies (12B8"); radioactive labeled anti - CD20 antibody ("Y2B8"); and immunologically active, chimeric anti - CD20 antibody ("C2B8"), obtained using a specific vector ("TCAE8") and variable regions generated from murine anti - CD20 monoclonal antibody (2B8").

1. Radioactively labeled anti-CD20 antibody 2B8

A. Receiving anti - CD20 monoclonal antibodies (masinag. Direct implantation and serial transplantation of human acute lymploblastic leukemia in hamsters, SB-2". Can. Res. 28: 1121- 1125 (1968); this cell line available from the American Tissue Culture Collection, Rockville, M. D. nog ATCC accession N=ATCC CCL 120), with weekly injections given over a period of 3-4 months. Were identified mouse detecting high serum titers of anti-CD20 antibodies, as determined by the known inhibition specific for CD20 antibodies (used antibodies Beckton Dickinson, San. Jose, CA, Cat. N 7670, and B1, Conlter Corp. Hiallah. FL. Cat. N 6602201); thereafter, removing the spleen of these mice. Cells of the spleen was merged with mouse myeloma SP2/0 in accordance with the Protocol described in Einfeld. D. A. et. al. (1988) EMBO 7:711 (SP 2/0 has ATCC accession N ATCC CRL 8006).

Test C 20 specificity was performed using radioimmunoassay. Briefly, purified anti-CD20 B1 radioactively marked I125according to the method of the iodine beads (iodobead), as described in Valentine. M. A. et. al. (1989), J. Biol. Chem. 264: 11282. (I125Sodium Iodide (sodium iodide), ICN Irvine, CA. Cat N 28665H). Hybridoma were subjected to screening by co-incubation, 0.05 ml of medium from each well to merge with 0.05 ml labeled I125anti-CD20 B1 (10 ng) in 1% BSA (bovine serum albumin) in PBS (pH 7,4) and 0.5 ml of the same buffer containing 100000 SB cells. After incubation for 1 hour at room temperature, the cells Vuh replications, containing unlabeled anti-CD20 B1 and wells that do not contain inhibiting antibodies were used as positive and negative controls, respectively. Wells that showed more than 50% inhibition, increased in volume and cloned. Antibodies that detect the highest inhibition were derived from a cloned cell line, called here "2B8",

B. Obtaining conjugate 2B8 - MX - DTPA i MX - DTPA

Labeled14C 1-isothiocyanatobenzene - 3-metallitron three-aminopentanoic acid ("14C labeled MX-DTPA") was used as a chelating agent for conjugation with a radioactive label 2B8. Manipulation of MX-DTPA was performed with preservation does not contain metal conditions, i.e. used contains no metal reagents and, if possible, polypropylene plastic containers (flasks, chemical beakers, graduated cylinders, pipette tips), washed with Alconox and rinsed Milli-Q water. MX-DTPA was obtained in the form of dry solids from Dr. Otto-Gansow (National Institute of Health, Bethesda, MD) and stored in a desiccator at 4oC (protected from light), the initial solutions were prepared in Milli-Q water at a concentration of 2-5 mm and stored at -70oC. MX-DTPA was obtained from Coulter Immunology (Hialeah, Florida) disodium with the MX-DTPA by transferring these antibodies do not contain metals 50 mm bizin - NAOff, pH 8,6 containing 150 mm NaCl, using repeatable shift buffer with METHOD 30TMspin filters (30000 MWCO; Amicon) Usually 50-200 µl of protein (10 mg/nl) was added to one filter, and then 2 ml bizin-buffer. The filter was centrifuged at 4oC in the rotor Sorval SS-34 (6000 rpm, 45 min). The volume of retentate was approximately 50 - 100 ál, this process was repeated twice using the same filter. Retentate was transferred into a polypropylene tube of 1.5 ml with screw cap, determined the protein was diluted to 10.0 mg/ml and kept at 4oC to use; protein similarly transferred in 50 mm sodium citrate, pH 5.5, containing 150 mm NaCl and 0.05% sodium azide, using the previous Protocol.

iii Conjugation 2B8 with MX-DTPA

Conjugation 2B8 with MX-DTPA was performed in polypropylene tubes at ambient temperature. Frozen original solutions of MX-DTPA was thawed immediately before use. 50 - 200 ml protein at 10 mg/ml was reacted with MX-DTPA at a molar ratio of MX-DTPA to 2B8 4:1. Reactions were initiated by addition of the initial solution MX-DTPA and careful mixing; conjugation occurred during the night (14 - 20 h) at ambient temperature. Unreacted MX-DTPA was removed from conjuga icny saline solution (0.9% weight/volume), containing 0.05% sodium azide. The protein concentration was brought to 10 mg/ml and the solution was stored at 4oC in a polypropylene tube to radioactive labeling.

in Determining the inclusion of MX-DTPA

MX-DTPA incorporation was determined using scintillation counting and comparing the values obtained with the purified conjugate, with specific activity of labeled14C MX-DTPA. For certain studies that used non-radioactive MX-DTPA (Coulter Immunology) the inclusion of MX-DTPA was assessed by incubation of the conjugate with an excess of radioactive solution - media yttrium - [90] a known concentration and specific activity.

The original solution of yttrium chloride of known concentration was prepared in not containing metals of 0.05 N HCl, to which was added not containing media yttrium - [90] (salt of hydrochloric acid). An aliquot of this solution was analyzed using a liquid scintillation account to determine the exact specific activity for this reagent. The amount of this reagent chloride yttrium containing 3 times the number of moles chelate than the number of moles, which must be attached to the antibodies (usually 2 mol/mol of antibody) was added to a polypropylene tube and the pH was brought to 4.0 to 4.5 2 M acetate hydroxide the Reaction was stopped by adding 20 mm EDTA to a final concentration of 1 mm and the pH was brought to about 6 2 M sodium acetate.

After 5 min incubation, the entire amount was purified using gel-filtration chromatography high resolution (described infra). Erwerbende containing protein fractions were combined, determine the concentration of protein in the aliquot was determined by the radioactivity. The inclusion of the chelate was calculated by the specific activity of the preparation of the chloride of yttrium - [90] and protein concentration.

v. Immunoreactivity 2B8 - MX-DTPA

Immunoreactivity conjugated 2B8 was assessed using ELISA for intact cells. SB cells in mid-logarithmic phase of growth were collected from the culture by centrifugation and washed two times with 1 × HBSS, the Cells were diluted to 1-2 106cells/ml in HBSS and took aliquots in 96-well polystyrene microtiter tablets in the 50,000 - 100,000 cells/well. The tablets were dried under vacuum for 2 h at 40 - 45oC for fixation of cells on plastic; the tablets were stored dry at -20oC before use. For the analysis of the tablets were heated to ambient temperature immediately prior to use, then blocked 1 PBS, pH of 7.2 to 7.4, containing 1% BSA (2 hours). Samples for analysis were diluted in 1 PBS/1% BSA, was applied to the plates and serially diluted (serial dilution) (1:2) in the same S. To the wells were added to the secondary antibody (goat anti-mouse IgG 1 conjugated with a specific HRP, 50 µl) (1: 1500 dilution in 1 x PBS 1% BSA) and incubated for 1 hour at ambient temperature. The tablets were washed 4 times with 1 x PBS, then added substrate ABTS solution (50 mm sodium citrate, pH 4.5, containing 0.01% of ATBS and 0.001% H2O2). The tablets were read at 405 nm after 15 - 30 min of incubation. Antigen-negative HSB cells included in the analysis to determine nonspecific binding. The immunoreactivity of the conjugate was calculated by applying the values of the absorption on the schedule against the respective dilution factor and compare them with the values obtained with the use of native antibodies (representing 100% immunoreactivity), tested on the same tablet; some values on the linear part tiralongo profile was compared and determined the average (data not shown).

vi Obtaining labeled with indium-[III]2B8 - MX-DTPA ("12B8")

The conjugates were labeled radioactively not containing media indium-[III]. An aliquot of the isotope (0.1 to 2 MCI/mg Titel) in 0.05 M HCl was transferred into a polypropylene tube and add approximately 1/10 of the volume does not contain metals 2 M HCl. After incubation for 5 min the tion of 0.5 mg 2B8 - MX-DTPA from the original solution of 10.0 mg/ml DTPA in normal saline or 50 mm sodium citrate /150 mm NaCl containing 0.05% sodium azide, and the solution immediately gently stirred. Checked the pH paper to determine the pH to ensure that the pH is 4.0 to 4.5, and the mixture is incubated at ambient temperature for 15 - 30 minutes Then the reaction was stopped by adding 20 mm EDTA to a final concentration of 1 mm and the reaction mixture is brought to approximately pH to 6.0 with 2 M sodium acetate.

After 5-10 min incubation complexional isotope was removed by gel-filtration chromatography. Node HPLC consisted of a Waters Model 6000 or TosoHaas TSK - 6110 delivery system solvent, supplied, respectively, of the injection valve Waters U6K or Rheodyne 700. Chromatographic separation was performed using a gel permeation column (BioRad SEC-250; 7.5 x 300 mm or comparable Toso Haas column) and a relief column SEC-250 (7.5 x 100 mm). The system was equipped with a collector fractions (Pharmacia Frac 200) and a UV monitor was equipped with a filter 280 nm (Pharmacia Model UV-1). The sample was applied and was suirable isocratically with the use of 1 x PBS, pH 7,4, when the speed of the current of 1.0 ml/min. was Collected fractions of 0.5 ml in a glass test tube and aliquots of these tubes was considered radioactively was calculated by summing the radioactivity related elyuirovaniya peak protein and dividing this number by the total radioactivity, elyuirovaniya from the column; this value was then expressed in percent (data not shown). In some cases, the incorporation of radioactivity was determined by instant thin-layer chromatography ("ITLC"). Radioactively labeled conjugate was diluted 1:10 or 1:20 in 1 x PBS containing 1 x PBS/1 mm DTPA, and then 1 μl was applied at a distance of 1.5 cm from one end of the strips 1 x 5 cm paper ITLC SG. The paper showed through ascending chromatography using 10% ammonium acetate in methanol : water (1 : 1, V/V). The strip was dried, cut a cross on the cross in half and the radioactivity associated with each section were determined by gamma-account. The radioactivity associated with the bottom strips (associated with protein radioactivity) was expressed in percentage of the total radioactivity determined by summing the values for the upper and lower halves (data not shown).

The specific activity was determined by measuring the radioactivity of a suitable aliquot of radioactively labeled conjugate. This value was corrected for the efficiency of the counter (usually 75%) and was attributed to the concentration of a protein conjugate as defined previously by the absorption of p is II] according to the above Protocol, but without purification with HPLC; this Protocol is called "mix-and-shoot" Protocol.

vii Obtaining labeled with yttrium - [90] 2B8 - MX-DTPA ("Y2B8")

The same Protocol described for obtaining 12B8, was applied to obtain labeled with yttrium - [90] V - MX-DTPA (Y2B8") conjugate, except that they had not used 2 ng HCl; all products labeled with yttrium conjugates were purified by gel-filtration chromatography as described above.

C. animal Studies (non-man)

i Biodistribution of radioactively labeled 2B8 - MX-DTPA

Estimated distribution V in tissues in 6 - 8 weeks BALB/c mice. Radioactively labeled conygar prepared using clinical V - MX-DTPA after the above Protocol "mix-and-shoot". The specific activity of the conjugate was 2.3 MCI/mg conjugate was prepared in PBS, pH 7,4 containing 50 mg/ml HSA (human serum albumin). Mice were intravenously injected with 100 μl W (approximately 21 µci) and groups of three mice were killed by cervical displacement at 0, 24, 48 and 72 hours. After killing had removed the tail, heart, lungs, kidneys, spleen, muscles and thigh, washed and weighed; a blood sample was also taken for analysis. The radioactivity associated with each image was determined using the account gam and through the blood, associated with individual organs, was not done.

In a separate Protocol aliquots V - MX-DTPA, incubated at 4oC and 30oC for 10 weeks, was radioactively labelled with indium [III] to a specific activity of 2.1 MCI/mg for both drugs. These conjugates were then used in the studies on the biodistribution, as described above.

For dosimetric definitions V - MX-DTPA was radioactively labelled with indium - [III] to a specific activity of 2.3 MCI/mg and about 1.1 µci were injected with each of 20 BaLB/c mice. Then groups of 5 mice were killed at 1, 24, 48 and 72 hours and their organs were removed and analyzed for analysis. In addition, part of the skin, muscles and bones were removed and processed for analysis, urine and faeces were also collected and analyzed for points 24 - 72 hours

Using this approach U - MX-DTPA also was radioactively labelled with yttrium -[90] and its biological distribution was evaluated in BALB/c mice during the 72-hour period. After purification by gel-filtration chromatography HPLC four groups of 5 mice were intravenously injected with approximately 1 µci clinically prepared conjugate (specific activity: 12.2 MCI/mg); these groups were then killed at 1, 24, 48 and 72 hours and their organs and tissues analizirovat the ow of energy in gamma-scintillation the counter. The magnitude of the activity was expressed as percent injected dose per gram tissue or in percent injected dose per organ. While the organs and other tissues repeatedly spolaskivanie to remove surface blood, the bodies inside were not rinsed. Thus not introduced amendments to the contribution of radioactivity due to contained inside the body the blood.

ii Localization V in tumor

The localization of radioactive labeled V - MX-DTPA was determined in deprived thymic mice bearing Ramos tumor B-cells. 6 - 8 weeks devoid of thymus mice were injected with subcutaneous (left rear side) of 0.1 ml of RPMI-1640 containing 1,2 107Ramos tumor cells that were pre-adapted to growth in deprived thymic mice. Tumors occurred within two weeks and had a weight of from 0.07 to 1.1, Mice were intravenously injected with 100 μl labeled with indium - [III] V - MX-DTPA (16,7 µci) and groups of three mice were killed by cervical displacement at 0, 24, 48 and 72 hours. After killing had removed the tail, heart, lungs, liver, kidneys, spleen, muscles, hip and swelling, washed, weighed, were also taken for analysis of blood samples. The radioactivity associated with each sample was determined using a gamma-account and determined the percentage injectionamikacin labeled V - MX-DTPA

After the above preliminary experiment bearsdley (Example I. B. viii, a) conjugated V was radioactively labelled with indium - [III] to a specific activity of 2.3 MCI/mg and about 1.1 µci were injected with each of the twenty BALB/c mice to determine bearsdley radioactively labelled material. Then groups of five mice were killed at 1, 24, 48 and 72 hours and their organs and parts of your skin, muscles and bones were removed and processed for analysis. In addition, the collected urine and faeces and analyzed at the points 24 to 72 hours. The level of radioactivity in the blood fell from 40.3% of the injected dose per gram at the first point (1 hour) to 18.9% at the point 72 hours (data not shown). Quantity for heart, kidney, muscle and spleen remained in the range of 0.7 - 9.8% in the entire experiment. The levels of radioactivity detected in the lungs, decreased from 14.2% in the first point (1 hour) to 7.6% at 72 hours: similarly, the corresponding values of the injected dose per gram lung were 10.3% and 9.9 per cent. These data were used to determine estimates of the absorbed dose of radiation V described below.

To determine the localization of the tumor was preparing W - MX-DTPA and was radioactively labelled with indium - [III] to a specific activity of 2.7 MCI/mg 100 ál mechaniczne tumors. The tumor weighed from 0.1 to 1.0 grams. At time points 0, 24, 48 and 72 hours after injection of 50 µl of blood was taken zaglaniczny puncture, mice were killed by cervical displacement and removed the tail, heart, lungs, liver, kidneys, spleen, muscles, thigh, and swelling. After processing and weighing tissues determined the radioactivity associated with each tissue image using a gamma counter and the values were expressed as percent injected dose per gram.

The results (not shown) showed that the concentration in tumor111In-2B8 - MX-DTPA increased steadily during the experiment. 13% of the injected dose accumulated in the tumour after 72 hours. Blood levels were falling in the course of the experiment from over 30% at 0 point to 13% at 72 hours. All other fabrics (except muscle) contained between 1.3 and 6% of the injected dose per gram tissue at the end of the experiment; muscle tissue contained approximately 13% of the injected dose per gram.

D. Studies on humans

i. 2B8 and V - MX-DTPA: immunohistological studies with human tissues.

The ability to react with different tissues of mouse monoclonal antibodies V was evaluated using a panel of 32 different human tissues, fixed with acetone. Antibodies V the notches in the lymphoid tissues, including lymphoid tissues of hematopoietic origin.

In the lymph node immunoreactivity was observed in the population of Mature cortical B-lymphocytes, as well as in proliferating cells in embryonic centers. Positive reactivity was also observed in peripheral blood B-cell zones of the tonsils, white pulp of the spleen and 40 - 70% of medullary lymphocytes detected in the thymus (thymus gland). Positive reactivity was also observed in the follicles of the lamina propria (of Meyerovich plaques) of the colon. Finally, units or scattered lymphoid cells in the stroma of various organs, including bladder, breast, cervix, esophagus, lung, parotid gland, prostate, small intestine and stomach were also positive with the antibody V (data not shown).

All cells single-layered epithelium, as well as multi-layered epithelium and the epithelium of the various bodies did not react with W. This way no reactivity with neuroectodermal cells, including the brain, spinal cord and peripheral nerves. Mesenchymal elements such as cells, skeletal and smooth muscle, fibroblast, endothelial cells and polymorphonuclear FOTS is V - MX-DTPA was evaluated using set (panel) 16 human tissues, fixed with acetone. As was shown previously with native antibody (data not shown), the conjugate V - MX-DTPA learned antigen CD20, which is found very restricted pattern of distribution: it was found only on the sub-cells of lymphoid origin. In the lymph node immunoreactivity was observed in the population of B-cells. Strong reactivity was observed in the white pulp of the spleen and in the medullary lymphocytes of the thymus. Immunoreactivity was also observed in scattered cells in the urinary bladder, heart, thick intestines, liver, lung, and uterus, as well as in inflammatory cells present in these tissues. As in the case of native antibody reactivity was not observed with cells neuroectoderm or mesenchymal elements (data not shown).

ii Clinical analysis 12B8 (tomography) and Y2B8 (therapy)

a. Clinical analysis of phase I-II 12B8 with the use of therapy with a single dose

Clinical research phase I-II 12B8 (MRI), followed by processing a single therapeutic dose of Y2B8 are currently in. For studies of therapy with the use of a single dose perform the following scheme:

B8 - therapy (three dose levels); and

4. The PSC transplantation or autologous BM (if necessary on the basis of the absolute account of neutrophils below 500 nm3for three consecutive days or platelets less than 20,000 per mm3in the absence of recovery of the bone marrow test bone marrow).

Level doses Y2B8 the following:

The dose - Dose (MCI)

1 - 20

2 - 30

3 - 40

To determine the maximum tolerated at dose (MTD) should be treated three patients at each dose level.

Tomographic (dosimetry) research carried out as follows: each patient participates in two studies in vivo distribution using 12B8. In the first study, 2 mg 12B8 (5 MCI) was injected as an intravenous (i.v.) within 1 h; after one week 2B8 (i.e. unconjugated antibody) is administered by i.v. at a speed of not more than 250 mg/h followed by immediate introduction 2 mg 12B8 (5 MCI) of i.v. within 1 h In both studies immediately after infusion 12B8 each patient is subjected to imaging and imaging is repeated at time t=14-18 h (if specified), t = 24 h; = 96 h (if specified). Determine the average retention time of the whole body for India - ([III]; such definitions are also deltares areas compared with the concentrations of the label of the whole body; on the basis of this comparison it is possible to evaluate the localization and to determine the concentration of Y2B8 using standard protocols. If the estimated cumulative dose of Y2B8 than 8 times higher than the dose of the whole body, or if a certain cumulative dose to the liver exceeds 1500 cGy, should not be the treatment of Y2B8.

If research tomography give acceptable results, either 0 or 1.0 mg/kg of body weight of the patient 2B8 administered by intravenous infusion at a speed not exceeding 250 mg/H. Then injected Y2B8 (10, 20 or 40 MCI) at a speed of i.v. injection of 20 MCI/h

b. Clinical trial phase I/II study of therapy with the use of multiple doses

Provide clinical analysis of phase I/II Y2B8. For studies with multiple doses, perform the following scheme:

1. Collection PSC or BM;

2. 12B8 - tomography;

3. Y2B8 therapy (three levels of dose for four doses or a total cumulative dose of 80 (MCI):

4. The PSC transplantation or autologous BM (on the basis of the decision of a health care worker).

Dose levels Y2B8 the following:

The dose - Dose (MCI)

1 - 10

2 - 15

3 - 20

To determine the MTD should be treated three patients at each dose level.

Tomographic (dosimetrical the ie 2B8) define with the first two patients. The first two patients receiving 100 mg of unlabeled 2B8 in 250 ml normal saline over 4 h and then 0.5 MCI 12B8. Blood samples taken for data on biodistribution at t=0, t=10 min t = 120 min, t = 24 h t = 48 hours Patients scanned using a gamma camera to obtain many local images at time points 2 h, 24 h, and 48 h After scanning at 48 h patients received 250 mg 2B8, as described, then 4.5 MCI 12B8. Sampling of blood and the scan should then, as described. If 100 mg 2B8 give excellent picture, these two patients receiving 50 mg 2B8, as described, then 0.5 MCI 12B8 and after 48 h 100 mg 2B8, then 4.5 MCI 12B8. If 250 mg 2B8 give excellent picture, these two patients receiving 250 mg 2B8, as described, then 0.5 MCI 12B8, after 48 h : 500 mg 2B8 and then 4.5 MCI 12B8. Subsequent patients treated with the lowest number of 2B8, which provides optimal image. The optimal image is characterized by: (1) the most effective image with the slow disappearance of antibodies; (2) the best distribution that reduce compartmentalization in one body; and (3) the best subjective resolution of damage (compared tumor/background).

For p the La subsequent patients first therapeutic dose of Y2B8 begin to give between 2 and 7 days after 12B8.

Before receiving Y2B8 for patients, except for the first four, 2B8 injected, as described, with the next i.v. the infusion of Y2B8 5-10 min) blood Samples for bearsdley taken at 0, 10 min, 120 min, 24 h, and 48 hours Ill give repeated doses Y2B8 (injected the same dose as in the first introduction) approximately every 6-8 weeks for a maximum of four doses or total cumulative dose of 80 trials. Most preferably, the patients had not received a subsequent dose of Y2B8 until WBC more than 3000 patients or equal to 3000, and AGC is greater than or equal to 100000.

After completion of the study of three dose levels determine the MTD. Then introduce additional study of patients who receive MTD

II. Receiving Anti-CD20 Antibodies ("C2B8")

A. Design of expressing the chimeric anti-CD20 immunoglobulin vector DNA

RNA was isolated from 2B8 murine hybridoma cells (as described in Chomczynki P. et. al. "Single stepmethod of RNA isolation by acid guanidimum thiocyanate - phenolchloroform extraction". Anal. Biochem. 162: 156-159 (1987) From this RNA was obtained cDNA. DNA variable region of the light chain of mouse immunoglobulin was isolated from cDNA using the polymerase chain reaction using a set of DNA primers having homology with murine signalname sequences of the light chain at the 5'-end 1. ULsemantic (SEQ ID No. 3)


Underlined part is the BglII site: the above part is istratovym a codon)

2. ULAntisense (SEQ ID # 4)


Underlined part is the BsiWI site) Cm. Fig. 1 and 2 for the respective sites BglII and BsiWII in TCAE 8 and Fig. 3 for the corresponding sites in anti-CD20 in TCAE 8.

These are obtained DNA fragments were cloned directly into the vector TCAE 8 ahead of the constant domain of the light chain of the Kappa man and sequenced. Certain DNA sequence for the variable region of murine light chain represented in Fig. 4. (SEQ ID No. 5), see also Fig. 3, nucleotides 978-1362. Fig. 4, in addition, gives the amino acid sequence of murine variable region to the area of the CDR and framework region. The variable region of the murine light chain of 2B8 refers to the murine family of Kappa VI Cm. Kabat, Supra.

The variable region of the murine heavy chain was similarly isolated and cloned ahead of the constant domains of human IgGI. The primers were as follows:

1. UHSemantic (SEQ ID No. 6)


Underlined part is the MluI site)

2. UHAntig. 1 and 2 for the respective sites MluI and Nhe I in TCAE 8 and Fig. 3 for the corresponding sites in anti-CD20 in TCAE8.

The sequence for murine heavy chain represented in Fig. 5 (SEQ ID No. 8); see also Fig. 3; nucleotides 2401-2820. Fig. 5 provides the amino acid sequence of this murine variable regions and CDRs of the frame. Murine variable region of the heavy chain of 2B8 refers to mouse VH family 2B. Cm. Kabatm, supra

B. Obtaining producing chimeric anti-CD20 antibody CHO and SP2/O transfected

The DG44 cells of the Chinese hamster ovary (CHO) were grown in medium SSFM II, not containing gipoksantin and thymidine (Gibco, Grand Island, NY, Form N 91-0456PK); cells of the mouse myeloma SP2/0 were grown in a modified Needle environment Delbanco ("DMEM") (Irvine Scientific, Santa Ana, Ca, Cat. N 9024) with 5% fetal bovine serum and 20 ml/l glutamine, added to the environment. Four million cells were electroporative or with 25 μg CHO, or with 50 μg of plasmid DNA SP 2/0, which was restriction using NotI using system BTX electroporation 600 (BTX, San Diego, CA), disposable cuvettes, 0.4 ml. Conditions were or 210 volts for CHO or 180 volts for P2/0, 400 microfarad, 13 Ohms. Each electroporation were sown in six 96-well plates (approximately 7,000 cells per well). In n the e, contained 50 μm gipoksantina and 8 μm thymidine), or 800 μg/ml for SP2/0 two days after electroporation and then 2 or 3 days, until there were colonies. The supernatant from the colonies were tested for the presence of chimeric immunoglobulin using E LISA, specific for human antibodies. Colonies producing the greatest quantity of immunoglobulin, increased in volume and were sown in 96-well plates, containing environment plus methotrexate (25 nm for SP 2/0 and 5 nm for CHO), and Wednesday was applied every two or three days. Supernatant tested as above, and explored the colony, producing the greatest amount of immunoglobulin. Chimeric anti - CD20 antibody was purified from the supernatant using protein A-affinity chromatography.

The purified chimeric anti - CD20 were analyzed by electrophoresis in polyacrylamide gels and appreciated that they had a purity of approximately 95%. Chimeric anti - CD20 antibodies tested in direct and competitive analysis of binding when compared to the murine anti - CD20 monoclonal antibody 2B8, found comparable affinity and specificity on the number of CD20 positive B - cell lines (data not shown). The average affinity constant ("CT") of the chimeric antibody was determined by the fetters of the grave 2B8 using the curve of Scatchard, estimated CT for producing chimeric anti - CD20 antibody CHO was 5,2 109M and for antibodies produced SP 2/0, 7,4 10-9M CT for 2B8 was 3.5 10-9.

Direct competition with the use of radioimmunoassay was used to confirm both the specificity and retention of immunoreactivity of chimeric antibodies by comparing its ability to compete effectively with 2B8. Needed almost an equivalent amount of a chimeric anti - CD20 and 2B8 antibody to obtain 50% inhibition of binding to the antigen CD20 on B-cells (data not shown), i.e., apparently, there was a minimal loss of inhibitory activity of anti - CD20 antibodies, due to hemeroteca.

The Results Of Example 11. To show inter alia that the chimeric anti - CD20 antibodies were obtained from CHO and SP 2/0-transfection using vector TCAE 8, and that these chimeric antibodies had basically the same specificity and affinity, and murine anti - CD20 monoclonal antibody 2B8.

C. determination of the immunological activity of chimeric anti-CD20 antibodies

i Analysis with human C1q

Chimeric anti - CD20 antibodies produced as CHO and SP2/0 cell lines were evaluated for binding the el, Mira Mesa CA, Prod N A 400 and FITC label (label) from Sigma, St. Louis MO. Prod. N=7-7250, FITC).

Tagging C1q was performed according to the Protocol described in Selected Methods In Cellular Immunology, Michel and Shiigi, Ed.(W. H. Freeman and Co, San Francisko, CA, 1980, p. 292). Analytical results were obtained using a flow cytometer Becton Dickinson FACScanTM(fluorescein was measured in the range of 515 - 545 nm). An equivalent amount of a chimeric anti - CD20 antibodies, human Ig GI, K myeloma protein (Binding Site, San Diego, CA, Prod. N VRO 78) and 2B8 incubated with an equivalent amount of CD20-positive SB cells were then washed in FACS buffer ( with 0.2% B S A in PB S, a pH of 7.4, with 0.02% sodium azide) to remove unattached antibodies, followed by incubation with FITC labeled C1q. After 30-60 min incubation, the cells were again washed. Analyzed three conditions, including FITC labeled C1q as a control, FACScaTMaccording to the manufacturer's instructions. The results are presented in Fig. 6.

As the results of Fig. 6, a significant increase in fluorescence was observed only for the case of chimeric anti - CD20 antibody, i.e., only B cells with attached chimeric anti - CD20 antibody C1q were positive, whereas other options gave the same result that was obtained in the control.

51Cr-labeled allogeneic target cells in vitro" . Immunology 14: 181 - 189 (1968). The results are presented in Fig. 7

Results Fig. 7 show that inter alia chimeric anti-CD20 antibody caused significant lysis (49%) under these conditions.

iii Effector analysis-dependent cellular cytotoxicity antibody

For this study used CD20 positive cells (SB) and CD20 negative cells (line T-cell leukemia HSB; see Adams, Richard, "Formal Discussion" Can. Res. 27: 2479 - 2482 (1967); ATS Deposit N ATCC CCL 120.1); their enthusiasm51Cr. Analysis was performed according to the Protocol described in Brunner K. T. et. al "Quantitative assay of the lytic action of immune lymphoid cells on51Cr-labeled allogeneic target cells in vitro, inhibition by isoantibody and druds " Immunology 14: 181 - 189 (1968); significant indirect dependent chimeric anti-CD20 antibody cell lysis of CD20 - positive B cell targets (51Cr-labeled) was observed at the end of the 4 h incubation at 37oC. This effect nalewajski peripheral lymphocytes; the ratio of effector cells to target cells was 100:1). Efficient lysis of target cells was obtained with a 3.9 µg/ml In contrast, under the same conditions murine anti-CD20 monoclonal antibody 2B8 was found statistically insignificant effect, and CD20 - negative. The cells were not lysed. The results are presented in Fig. 8.

The results of Example 11 show inter alia that the chimeric anti-CD20 antibodies of Example 1 were immunologically active.

III Depletion of B Cells in vivo using chimeric anti - CD20 Antibodies

A. Studies in primates (non-human)

Conducted three separate studies in primates (non-human). For convenience they are called here: "Chimeric anti-D20 : CHO and SP 2/0"; Chimeric anti-CD20 : CHO"; and "High doses of chimeric anti-CD20 ". Conditions were as follows:

Chimeric anti-CD20 : CHO and SP2/0

Six cynomolgus monkeys weighing in the range from 4.5 to 7 kg (White Sands Research Center, Alamogordo, NM) were divided into three groups of two monkeys each. Both animals of each group received the same dose of immunologically active chimeric anti-CD20 antibodies. One animal in each group received the purified antibody produced by CHO-transfective; the other received the antibody's unstable past also produces the Yan during 4 consecutive days. Immunologically active chimeric anti-CD20 antibody is mixed with sterile saline, was injected intravenously influence; before each infusion took blood samples. Additional blood samples were taken at 24 h after the last injection (T = 0) and then in the days of the 1st, 3rd, 7th, 14th and 28th; blood samples were also taken after that at intervals of two weeks prior to the completion of the study 90-day.

Approximately 5 ml of whole blood from each animal was centrifuged at 2000 rpm for 5 minutes, the Plasma was removed for analysis of the levels of soluble chimeric anti-CD20 antibodies. The precipitate (containing peripheral blood leukocytes and erythrocytes) resuspendable in fetal calf serum for analysis labeled with fluorescein antibodies (see "Fluorescent Antilogy Labeling of Lymphoid Cell Population" infra" Chimeric anti-CD20: CHO

Six cynomolgus monkeys weighing from 4 to 6 kg (White Sands) were divided into three groups of two monkeys each. All animals were injected immunologically active chimeric anti-CD20 antibodies derived from CHO

transfection (in sterile saline). Three groups differed in the following way: sub-group 1 received daily intravenous injections of 0.01 mg/kg of the antibodies within 4 days; sub-group 2 received daily /kg of antibody.

All three subgroups took a blood sample before treatment; additional blood samples were taken at T= 0; 1, 3, 7, 14 and 28 days after the last injection, as described above, and these samples were processed for analysis of fluorescently labeled antibodies (see "Fluorescent Antibody Labeling, infra). In addition to the quantitative analysis of B-cells in the blood, took a biopsy of the lymph nodes on the 7th, 14th and 28th days after the last injection and single cell preparation was stained to determine the number of populations of lymphocytes using flow cytometry.

High doses of chimeric anti-CD0 antibodies

Two cynomolgus monkeys (White Sands) was poured to 16.8 mg/kg immunologically active chimeric anti-CD20 antibodies from CHO-transfected (in sterile saline) weekly for a period of four consecutive weeks. At the end of the experiment both animals were anestesiologi to remove bone marrow; also took a biopsy of the lymph nodes. Both sets of tissues were stained for the presence of B-lymphocytes using Leu 16 flow cytometry according to the Protocol described in Ling, N. R. et. al. "B-cell and plasma cell antigens" Leucocyte Typing III, White Cell Diffeventiation Antigens, A. J. Mc Michael, Ed. (Oxford University Press, Oxford UK, 1987, p. 302). Tagging fluorescent antibody population of lymphoid cells

Psme volume of fetal calf serum (heat inactivated at 56oC for 30 minutes). A volume of 0.1 ml of the cell preparation was distributed in each of the six conical centrifuge tubes 15 ml Labeled with fluorescein monoclonal antibodies specific for surface markers CD20 human lymphocytes (AMAC, Westbrook, ME), CD20 (Becton Dickinson) and human IgG (the Binding Site, San Diego CA) was added to three of the tubes to identify populations of T - B - lymphocytes. All reagents were pre-tested as positive in relation to the corresponding antigens of lymphocytes of monkeys. Chimeric anti-CD20 antibody associated with CD20 surface of B - cells of monkeys, were measured in the fourth test tube using a polyclonal goat anti-human IgG coupled to phycoerythrin (AMAC). This reagent was previously adsorbing on sepharose column with immunoglobulin monkeys to prevent cross-reactivity with immunoglobulin monkeys, which made possible the specific detection and quantification of chimeric anti-CD20 antibodies bound to the cells. The fifth test tube contained both anti-IgM and anti-human IgG for double-stained populations of B-cells. The sixth sample was included without reagents to determine the autofluorescence. Cells were incubated with fluorescein, a pH of 7.4) and analyzed on the instrument Becton Duckinson FACTM. Populations of lymphocytes source identified by the scattering of light (forward versus right angle) in the dot-plotdevice images with the unlabeled leukocytes. Then allocated the total population of lymphocytes, discarding all other events. Subsequent measurement of fluorescence reflects only specific for lymphocytes events.

Depletion of B-lymphocytes in peripheral blood

Not found the observed difference between the efficiency of CHO and SP 2/0 produced antibodies in the depletion of B-cells in vivo, although a weak increase in the recovery of B-cells, beginning after the 7th day in monkeys injected chimeric anti-CD20 antibodies derived from CHO-transfection, at doses of 1.6 mg/kg and 6.4 mg/kg was observed, as well as for the monkeys injected produced SP 2/0 antibodies at the dose level of 0.4 mg/kg Fig. 9A, B and C give the results obtained from the study "chimeric anti-CD20: CHO and SP 2/0": Fig. 9A depicts the dose level of 0.4 mg/kg; Fig. 9B depicts the dose of 1.6 mg/kg and Fig. 9C the dose of 6.4 mg/kg

As can be seen from Fig. 9, the observed strong reduction (>95%) in the levels of B-cells in peripheral blood after therapeutic treatment in all IP is the axis of recovering B-cells and the start time of the recovery is not dependent on the level of dose.

In the study, a chimeric anti-CD20: CHO: used 10 times lower dose concentration (0.01 mg/kg) during the four days of daily injections (0.04 mg/kg total). Fig. 10 gives the results of this study. This dose drain the population of B-cells peripheral blood approximately 50% of the normal levels that were assessed either with IqM against the surface of cells, or Leu 16 antibodies. The results also show that the saturation of the CD20 antigen on the population of B-lymphocytes is not achieved with immunologically active anti-CD20 antibody at this dose concentration during this time period in primates; B-lymphocytes, are covered by these antibodies were detected in blood samples during the initial three days after therapeutic treatment. However, on the 7th day of antibody coated cells were not detected.

Table I summarizes the results of single and multiple doses of immunologically active anti-CD20 antibodies in the population of peripheral blood; a single dose was 6.4 mg/kg; repeated doses were 0.4 kg/kg for four consecutive days (these results were obtained on the above monkeys).

The data summarized in table I, while the actively, regardless of single or multiple doses. In addition, depletion observed for at least seven days after the last injection, with partial recovery of B-cells observed on day 21.

Table II summarizes the effect of immunologically active chimeric anti - CD20 antibodies on the cell population of lymph nodes using treatment table I (4 daily doses of 0.4 mg/kg; I dose of 6.4 mg/kg); also provides comparative values for normal lymph nodes (control monkeys, axillary and inguinal lymph nodes) and normal bone marrow (two monkeys).

The results of table II show the effective depletion of B-lymphocytes for both treatment regimens. Further, table II shows that for primates, non-human, full saturation of B-cells in lymph tissue immunologically active chimeric anti - CD20 antibody is not achieved; in addition, antibody coated cells was observed after 7 days after treatment, with subsequent noticeable depletion of B-cells in the lymph nodes observed on the 14th day.

On the basis of these data was performed by the above-mentioned study of high doses of chimeric anti - CD20, such is licnosti, associated with the introduction of chimeric antibodies, as well as the efficiency of depletion of B-cells in the lymph nodes, peripheral blood and bone marrow. In addition, because the data of table II show that for this study a large part III cells of the lymph nodes is depleted between 7 and 14 days after treatment, the mode with weekly dosing may be more effective. Table III summarizes the results of a study of high doses of chimeric anti-CD20 antibodies.

Both animals examined at 22 days after cessation of treatment, contained less than 5% of B-cells compared with 40% in the control lymph nodes (see Table II, Supra). Similarly, in the bone marrow of animals treated with chimeric anti-CD20 antibody, the levels of CD20 - positive cells were below 3% compared with 11 - 15% in normal animals (see table II, Supra). In animals examined at 36 days after cessation of treatment, one of their animals (H) had approximately 12% of B-cells in the lymph node and 4.4% of B-cells in bone marrow, whereas other animal (C) had approximately 5% of B-cells in the lymph node and 0.8% in the bone marrow. These data show significant depletion of B-cells.

The results of Example Osenniy B-cells in the peripheral blood of primates. These data also show that a significant depletion of the population of B-cells was achieved in the peripheral lymph nodes and bone marrow with the introduction of repeated high doses of the antibody. Prolonged examination of test animals showed that even such a strong depletion of peripheral B-lymphocytes during the first week of treatment did not lead to adverse health effects. In addition, when observed the restoration of the population of B-cells, it was concluded that pluripotent stem cells these primates were not fatally damaged by this treatment.

B. Clinical analysis C2B8

i Clinical trial phase I/II C2B8: a study of therapy with the use of a single dose

Fifteen patients with histologically documented recurrent B-cell lymphoma were treated C2B8 in a clinical trial phases I-II. Each patient received one dose of C2B8 in the study with increasing doses; three patients experienced for doses: 10 mg/m2; 50 mg/m2; 100 kg/m2; 250 mg/m2and 500 mg/m2. The introduction was performed by intravenous infusion over to 0.22 micron filter, and C2B8 was diluted in a final volume of 250 ml or at a maximum concentration of 1 is sicnosti, the speed of dosing was increased up to a maximum of 200 ml/h

Toxicity (as indicated by the Clinician) ranged from "no" to "heat" to "moderate" (two patients), to "severe" (one patient); all patients underwent a complete therapeutic treatment. To determine inter alia the steps C2B8 on T-cells and B-cells were analyzed peripheral blood lymphocytes. In all patients, peripheral blood lymphocytes were depleted after infusion C2B8 and this depletion was maintained for more than two weeks.

One patient (receiving 100 mg/m2C2B8, found a partial response to the processing of C2B8 (reduced by more than 50% of the sum of the perpendicular diameters of all measurable indicator of damage, lasting more than four weeks, during which no new damage and can't grow old damage); at least one patient (receiving 500 mg/m2) found a weak response to the processing of C2B8 (reduction of less than 50%, but not less than 25% in the sum of the measurements of the two longest perpendicular diameters of all measurable indicator of damage). To represent the effectiveness of the results of PB is shown in Fig. 14A; for the patient, detecting small B - cell markers CD20 and CD19, Kappa and Lambda were depleted for more than two weeks, whereas it was weak initial decrease in the number of T-cells, which were returned to the background level in a relatively short period of time.

ii Clinical trial phase I/II C2B8; study of therapy with the use of multiple doses

Patients with histologically confirmed B-cell lymphoma with measurable progressive disease is desirable for this study, which was divided into two phases; in phase I, consisting of dose escalation (increasing dose), to characterize the rate-limiting toxicities and determination of biologically active tolerated dose, groups of three patients will receive weekly intravenous infusion C2B8, a total of four separate infusion. The cumulative dose for each of the three levels will be: 500 mg/m2(125 mg/m21 infusion); 1000 mg/m2(250 mg/m21 infusion); 1500 mg/m2(375 mg/m21 infusion. Biologically active tolerated dose is defined as the lowest dose with tolerable toxicity and adequate activity); phase 11 additional patients receive biologically active tolerated dose, and attach great importance to the determination of the activity of four doses Cm C2B8 and Y2B8 was studied in a model with xenotransplantation mouse (PI/PI mouse female, approximately 10 weeks of age), using B-cell lymphoblastic tumors (Ramos tumor cells). For comparative purposes, additional mice were treated C2B8 and Y2B8.

Ramos tumor cells (ATSC, CRL-1596) were maintained in culture using RPMI - 1640 with the addition of 10% fetal calf serum and glutamine at 37oC and 5% CO2. Tumors initiated in 9 females "Nude" mice approximately 7 to 10 weeks of age using a subcutaneous injection of 1.7 106Ramos cells in a volume of 0.10 ml (HBSS) with the use of the syringe 1 ml with needle 25, With all animals were manipulated in a laminar box and all cages, bedding, feed and water were autoclavability. Tumor cells were obtained by cutting out tumors and conducting them through a sieve of 40 mesh; cells were washed twice 1xHBSS (50 ml) by centrifugation (1300 rpm), resuspendable 1 x HBSS 10 106cells/ml and frozen at -70oC until use.

For experimental conditions, cells from several parties were thawed, besieged by centrifugation (1300 rpm) and washed twice in 1 x HBSS. The cells are then resuspendable approximately 2.0 to 106cells/ml Approximately 9 to 12 mice were injected with 0.10 ml of this cell su is approximately in the middle part. Tumors developed in approximately two weeks. Tumors were excised and processed as described above. Mice used for research were injected with, as described above, using 1,67 106cells in 0.10 ml HBSS.

On the basis of preliminary experiments on dosing was determined that for this study, use 200 mcg C2B8 and 100 µci Y2B8. 90 females PI/PI mice (naked mouse, i.e., the Nude mouse with a mutation in the gene nude) (approximately 10 weeks of age) were injected with tumor cells.

After approximately 10 days, 24 mice were determined for the four studied groups (six mice per group) and tried to maintain a comparable distribution of tumor size in each group (mean tumor size was expressed as the product of the length to the width of the tumor was approximately 80 mm2). The following groups were treated, as indicated, through injection into the tail vein using microspace Hamilton with needle 25 g:

A. Normal saline

B. Y2B8 (100 µci)

C. C2B8 (200 mg)

D. Y2B8 (100 µci) + C2B8 (200 mg)

The groups tested with C2B8, were given a second injection of C2B8 (200 mg/mouse) 7 days after the initial injection. Measurement of tumor provodimostei with the following protocols:

A. Preparation of Y2B8

Chloride of yttrium - [90] (6 MCI) was made in polypropylene test tube and the pH was brought to 4.1 and 4.4 using does not contain metals 2 M sodium acetate. Added 3B8 - MX-DTPA (0.3 mg in normal saline (physiological) solution, see above for preparation of 2B8-MX-DTPA) and gently mixed using a vortex. After 15 min incubation the reaction was stopped by adding 0.05 x volume of 20 mm EDTA and 0.05 x volume of 2 M sodium acetate. The concentration of radioactivity was determined by dilution of 5.0 μl of the reaction mixture 2.5 ml of 1 x PBS containing 75 mg/ml HSA and 1 DTPA ("composite buffer"); radioactivity counted by the addition of 10.0 ál to 20 ml scintillation mixture EcolumeTM. The remainder of the reaction mixture was added to 3.0 ml of the composite buffer, sterile filtered and stored at 2 - 8oC before use. Specific radioactivity (14 MCI/mg at the time of injection) was calculated based on the radioactive concentration and the calculated concentration of protein (based on the number added to the reaction mixture of antibodies). Associated with protein radioactivity was determined using a rapid thin-layer chromatography. Incorporation of radioactivity was 95%. Y2B8 was diluted in composite buffer immediately before ispgateway C2B8

C2B8 was prepared as described above. He was received in the form of sterile reagent in normal saline solution at 5.0 mg/ml Before injection C2B8 was diluted in normal saline to 2.0 mg/ml and sterile filtered.

C. Results

After treatment, the tumor size was expressed as the product of length and width measurements were made in the days indicated in Fig. 11 (Y2B8 vs. salt solution); Fig. 12 (C2B8 compared with saline) and Fig. 13 (Y2B8 + C2B8 compared with saline). Also determined the standard error.

As shown in Fig. 13, the combination of Y2B8 and C2B8 gave destroying tumor effects comparable to the effects obtained separately with Y2B8 or C2B8.

V. Alternative therapy strategy

Alternative therapy strategy is obvious, if we take into account the previous examples. One of these strategies involves the use of therapeutic doses of C2B8 followed (within about one week) by combining or 2B8 and radioactively labeled 2B8 (for example, Y2B8) or 2B8, C2B8 and, for example, Y2B8; or SV and, for example, UV. An additional strategy is by using radioactively labeled SV: this strategy allows you to use the benefits of the active tags include yttrium - 90 due to the larger half-life SV compared with murine antibodies B. Due to the ability SV to Deplete B-cells and the advantages arising from the use of radioactive labels, preferred an alternative strategy involving treatment of the patient using SV (using a single dose or multiple doses), so that the majority of B-cells, if not all B-cells are removed. This could be accompanied by the use of radioactive W; due to the removal of peripheral B-cells radioactively labeled V has increased the possibility of penetration into tumor cells. Preferably used labeled with iodine - [131] W, based on the nature of the results reported in the literature for this label (see Kaminski). An alternative preference is given to the use of radioactively labeled V (or SV) initially in an attempt to increase the permeability of the tumor, with subsequent single or multiple treatments SV; this strategy is designed to increase the chances of getting SW as to the outer part swollen, and inside the tumor. A further strategy involves the use of chemotherapeutic agents in combination with SV. These strategies include nazyvaemye this Protocol. Alternatively, a suitable source processing one or more doses SW, followed by chemotherapy treatment. Preferred chemotherapeutic agents include, but are not limited to) cyclophosphamide, doxorubicin; veneration; prednisone, see Armitage, Y. O. et. al. Cancer 50 : 1595 (1982), incorporated herein by reference.

Previous alternative therapy strategy are not intended to limit the invention, but rather are presented as representative strategies.

VI. Information about Deposit

Anti-CD20 in TCAE 8 (transformed into E. coli for the purpose of Deposit) was deposited in the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 under the provisions of the Treaty Budapest Treaty for the International Recognition of the Deposit of Microorganisms for purposes of Patent procedure ("Budapest Treaty"). This microorganism was tested in ATSC 9 November 1992 and determined that at the time of testing it was viable. ATS identified this organism for ATSS Deposit rooms: ATS 60119 (anti-CD20 in TCAE 8). Hybridoma V was placed in ATS 22 June 1993 on the provisions of the Viability of the culture was determined on 25 June 1993, and ATSS identified for this hybridoma Deposit ATSS room HB 11388.

1. A method of treating b-cell Li is an effective amount of at least one immunologically active chimeric antibody, obtained from trasferta containing anti-CD 20 in TGAE 8 depositional number 69119 in ATSS.

2. The method according to p. 1, wherein therapeutically effective amount is 0.001-30 mg/kg body weight.

3. The method according to p. 1, characterized in that it additionally includes the stage of introduction of the second therapeutically effective amount of at least one immunologically active chimeric anti-CD-20 antibodies.

4. The method according to p. 3, characterized in that the introduction of spend within 7 days from the first injection of this antibody man.

5. A method of treating b-cell lymphomas, including the stage of introduction of the human anti-CD-20 antibody, characterized in that in the first stage, carry out the introduction of the first therapeutically effective amount of immunologically active chimeric antibody derived from transfection containing anti-CD 20 in TGAE 8 with a Deposit number 69119, in the second stage, the second therapeutically effective amount of the antibody and the third stage - third therapeutically effective amount of the antibody.

6. The method according to p. 5, wherein the first, second and third therapeutically effective amount of antibody is 0,001 - the first period of introduction.

8. The method according to p. 5, wherein the third period of introduction is within 14 days from the first period of introduction.

9. Immunologically active chimeric anti-CD-20 antibody, characterized in that it has a constant region of human and murine variable region having antigenic specificity against CD-20 antibodies on the cell surface of human lymphocytes, obtained from transfection containing anti-CD 20 in TGAE (Deposit number 69119 in ATS).

10. Hybridoma, secreting specific CD-20 antigen antibody derived by fusion of anti-CD-20 - mouse antibodies with SP 2/0 identified ADS Deposit number HB 11388.

11. Monoclonal antibody 2B 8 specific against CD-20 antigen of human lymphocytes, Sekretareva hybridomas under item 10.

12. Radioactive labeled antibody, representing monoclonal 2B 8 antibody on p. 11, conjugated with a radioactive label selected from the group consisting of yttrium (90), indium (III) and iodine (131).

13. A method of treating b-cell lymphoma, characterized in that it includes the introduction phase to the person a therapeutically effective amount of the antibody under item 12.

14. The method according to p. 13, characterized in, waiting for the stage of introduction of the human anti-CD-20 antibody, characterized in that exercise introduction in the first stage of immunologically active chimeric antibody derived from transfection containing anti-CD 20 TGAE 8 depositional number 69119 in ATS, and the second stage is radioactively labeled monoclonal anti-CD-20 antibody secreted by hybridoma identified by a Deposit number HB 11388 in ADS".

Priority points:

13.11.92 on PP.1-9;

03.11.93 on PP.10-15.


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