Treatment of b-cellular malignant tumors using anti-cd40l antibodies in combination with anti-cd20 antibodies and/or by chemotherapy and radiotherapy

FIELD: oncology.

SUBSTANCE: invention characterizes compositions, their employment, and embodiments of a method of treatment of B-cellular lymphomas, leucosis, and other malignant tumors CD40+. Principal active therapeutical agent is anti-CD40L antibody or another CD40L antagonist inhibiting CD40-CD40L intermediate. In combination or composition with indicated CD40L antagonist any one or several of the following components can be additionally used: anti-CD20 antibody, a chemotherapeutical agent or a combination thereof, and radiotherapy.

EFFECT: enhanced mechanisms of apoptosis of tumor CD40+ cells due to sensitization of these earlier destruction-resistant cells.

88 cl, 4 dwg, 6 tbl, 8 ex

 

The scope of the invention

The invention relates to a method of treatment and combination therapy for the treatment of b-cell lymphomas and leukemias and other malignancies CD40+by regulating the interaction between CD40 and its ligand, CD40L, or regulate the transmission of the signal through CD40. In particular, the interaction can inhibit antibody against CD40L, to prevent binding of CD40L to CD40. These antibodies or other agents that can inhibit the interaction CD40/CD40L, in addition, can be combined with chemotherapy, radiation and/or antibodies against CD20 and antibodies against CD40.

Background of the invention

Lymphomas are tumors of lymphocytes. Ninety percent of lymphomas has b-cell origin, the remaining ten percent of lymphomas of T-cell origin. Most patients diagnosed with either Hodgkin's disease (HD), or lymphoma nehodgkinski type (NHL).

Depending on the diagnosed lymphoma treatment options include radiation therapy, chemotherapy and application of monoclonal antibodies.

A. Antibodies against CD20.

CD20 is a cell surface antigen, expressed by more than 90% of b-cell lymphomas, which disappears and is not modulated in neoplastic cells (McLaughlin et al., J. Clin. Oncol. 16: 2825-2833 (1998b). The CD20 antigen is deglycosylation membrane protein In cells with a molecular mass of 35 kDa, involved in intracellular signal transmission, the differentiation of b-cells and mobilization of calcium channels (Clark et al., Adv. Cancer Res. 52: 81-149 (1989); Tedder et al., Immunology Today 15: 450-454 (1994)). The antigen appears as an early marker of differentiation In cell lines and is widely expressed with different antigenic density of both normal and malignant populations of b-cells. However, the antigen is absent in the fully Mature b cells (e.g., plasma cells), early populations of b-cells and stem cells, making it a suitable target for indirect antibody therapy.

Were obtained antibodies against CD20 for use in both research and therapy. One antibody against CD20 is a monoclonal antibody B1 (U.S. patent No. 5843398). Antibodies against CD20 were also obtained in the form of radionuclides for the treatment of b-cell lymphoma (e.g., an antibody against CD20, labeled131I), and also in the form of labelled89Sr for pain relief in the bones caused by metastases in patients with prostate cancer and breast cancer (Endo, Gan To Depending Ryoho 26: 744-748 (1999)).

Mouse monoclonal antibody 1F5 (antibody against CD20), reportedly, was administered by continuous intravenous infusion to patients is b-cell lymphoma. However, according to the reports, required extremely high levels (>2 grams) 1F5, to reduce the number of circulating tumor cells, and the result was described as "temporary" (Press et al., Blood 69: 584-591 (1987)). Possible problem with the use of monoclonal antibodies in therapy lies in the fact that the monoclonal antibodies of human and animals (e.g., murine monoclonal antibodies), there is typically no effector functions of human antibodies, for example, among other things, they are not able to mediate complementability lysis or to lyse target cells of a person due to antibody-dependent cellular toxicity or indirect Fc-receptor phagocytosis. In addition, the monoclonal antibodies are not humans, and animals, can be recognized by the master of man as alien protein; therefore, repeated injections of such alien antibodies can lead to the induction of immune responses, leading to a dangerous hypersensitivity reactions. For monoclonal antibodies derived from mice, this phenomenon is often called the response through the development of artemisinin antibodies in humans or answer "NAMA". In addition, these "alien" antibodies can be attacked by the immune system of the host so that in fact they are neutralized before reached its target.

RITUXAN®. ITUXAN ®(also known as Rituximab, MabThera®, IDEC-C2B8 and SW) was the first FDA-approved monoclonal antibody was developed in IDEC Pharmaceuticals (see U.S. patent No. 5843439; 5776456 and 5736137) for the treatment of b-cell lymphoma person (Reff et al., Blood 83: 435-445 (1994)). RITUXAN®is a chimeric monoclonal antibody (MAB) against CD20, which inhibits growth and, reportedly, sencibilisiruet some lines lymphoma cells against apoptosis under the action of chemotherapeutic agents in vitro (Demidem et al., Cancer Biotherapy and Radiophannaceuticals 12: 177-(1997)). RITUXAN®also shows antitumor activity when tested in vivo using animal models of mouse xenografts. RITUXAN®effectively communicates with the human complement, has a strong binding to FcR, and can effectively kill human lymphocytes in vitro by how dependent compliment (CDC)and antibody-dependent (ADCC) mechanisms (Reff et al., Blood 83: 435-445 (1994)). In macaques, the antibody selectively depletes the amount of normal b-cells in blood and lymph nodes.

RITUXAN®it was recommended for the treatment of patients with non-Hodgkin lymphoma low-grade or follicular b-cell non-Hodgkin lymphoma (McLaughlin et al., Oncology (Huntingt) 12: 1763-1777 (1998a); Maloney et al., Oncology 12: size 63-76 (1998); Leget et al., Curr. Opin. Oncol. 10: 548-551 (1998). In Europe RITUXAN® was approved for the treatment of relapsed follicular lymphoma stage III/IV (White et al., Pharm. Sci. Technol. Today 2: 95-101 (1999)) and, reportedly effective against lymphoma cells follicular centers (FCC) (Nguyen et al., Eur. J. Haematol 62: 76-82 (1999)). Other disorders that are treated with RITUXAN®include lymphoma cells follicular centres (FCC), of lymphoma mantle cell (MCL), diffuse lymphoma large b-cell (DLCL) and small cell lymphocytic lymphoma/chronic lymphocytic leukemia (SSL/CLL) (Nguyen et al., 1999)). Patients with refractory or incurable NHL reportedly responded to combined therapy with RITUXAN®and CHOP (for example, cyclophosphamide, vincristine, prednisone, and doxorubicin) (Ohnishi et al., Gan To Depending Ryoho 25: 2223-8 (1998)). RITUXAN®showed minimal toxicity and significant therapeutic activity in non-Hodgkin lymphoma (NHL), low grade in clinical studies of phases I and II (Berinstein et al., Ann. Oncol. 9: 995-1001 (1998)).

RITUXAN®used separately for the treatment of b-cell NHL is usually a weekly dose of 375 mg/m2for four weeks with relapsed or refractory NHL low-grade or follicular NHL, was well tolerated and had significant clinical activity (Piro et al., Ann.Oncol. 10:655-61 (1999); Nguyen et al., (1999); and Coiffer et al., Blood 92: 1927-1932 (1998)). However, in the tests using antibodies also were administered doses up to 500 mg/m2within four weeks (Maloney et al., Blood 90: 2188-2195 (1997)). RITUXAN®also combined with chemotherapeutics, such as CHOP (for example, cyclophosphamide, doxorubicin, vincristine and prednisone), to treat patients with non-Hodgkin lymphoma low-grade or follicular b-cell non-Hodgkin lymphoma (Czuczman et al., J. Clin. Oncol. 17: 268-76 (1999); and McLaughlin et al., (1998a)).

B. CD40 and CD40L.

CD40 is expressed on the cell surface of Mature b cells, as well as leukemia and lymphocytic B-cells and on cells of Hodgkin and reed-Sternberg (PC) Hodgkin's disease (HD) (Valle et al., Eur. J. Immunol. 19: 1463-1467 (1989); and Gruss et al., Leuk. Lymphoma 24: 393-422 (1997)). CD40 is a receptor In cells, leading to activation and survival of normal and malignant b-cells, such as cell non-Hodgkin follicular lymphoma (Johnson et al., Blood 82: 1848-1857 (1993); and Metkar et al., Cancer Immunol. Immunother. 47: 104 (1998)). Signaling through the CD40 receptor protects immature b-cells and b-cell lymphoma from apoptosis induced IgM or Fas (Wang et al., J. Immunology 155: 3722-3725 (1995)). Similarly cell lymphoma mantle cells have high levels of CD40, and the addition of exogenous CD40L increased their survival and saved them from apoptosis in Zirovnica with fludarabine (Clodi et al., Brit. J. Haematol. 103: 217-219 (1998)). In contrast, other authors reported that CD40 stimulation can inhibit neoplastic growth of cells in vitro (Funakoshi et al., Blood 83: 2787-2794 (1994))and in vivo (Murphy et al., Blood 86: 1946-1953 (1995)).

Antibodies against CD40 (see U.S. patent No. 5874082 and 5667165), administered to mice increased the survival of mice with b-cell lymphomas person (Funakoshi et al. (1994); Tutt et al., J. Immunol. 161: 3176-3185 (1998)). Methods of treatment of neoplasm, including b-cell lymphoma and EBV-induced lymphoma using anti-CD40 antibodies that mimic the effect of CD40L and therefore delivers a signal of death, described in U.S. patent No. 5674492 (1997), which is included in this description by reference in full. Signaling through CD40 is also associated with a synergistic interaction with CD20 (Ledbetter et al., Circ. Shock 44: 67-72 (1994)). Additional references describing the preparation and use of anti-CD40 antibodies include U.S. patents№ 5874085 (1999), 5874082 (1999), 5801227 (1998), 5674492 (1997) and 5667165 (1997), which are incorporated in this description by reference in full.

The CD40 ligand, gp39 (also known as CD40 ligand, CD40L or CD154), is expressed on activated, but not resting Th cells CD4+(Spriggs et al., J. Exp. Med. 176: 1543-1550 (1992); Lane et al., Eur. J. Immunol. 22: 2573-2578 (1992) and Roy et al., J. Immunol. 151: 1-14 (1993)). As CD40 and CD40L have been cloned and characterized (Stamenkovi et al., EMBO J. 8: 1403-1410 (1989); Armitage et al., Nature 357: 80-82 (1992); Lederman et al., J. Exp. Med. 175: 1091-1101 (1992); and Hollenbaugh et al., EMBO J. 11: 4313-4321 (1992)). CD40L also described in U.S. patent No. 5945513. Cells transfetsirovannyh CD40L gene and expressing the protein CD40L on their surface, can run the proliferation of b cells and, along with other stimulating signals can induce the production of antibodies (Armitage et al. (1992); and U.S. patent No. 5945513). CD40L can play an important role are dependent on cell contact interaction of tumor b cells (CD40+) with neoplastic follicles or cells reed-Sternberg (CD40+in the areas of disease Hodgkin (Carbone et al., Am. J. Pathol. 147: 912-922 (1995)). Monoclonal antibodies against CD40L, reportedly effectively used to inhibit the induction of murine AIDS (MAIDS) in mice infected with LP-BM5 (Green et al., Virology 241: 260-268 (1998)). However, the mechanism of signal transmission CD40L-CD40, leading to survival instead of answers in the form of cell death In malignant cells, is unknown. For example, in cells of a follicular lymphoma downward regulation inducing apoptosis molecules TRAIL (APO-2L) (Ribeiro et al., British J. Haematol. 103: 684-689 (1998)and overexpression of BCL-2, and in the case of B-CLL, decreasing regulation of CD95 (Fas/APO-1) (Laytragoon-Lewin et al., Eur. J. Haematol. 61: 266-271 (1998)), have been proposed as mechanisms of survival. On the contrary, relative to follicular lymphoma, there is evidence that activation D40 leads to increasing regulation of TNF (Worm et al., International Immunol. 6: 1883-1890 (1994)), CD95 molecules (Plumas et al., Blood 91: 2875-2885 (1998)).

Also received anti-CD40 antibodies to prevent or treat mediated by antibodies diseases such as allergies and autoimmune diseases, as described in U.S. patent No. 5874082 (1999). Anti-CD40 antibodies, reportedly, effectively combined with anti-CD20-antibody, receiving an additive effect in the inhibition of growth of non-Hodgkin b-cell lymphomas in cell culture (Benoit et al., Immunopharmacology 35: 129-139 (1996)). In vivo studies in mice, in fact, showed that anti-CD20 antibodies were more effective than anti-CD40 antibodies, entered separately, the stimulation of survival of mice carrying some, but not all, lines of lymphoma (Funakoshi et al., J. Immunother. Emphasis Tumor Immunol. 19: 93-101 (1996)). Anti-SU antibodies, reportedly, is also effective in vivo in the treatment of two b-cell lymphomas syngeneic mice, BCL1 and A31 (Tutt et al. (1998)). Also described antibodies to CD40L for use in the treatment of disorders associated with activation of b-cells (European patent No. 555880 (1993)). Anti-CD40L antibodies include monoclonal antibodies E, N, N, 4D9-8 AND 4D9-9, 24-31, 24-43, 89-76 and 89-79, which are described in U.S. patent No. 57474037 (1998), and anti-CD40L antibodies described in U.S. patent No. 5876718 (1999), used to treat disease graft-versus-host.

Therefore, without contradicting the fact that is was Balos in the literature previously, you can talk about the need to improve methods of treatment and combination therapy for the treatment of b-cell lymphomas and leukemias. The use of compositions containing anti-CD40L antibodies and other agents that counteract the interactions of CD40-CD40L, opens a new way of treating cancer patients, which may be less toxic than existing therapy. In particular, methods and compositions for inhibition of CD40 stimulation in order to prevent the transformation of cancer cells resistant to programmed cell death induced by chemotherapy or other treatments for cancer, reveals a previously unknown method of strengthening therapy of cancer and reduce the likelihood that developing cells will be resistant to therapy.

The invention

The object of the invention is a method of treating b-cell lymphomas, b-cell leukemia and other malignant tumors CD40+that includes the introduction of a therapeutically effective amount of the antibody or antibody fragment that binds to CD40L. B-cell lymphomas include Hodgkin's and non-Hodgkin lymphomas of any degree.

Another object of the invention is a combination therapy for the treatment of b-cell lymphoma or b-cell leukemia, comprising anti-CD40L-antibody or a fragment of an is and the body or CD40L antagonist and at least one of the following components: (a) a chemotherapeutic agent or combination of chemotherapeutic agents, (b) radiation therapy, (C) anti-CD20-antibody or its fragment, and (d) anti-CD40-antibody or its fragment.

Brief description of drawings

Figure 1. The sensitivity of cells In the lymphoma to adriamycin after 4 hours of exposure.

Figure 2. (Panel A), Anti-CD40L (IDEC-131) cancels CD40L mediated resistance cells In the lymphoma to death under the action of ADM. (Panel) the Effect of RITUXAN®on normal and pre-treated SCD40L cells DHL-4.

Figure 3. (Panel A) Blocking CD40L mediated survival of B-CLL using an anti-CD40L antibody (IDEC-131). (Panel C) Blocking CD40L mediated survival of B-CLL using SW IDEC.

Figure 4. FACS-analysis to compare the expression of HLA-DR cells CD19+-CLL, cultivated with sCD40L, and the cells not cultured with sCD40L.

Detailed description of the invention

In another aspect the invention relates to compositions for the treatment of leukemias and lymphomas and other malignant tumors that Express CD40. The preferred option of the invention are compositions and methods of their use for the treatment of lymphomas and leukemias, b-cell lines. The composition may contain funds that are antagonistic effect on signaling through CD40 or interaction between CD40 and CD40L. Funds may not necessarily contain one active agent, such as anti-CD40L-antibody or the th fragment, as well as peptide fragments, peptide mimetics, or chemical compounds. Alternatively the composition may contain multiple active agents, the types of targets which in malignant tumors other than signaling through CD40 or the interaction of CD40/CD40L, such as chemotherapeutic agents, other antibodies, and/or composition can be introduced in combination with radiation therapy.

A. Definition.

In the sense used here assumes that the term CD40L-antibody" includes antibodies and fragments thereof, that react with specific protein CD40L or peptide, or a fused protein CD40L. Antibodies to CD40L may include human antibodies, primaryservername antibodies, chimeric antibodies, bispecific antibodies and humanized antibodies.

In the sense used here, the term "CD40-antibody" means that it includes immunoglobulins and fragments thereof, that react with specific CD40 protein or peptide, or a fused protein CD40. Antibodies to CD40 can include human antibodies, primaryservername antibodies, chimeric antibodies, bispecific antibodies and humanized antibodies.

In the sense used here under the "CD20-antibody" is meant that the term includes immunoglobulins and fragments thereof, that react with specific proteins C20 or peptide, or fused protein CD20. Antibodies to CD20 may include human antibodies, primaryservername antibodies, chimeric antibodies, bispecific antibodies and humanized antibodies. Anti-CD20-antibody is a monoclonal antibody B1 and RITUXAN®.

By "humanized antibody" refers to an antibody derived from antibodies are not human, and animal, usually a mouse antibody, that retains or substantially retains antigennegative the properties of the original antibody, but which is less immunogene people. This can be achieved in various ways, including (a) transplant complete variable domains of antibodies are not human constant region of human antibodies to create a chimeric antibody; (b) transplanting only areas, complementarity determining (CDR)of the antibody does not person to frame and constant parts of the human antibodies with or without saving the critical residues of the framework, and (C) the transplantation of a complete variable domains of antibodies are not human, but they are "hiding" areas, like parts of the human antibodies by replacement of surface residues. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81: 6851-5 (1984); Morrison et al., Adv. Immunol. 44: 65-92 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988); Padlan, Molec. Immun. 28: 489-498 (1991); and Padlan, Molec. Immun. 31: 169-217 (1994), all work on the us in this description by reference in full. Humanized anti-CD40L antibodies can be obtained, as described in the application for the grant of U.S. patent No. 08/554840, filed November 7, 1995, also included in this description by reference in full.

The term "human antibody" refers to an antibody containing human light and heavy chain and the constant region derived from any of the known standard methods.

Under "primaryservername antibody" refers to a recombinant antibody, which was designed so that it contains the variable domains of the heavy and light chains of antibodies monkeys (or other Primate), in particular antibodies macaques of having, and which contains the sequence constant domains of human antibodies, preferably constant domain of a human immunoglobulin gamma 1 or gamma 4 (or RE). The receipt of such antibodies is described in Newman et al., Biotechnology, 10: 1458-1460 (1992); and in co-assigned applications 08/379072, 08/487550 or 08/746361, which are all included in this description by reference in full. It was reported that these antibodies exhibit a high degree of homology with human antibodies, i.e. 85-98%exhibit effector functions of antibodies man, have low immunogenicity and can exhibit a high affinity to antigens person.

Under "frag entom antibody" refers to a fragment of the antibody, as Fab, F(ab′)2, Fab′ and scFv.

By "chimeric antibody" refers to an antibody containing sequences derived from two different antibodies, which are usually antibodies of different species. Often chimeric antibodies include antibody fragments of human and mouse and in most cases, a constant region of human and variable regions of a mouse.

Under "bispecific antibody" refers to an antibody molecule with one antigennegative site, specific for one antigen, and other antigennegative site specific for a different antigen.

Under "immunogenetic" refers to the ability directed to the target protein or therapeutic component to induce an immune response (e.g. humoral or cellular) when administered to a subject.

Particularly preferred is the preparation of parenteral compositions in dosage form for ease of administration and consistent dosing. "Dosage form" is used in this sense refers to physically discrete units suitable for single doses for subjects mammals, which need to be treated; they expect each unit containing a predetermined quantity of active compound in order to obtain the required terap whitesky effect in Association with the required pharmaceutical carrier. Specific determination of the dosage forms according to the invention are dictated by and directly dependent on (A) the unique characteristics of the active compound and the particular therapeutic action that you want to achieve; and (B) the limitations inherent in the design of compounding such an active compound for the treatment associated with the sensitivity of individuals.

Century CD40L Antagonists.

According to the methods of the invention CD40L antagonist is administered to the subject in order to prevent the interaction of CD40L and his partner linking CD40. "CD40L antagonist" is defined as a molecule that prevents this interaction. The CD40L antagonist can be an antibody directed against CD40L (e.g., monoclonal antibody against CD40L), a fragment or derivative of the antibody against CD40L (e.g., Fab or F(ab)′2-fragments), chimeric antibodies or humanizirovanne antibodies, soluble form of CD40, soluble form of fused protein containing CD40, or pharmaceutical agents that destroy or inhibit the interaction of CD40L-CD40 or prevent the transmission of the signal through CD40.

The antibodies. To get anti-CD40L antibody of a mammal (e.g., mouse, hamster, rabbit, or hoofed animal), it is possible to immunize immunol the authorized form of the protein or protein fragment of CD40L (e.g., peptide fragment), which causes the humoral response in the animal. As immunogen you can also use a cell expressing CD40L on their surface. Alternative immunogen include purified protein CD40L or purified protein fragments. CD40L can be cleared from cells expressing CD40L, standard purification methods (Armitage et al. (1992); Lederman et al. (1992); and Hollenbaugh et al. (1992)). Alternative peptides CD40L can be obtained on the basis of the amino acid sequence of CD40L, as shown by Armitage et al. (1992). The ways of giving the immunogenicity of protein include conjugation to carriers or other means well known in the field. For example, the protein can be introduced in the presence of adjuvant. The process of immunization can be monitored by determining the antibody titer in serum or plasma. You can use the standard ELISA method or another immunoalkaline with the immunogen as antigen to assess the levels of antibodies. After immunization, you can get anticigarette and isolate polyclonal antibodies. To obtain monoclonal antibodies can be collected antitelomerase cells and merge with myeloma cells using standard methods of fusion of somatic cells, as described in U.S. patent No. 5833987 (1998) and 5747037 (1997).

Antibodies can be fragmented using standard methods, and the can is to hold a screening of fragments in terms of applicability in the same way, that is described above for whole antibodies. For example, F(ab')2fragments can be created during the processing of antibodies with pepsin. The resulting F(ab')2the fragment can be treated to restore the disulfide bridges and get Fab'-fragments. Further antibody fragments include Fab and scFv.

One way to minimize the detection of antibodies collective therapeutic use in humans other than the General immunosuppression, is to obtain chimeric derivatives of antibodies, i.e. molecules, antibodies, combined with the variable region of the antibody is not human, and animal, and constant region of human antibodies. Molecules of humanized chimeric antibodies, for example, may contain antigennegative domain from an antibody of a mouse, rat or other species and the constant region of human antibodies. Methods of obtaining these humanized chimeric antibodies include the methods described in the references cited in U.S. patent No. 5833987 (1998).

For therapy of human antibodies specific reactive with the protein or peptide CD40L, can be further humanize the receipt of chimeras variable regions of human antibodies in which parts of the variable regions, especially the conservative frame areas antigennegative domain, are of human origin is, and only the hypervariable regions are not of human origin. Such modified molecules of the immunoglobulin can be obtained in any of several ways known in this field (e.g., Teng et al., Proc. Natl. Acad. Sci. USA 80: 7308-7312 (1983); Kozbor et al., Immunology Today 4: 7279 (1983); Olsson et al., Meth. Enzymol. 92: 3-16 (1982)), and preferably receive in accordance with the methods published PCT application WO 92/06193 or EP 0239400. Humanized antibodies can be obtained commercially, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain. Preferred humanitariannet gp 39(CD40L)antibody, IDEC-131, described in the agreed application for the grant of a U.S. patent 08/554840 included in this description by reference in full.

Another way of generating specific antibodies, or fragments of antibodies reactive against the protein or peptide CD40L (e.g., such as protein gp 39, described in U.S. patent No. 5945513), is screening expressing libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria, protein or polypeptide CD40L. For example, complete Fab fragments, VHareas and Fv-areas can be Express in bacteria using phage expressing the library. See, for example. Ward et al., Nature 341: 544-546 (1989); Huse et al., Science 246: 1275-1281 (1989); and McCafferty et al., Nature 348: 552-554 (1990). By screening such libraries with, for example with the aid of the d peptide CD40L, you can identify the fragments of immunoglobulins, reactive with CD40L. Alternative you can use mice SCID-hu (available from Genpharm), to obtain antibodies or fragments thereof.

Methods of obtaining monoclonal antibodies (MAB)directed against CD40L, including CD40L human and mouse CD40L, and suitable antibodies for use in methods according to the invention described in the application for the grant of a patent PCT No. WO 95/06666, entitled "Anti-gp 39 Antibodies and Uses Therefor"; guidelines which are included in this description by reference in full. Particularly preferred antibodies against CD40L man according to the invention are Mat 24-31 and 89-76, obtained respectively by using a hybrid 24-31 and 89-76. Hybridoma 89-76 and 24-31, respectively producing antibodies 89-76 and 24-31, were deposited under the terms of the Budapest Treaty in the American type culture collection (ATSS), 10801 University Blvd., Manassas, VA 20110-2209, September 2, 1994, Hybridoma 89-76 assigned inventory number ATSS NV, and hybridoma 24-31 assigned inventory number ATSS NV.

Recombinant anti-CD40L antibodies, such as chimeric and humanized antibodies, can be obtained by processing a nucleic acid (e.g. DNA or cDNA)that encodes anti-CD40L antibodies, in accordance with standard methods of recombinant DNA. Thus, another aspect of this image is to be placed pertains to isolated nucleic acid molecules, encoding the heavy or light chains of immunoglobulins or parts thereof, reacting with CD40L, in particular human CD40L. Nucleic acid encoding the immunoglobulin may encode the variable region of light (VL) or heavy (VN) chain immunoglobulin with or without an associated constant region of the heavy or light chain (or portion thereof). Such nucleic acids can be isolated from cells (e.g., hybridoma), producing MAB against human CD40L, standard ways. For example, nucleic acids encoding the Mat 24-31 or 89-76, you can select accordingly from the hybrid 24-31 or 89-76, by screening the cDNA library, PCR amplification, or other standard methods. In addition, nucleic acids encoding CD40L MAB against human, you can include in expressing vector and type in a suitable cell host, to ensure the expression and production of recombinant forms of antibodies against CD40L person.

Primaryservername antibodies. Other highly effective methods of creating recombinant antibodies presents Newnan, Biotechnology, 10: 1455-1460 (1992). More specifically, the result of this method is the creation of pimatisiwin antibodies, which contain the variable domains of monkeys and a constant sequence of human rights. This publication is incorporated in this description by reference to the floor the second volume. Moreover, the method is also described in co-assigned application for the issuance of U.S. patent No. 08/379072, filed January 25, 1995, which is a continuation application for the grant of a U.S. patent registration No. 07/912292, filed July 10, 1992, which is a partial continuation of the application for the grant of a U.S. patent registration No. 07/856281 filed March 23, 1992, which, finally, is a partial continuation of patent application U.S. registration No. 07/735064 filed July 25, 1991. Application 08/379072 and all original applications included in this description as references in full.

In this way modify the antibodies so that they are not rejected because of their antigenic properties when administered to humans. This method is based on immunization of macaques-Griboedov antigens or receptors person. This method was developed in order to generate monoclonal antibodies of high affinity towards the surface antigens of human cells.

Identification of macaque antibodies to CD40L person by screening libraries based on phage display or heterohybridomas monkeys obtained using b-lymphocytes of monkeys immunized with CD40L, can be accomplished using the methods described in co-assigned application for U.S. patent No. 08/487550, filed June 7, 1995, are included in this description is of as links in full.

Earlier it was reported that antibodies obtained using the methods described in these applications are the effector function of human antibodies have reduced immunogenetic and prolonged half-life in serum. The technology is based on the fact that, despite the fact that macaques-kraboedy phylogenetically similar to people, they still recognize many proteins human as alien and therefore produce an immune response. In addition, since macaques-kraboedy phylogenetically close to humans, it has been found that antibodies produced in these monkeys have a high degree of amino acid homology with antibodies obtained in humans. Indeed, after the sequencing of the genes for the variable regions of light and heavy chains of immunoglobulin macaques was found that the sequence of each gene family in 85-98% homologous to the sequence equivalent man (Newman et al., 1992). First created thus antibody, anti-SW-antibody was 91-92% homologous to the consensus sequence of frame regions of human immunoglobulin (Newman et al., 1992).

As described above, the present invention is due in part to the identification of monoclonal antibodies or their pimatisiwin forms that are specific against the antigen CD40L person and is able to inhibit transfer the signal through CD40 or to inhibit the interaction CD40/CD40L. Blocking the site of the primary activation between CD40 and CD40L identified by the antibody (or a therapeutically effective fragment), providing the possibility of combined antagonistic action on positive costimulation with an agnostic negative effect on signal transmission, will be suitable therapeutic approach impact on recurrent forms of malignancies, especially b-cell lymphoma and leukemia. The functional activity of the identified antibodies determined by the blocking of CD40 signals, allowing the cell to survive and escape apoptosis induced IgM or Fas.

The production of new monoclonal antibodies monkeys that are specific bind CD40L or CD40 person, and obtained from them pimatisiwin antibodies can be accomplished using the methods described in the application for the grant of a U.S. patent, filed for joint consideration with registration No. 08/487550, and as is indicated here. These antibodies have high affinity for CD40L and therefore they can be used as immunosuppressive drugs, which inhibit the path of the CD40L/CD40.

Obtaining monoclonal antibodies monkeys preferably be carried out by screening libraries based on phage display or by obtaining heterohybridomas monkeys using b-lymphocytes, the scientists from monkeys, immunized CD40L (e.g., CD40 person). CD40 person may also be of the fused protein, is described in U.S. patent No. 5945513.

As indicated, the first method of creating an anti-CD40L antibodies lies in the technology of recombinant phage display. This method generally described above.

Essentially the way will include the synthesis of recombinant libraries of antibodies against CD40L antigen exposed on the surface of filamentous phage, and the selection of phage that secrete antibodies with high affinity to the antigen CD40L. As noted above, preferably will be selected antibodies that bind with CD40L and CD40 person. To implement this methodology, the authors present invention, to obtain a library of monkeys, has created a unique library, which reduced the possibility of recombination and improved stability.

Essentially, in order to take advantage of phage display for use in the case of libraries monkeys, this vector contains specific primers for PCR amplification of immunoglobulin genes monkeys. These primers based on the sequences macaques learned from the development of technology primatiale, and databases containing sequences person.

Suitable primers are presented in co-assigned application 08/379072, VK is uchennai in this description by reference.

The second method is to immunization of monkeys, such as macaques, anti-CD40L antigen person. The advantages inherent makaka associated with obtaining monoclonal antibodies discussed above. Such monkeys, such as macaques-Griboedov, it is possible to immunize against antigens or receptors person. In addition, the resulting antibodies can be used to create pimatisiwin antibodies according to the method of Newman et al. (1992) and Newman et al., jointly assigned to the application for the grant of a U.S. patent registration No. 08/379072, filed January 25, 1995, and these works are included in this description as references in full.

A significant advantage of the antibodies obtained from macaques-Griboedov, is that monkeys recognize many proteins human as alien and therefore provide the formation of antibodies, some of which have high affinity for the desired antigens person, such as surface proteins and cell receptors person. Moreover, because they are phylogenetically close to humans, the resulting antibodies show a high degree of amino acid homology with antibodies obtained in humans. As noted above, after the sequencing of the genes for the variable regions of light and heavy chains of immunoglobulin macaques was found ujena, each family of genes was 85-88% homologous to their counterparts in humans (Newman et al., 1992).

Essentially, makaka-rabadam injected antigen CD40L people, including isolated In cells, such as animals take a biopsy of the lymph node, and then b-lymphocytes merge with cells heteromyinae KN/B5 (mouse × people), using polyethylene glycol (PEG). Then identify heterohybridomas, secreting antibodies that bind the antigen CD40L person.

Desirable antibodies that bind to CD40L or CD40 in a way that stops or regulates the transmission of the signal through CD40, because such antibodies can potentially be used to inhibit the interaction of CD40L with CD40, with their counter receptors. If you can produce antibodies against more than one epitope on CD40L or CD40 and use antibodies together, their combined activity could potentially give a synergistic effect.

The invention consists in applying to the animal that primerano to the production of specific antibodies (e.g., primates, such as orangutans, baboons, monkeys and macaques-kraboedy). Other animals that can be used to generate antibodies to CD40L person, include, but are not limited to, the following animals: mice, rats, Guinea pigs, hamsters, monkeys, swine is, goats and rabbits.

The preferred method to produce antibodies of the person using SCID mice disclosed in co-owned, filed for joint consideration of the application for the grant of a U.S. patent registration No. 08/488376.

Human genes encoding antigens, CD40, CD40L and CD20, cloned and sequenced, and therefore can easily be obtained by recombinant methods.

Preferably the antigens CD40L, CD40 or CD20 person should be entered in soluble form, for example, in the expression of the gene encoding the antigen, which removed the transmembrane and cytoplasmic domains, and left only the extracellular part, i.e. the extracellular domains like superfamily V and C.

Macaques subjected to immunization CD40L antigen, preferably the soluble form, the conditions that lead to product-specific antibody. Preferably soluble CD40L antigen person should be introduced in combination with an adjuvant, such as complete adjuvant's adjuvant (CFA), alum, saponin or other known adjuvants, as well as their combinations. Typically, you will need to re-immunization, for example, by re-injection in a few months. For example, the introduction of soluble CD40L antigen of human conduct in Freund with booster-immunization during the period of time is from 3 to 4 months, getting in the serum containing antibodies which bind the antigen CD40L person.

After immunization collect In cells, for example, by taking biopsies of lymph nodes from immunized animals, and b-lymphocytes merge with cells heteromyinae KN/B5 (mouse × people), using polyethylene glycol. Methods of obtaining such heteromalla known and can be found in the application for the grant of a U.S. patent registration No. 08/379072, Newman et al., filed January 25, 1995 and is incorporated in this description by reference.

Then identify heterohybridomas that secrete antibodies that bind human CD40L. This can be done by the method of ELISA or radioimmunoassay using CD40L antigen person labeled with an enzyme or a radionuclide.

Then the line of cells that secrete antibodies with desired specificity towards the antigen CD40L person, subcloning to monoclonality.

Cell lines that Express antibodies that are specific contact CD40L antigen person, then used to clone sequences of the variable domains to obtain pimatisiwin antibodies, mainly as described by Newman et al., (1992) and Newman et al., U.S. patent registration No. 379072, filed January 25, 1995, both works are included in this opisanie as a reference. Essentially this is due to the extraction of RNA from cells to turn into cDNA and the amplification of cDNA by PCR using primers specific for Ig. Suitable primers are described in Newman et al., 1992 and in U.S. patent registration No. 379072.

The cloned variable genes monkeys then embed in expressing vector, which contains the genes for the constant regions of the heavy and light chains of human rights. Preferably this is carried out using patented expressing vector IDEC, Inc., named NEOSPLA. The specified vector contains the promoter/enhancer of cytomegalovirus, the main promoter of the beta-globin mouse, the beginning of SV40 replication, polyadenylation sequence of the growth hormone bull, exon 1 and exon 2 neomycinphosphotransferase, the constant region of human immunoglobulin Kappa - or lambda-type gene digidrofolatreduktazy, constant region RE of the human immunoglobulin gamma 1 or gamma 4 and the sequence of the leader. It was found that this vector leads to high expression pimatisiwin antibodies enabling gene variable regions of the apes, transfection into cells SNO and subsequent selection in medium containing G418, and amplification in the presence of methotrexate.

For example, the previously stated this expression system, in order to get primaryservername ant the body, with high avidity (Kd≤10-10M) with respect to CD4 and other surface receptors on human cells. In addition, it was found that the antibodies exhibit the same affinity, specificity and functional activity as the source of antibodies monkeys. The specified vector system largely disclosed in co-assigned U.S. patent registration No. 379072 included in this description by reference, as well as in the application for the grant of a U.S. patent registration No. 08/149099, filed November 3, 1993, also included in this description by reference in full. This system provides high levels of expression, ie > 30 PG/cell/day.

The number of antibodies suitable for a therapeutic effect can be determined by standard methods, well known to specialists in this field. Antibodies will be mostly prepared in the standard way with a pharmaceutically acceptable buffer, and you can enter them in any desirable way. Due to the effectiveness of the inventive antibodies and tolerance to them man these antibodies can be administered repeatedly to struggle with various diseases or pathological conditions in humans.

The person skilled in the art will be able by ordinary experimentation to determine what should be in the order of induce the Finance immunosuppression effective non-toxic amount of antibodies. However, as a rule, the effective dose will be in the range from about 0.05 to 100 milligrams per kilogram of body weight per day.

The antibodies (or fragments thereof) according to this invention should also be suitable for treatment of tumors in mammals. More specifically, they must be suitable for reducing tumor size, inhibiting tumor growth and/or prolonging the time of survival of animals bearing tumor. Accordingly, this invention also relates to a method of treating tumors in a human or other animal by introducing such human or animal an effective non-toxic amount of antibodies. The person skilled in the art will be able by ordinary experimentation to determine what should be in order to treat carcinogenic tumors effective non-toxic amount of anti-CD40L antibodies. However, as a rule, it is assumed that the effective dose will be concluded within approximately from 0.05 to 100 milligrams per kilogram of body weight per day.

Antibodies according to the invention can enter the human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce such an effect on therapeutic or prophylactic level. Such antibodies according to the invention it is possible to enter a specified person or other animal is in a conventional dosage form, prepared by combining the antibody according to the invention with a conventional pharmaceutically acceptable carrier or diluent according to known methods. The person skilled in the art it will be obvious that the form and nature of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it must be combined, the route of administration and other well-known variables.

Route of administration of the antibody (or fragment) according to the invention may be oral, parenteral, by inhalation or local. The term parenteral in the sense used here includes intravenous, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal doing. As a rule, preferred subcutaneous and intramuscular forms of parenteral administration.

Daily dosing regimens of parenteral and oral administration for the compounds according to the invention to prophylactically or therapeutically to induce immunosuppression or to treat carcinogenic tumors, as a rule, will be concluded in the range of about 0.05 to 100, but preferably from about 0.5 to about 10 milligrams per kilogram of body weight per day.

Antibodies according to the invention can also be entered through the galazii. Under "inhalation" understand intranasal or oral inhalation administration. Appropriate dosage forms for such administration, such as aerosol composition or dosing inhaler, can be obtained by standard methods. The preferred dose amount of the compounds according to the invention generally is in the range of about 10 to 100 milligrams.

Antibodies according to the invention can also be entered locally. Under the local introduction is meant non-systemic introduction, and it involves the use of compounds of the antibody (or fragment) according to the invention externally to the epidermis, to the buccal cavity and instillation of such antibodies in the ear, eyes and nose, and where the antibody does not penetrate substantially into the blood stream. Under the system the introduction refers to oral, intravenous, intraperitoneal, and intramuscular. The number of antibodies required for therapeutic or prophylactic action, of course, will vary depending on the selected antibodies, the nature and severity of the condition being treated and the animal undergoing treatment, and, ultimately, is at the discretion of the physician. Suitable local dose of the antibodies according to the invention will be generally in the range of about 1 to 100 milligrams on kilogram of body weight daily.

C. Soluble ligand CD40L.

In addition to antibodies that recognize and bind to CD40L and inhibit its interaction with CD40, provide for the use of other CD40L antagonists for the treatment of b-cell lymphomas and leukemias, either individually or in combination with other therapies (e.g. radiation or chemotherapy). Other CD40L antagonists represent a soluble form of the ligand CD40L. Monovalent soluble ligand CD40L, such as soluble CD40, can bind CD40L, thereby inhibiting the interaction of CD40L with CD40 expressed In cells. The term "soluble" indicates that the ligand is not constantly associated with the cell membrane. Soluble ligand CD40L can be obtained by chemical synthesis or preferably, by means of recombinant DNA, for example by expression of only the extracellular domain (in the absence of the transmembrane and cytoplasmic domains) ligand. Preferred soluble ligand is soluble CD40L CD40. Alternative soluble ligand CD40L may be in the form of a fused protein. This protein contains at least part of the ligand CD40L associated with the second molecule. For example, CD40 can be Express in the form of a fused protein with immunoglobulin (i.e. CD40-Ig-protein). In one embodiment, the receive protein containing amino acid residues hour and molecules CD40, the corresponding extracellular domain connected with amino acid residues of the sequence corresponding to the hinge area, areas WithN2, and CN3, the heavy chain of the immunoglobulin, e.g., Cα1, to form a fused protein CD40Ig (see, e.g., Linsley et al., J. Exp. Med. 1783: 721-730 (1991); Capon et al., Nature 337: 525-531 (1989) and U.S. patent No. 5116964 (1992)). Such fused proteins can be obtained by chemical synthesis or preferably, by means of recombinant DNA-based cDNA CD40 (Stamenkovic et al., EMBO J. 8: 1403-10 (1989)).

D. the Introduction of anti-CD40L.

The CD40L antagonist is administered to the subject in a biologically compatible form suitable for pharmaceutical injection in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the antagonist, which must be entered for which therapeutic effect of protein is superior to any toxic effects. It is implied that the term "subject"used in the description, includes living organisms in which it is possible to induce an immune response, for example, mammals. The preferred examples of subjects include humans, dogs, cats, horses, hoofed animals, cows, pigs, goats, sheep, mice, rats, and transgenic species. The CD40L antagonist can be entered in any pharmacological form, optionally in a pharmaceutically acceptable carrier. Introduction terapeuticas the effective amount of the antagonist is defined as a number, effective at dosages and for periods of time necessary to achieve the desired result (for example, inhibition of progression or proliferation of lymphoma, which is subjected to treatment). For example, a therapeutically active amount of CD40L antagonist may vary according to factors such as stage of disease (for example, stage I compared with stage IV), age, sex, medical complications (e.g., lymphoma related to or resulting from AIDS or other immunosuppressive conditions or diseases) and weight of the subject, and the ability of the antagonist to cause the desired response in the subject. The dosage can be adjusted to provide the optimum therapeutic response. For example, every day you can enter multiple fractional dose, or the dose may be proportionally reduced in accordance with the requirements of therapeutic situation.

Active connection, such as anti-CD40L-antibody, alone or in combination with other active agents, you can enter a convenient way, such as by injection (subcutaneous, intramuscular, intravenous, etc.), oral administration of inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active compound may be coated with a material to protect the compound from the action of enzymes, acids and other natures is the R conditions, that may inactivate the compound. The preferred route of administration is intravenous (IV) injection.

To introduce the antagonist CD40L other way other than parenteral administration, it may be necessary floor antagonist or joint introduction of the antagonist with a material that prevents inactivation. For example, the antagonist can enter the individual in an appropriate carrier or diluent, co-type with enzyme inhibitors or in an appropriate carrier or vector, such as a liposome. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Inhibitors of enzymes include trypsin inhibitor pancreas, diisopropylfluorophosphate (DEP) and the drug. Liposomes include emulsions of the type water-in-oil-water, as well as conventional liposomes (Strejan et al., J. Neuroimmunol. 7: 27-41 (1984)). Additional pharmaceutically acceptable carriers and excipients are known in this field.

An active connection, you can also enter parenterally or intraperitoneally. It is also possible to prepare dispersions in glycerol, liquid polietilenglikolja and their mixtures or oils. Under normal conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical to the position, suitable for injectable use include sterile aqueous solutions (in the case of water-soluble compounds) or dispersions and sterile powders for the preparation immediately before use sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent at which it easily passes through the syringe. The composition should be stable under conditions of manufacture and storage and must be preserved against the contaminating influence of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, high molecular weight alcohol (e.g. glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures. Proper fluidity can, for example, to support using a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by using surfactants. Prevention of the action of microorganisms can be achieved in various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, the preferred is the incorporation in the composition of isoto the practical agents, for example sugars, polyalcohol, such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be called by the inclusion in the composition an agent that slows down the absorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporation of active compounds (for example, CD40L antagonist as such or in combination with other active agents) in the required amount in an appropriate solvent, if necessary with a single ingredient or a combination listed in this description, with subsequent filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile filler, which contains the basic dispersion medium and the other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which give a powder of the active ingredient plus any additional desired ingredient from their previously sterile-filtered solution.

In that case, if the active connection is suitably protected, as described above, the protein can be administered orally, for example, with inert the second diluent or assimilable edible carrier. In the sense used here "pharmaceutically acceptable carrier" includes any and all solvents, dispersion medium, covering materials, antibacterial and antifungal agents, isotonic and slows the absorption means and the like. The use of such media and agents for pharmaceutically active substances is well known in this field. In addition to cases in which any conventional medium or agent is incompatible with the active compound, their use is expected in therapeutic compositions. All discussed above composition for use with CD40L antagonists can also include in the composition an additional active compounds (for example, chemotherapeutic drugs, anti-CD20 antibodies).

That is, the Introduction of anti-CD40L with other means.

Disease Kockica. About 7500 new cases of Hodgkin's disease (HD) are diagnosed annually in the United States. Used separately radiation therapy for the treatment of BH in stage I, II, and even in stage III. Also used radiation in combination with chemotherapy (e.g., ABVD and MORR). Cm. V..DeVita et al., "Hodgkin′'s Disease," IN CANCER: PRINCIPLES AND PRACTICE OF ONCOLOGY, vol.2, 2142-2283 (DeVita et al., eds., 5thed. 1997) and references in this work in connection with the introduction of radiation and chemotherapy protocols for the treatment of BH.

Drugs for chemotherapy, suitable for Leche is of BH, include alkylating agents, Vinca alkaloids (e.g. vincristine and vinblastine), procarbazine, methotrexate and prednisone. The four-drug combination of MORR (mechlorethamine (nitrogen mustard), vincristine (oncovin), procarbazine and prednisone) is very effective in the treatment of BH. For patients resistant to MORR, you can use a combination of ABVD (e.g., adriamycin, bleomycin, vinblastine and dacarbazine), ChlVPP (chlorambucil, vinblastine, procarbazine and prednisone), CABS (lomustin, doxorubicin, bleomycin and streptozotocin), MORR plus ABVD, MOPP plus ABV (doxorubicin, bleomycin and vinblastine) or BCVPP (carmustine, cyclophosphamide, vinblastine, procarbazine and prednisone). Arnold S. Freedman and Lee M. Nadler, Malignant Lymphomas, in HARRISON′S PRINCIPLES OF INTERNAL MEDICINE 1774-1788 (Kurt J. Isselbacher et al., eds., 13thed. 1994) and V..DeVita et al., (1997) and references cited in these works in connection with a standard dosage and consumption patterns. These methods of treatment can be used without changing or need to change for a particular patient, in combination with CD40L antagonists as such, or in combination with anti-CD20-antibody or fragments.

For recurrent or resistant BH simple combined dose regimes "salvage therapy" to use in combination in an anti-CD40L-antibodies, alone or together with anti-CD20-antibody. An example of the conventional combined dose regimes "salvage therapy" when BH include VABCD (vinblastine, doxorubicin, dacarbazine, lomustin, and bleomycin), ABDIC (doxorubicin, bleomycin, dacarbazine, lomustin, prednisone), CBVD (lomustin, bleomycin, vinblastine, and dexamethasone), PCVP (vinblastine, procarbazine, cyclophosphamide, and prednisone), SER (lomustin, etoposide and prednimustine), EVA (Etoposid, vinblastine and doxorubicin), MOPLACE (cyclophosphamide, etoposide, prednisone, methotrexate, cytarabine and vincristine), MIME (methyl-GAG, ifosfamide, methotrexate and etoposide), MINE (mitoguazone, ifosfamide, vinorelbine, and etoposide)STC-CHOP (methotrexate and CHOP), SEM (lomustin, etoposide and methotrexate)CAVP (lomustin, melphalan, etoposide and prednisone), EVAP (etoposide, vinblastine, cytarabine and cisplatin), EPOCH (etoposide, vincristine, doxorubicin, cyclophosphamide, and prednisone) using doses and consumption patterns, which are described in V. T. DeVita et al., (1997).

Nahodkinskaja lymphoma (KHL). An estimated 40,000 new cases of NHL are diagnosed each year in the United States, and, apparently, this number increases. Moreover, the NHL ranks fourth in the total number of people who die each year from cancer. The NHL includes several subtypes of lymphomas with unique clinical manifestation and natural course of development. Classification of subtypes of NHL are set out according to the generally accepted classification of NHL, working classification. Table 1 presents three degrees working classification.

Table 1
The degree of malignancyThe subtype of NHL
LowSmall cell lymphocytic

Follicular, predominantly from

small cells with cleaved nuclei

Follicular from a mixture of small cells with

split nuclei and large cell
Intermediate degreeFollicular, predominantly large cell

Diffuse small cells with cleaved nuclei

Diffuse from a mixture of small and large cells

Diffuse both
A high degreeBoth immunoblast

Lymphoblastic

From small cells with unsplit cores type Burkitt and non-Burkitt
Other typesLymphoma, AIDS-related

Cutaneous T-cell lymphoma

T-cell leukemia/lymphoma adult

Angioimmunoblastic lymphadenopathy

Monocytoid b-cell lymphoma

In cell types of NHL include: small cell lymphocytic lymphoma/b-cell chronic lymphocytic leukemia (SLL/B-CLL), lymphoplasmacytoid lymphoma (LPL), lymphoma mantle cell (MCL), follicular lymphoma (FL), diffuse lymphoma large cell (DLCL) and Burkitt's lymphoma (BL). Cm. Gaiano et al., "Lymphomas," IN CANCER: PRINCIPLES AND PRACTICE OF ONCOLOGY, vol. 2, 2131-2145 (DeVita et al., eds., 5thed. 1997). Two other classifications (e.g., the Kiel classification and revised Euro-American classification of lymphomas, or REAL) are also used in Oncology, and the names of NHL may vary in the two classification systems. Cm. M. A. Shipp et al., "Non-Hodgkin's Lymphomas, "IN CANCER: PRINCIPLES AND PRACTICE OF ONCOLOGY vol. 2. 2165-2220 (DeVita et al., eds., 5thed. 1997). NHL-lymphoma, can also be classified according to the age of the patient at which it is diagnosed:

Table 2
B-cell lymphoma adultB-cell lymphoma of childhood.
Follicular lymphoma Diffuse b-cell lymphoma large cell Lymphoma of mantle cell B-CLL/SLLBurkitt's lymphoma

Diffuse b-cell lymphoma large cell

Follicular lymphoma

Lymphoma of the predecessors In-

LBL
Immunocytoma/waldenstrom
The type of MALT/monocytoid b-cell

Cm. M.A. Shipp et al., (1997).

Radiation therapy is usually limited to the treatment of patients diagnosed with stage I or II NHL low-grade malignancy, and as a possible remedy for the patients who develop aggressive is Tadei disease. This invention covers the combination of CD40L antagonist with radiation therapy and other cancer therapies for the treatment of NHL.

Chemotherapy is used for most patients with stage II and for all patients with III and IV stages of the disease. The modes include the use of single alkylating agents such as cyclophosphamide or chlorambucil, or combinations, such as CVP (cyclophosphamide, vincristine and prednisone), CHOP (CVP and doxorubicin), C-MOR (cyclophosphamide, vincristine, prednisone and procarbazine), CAP-BOP (CHOP plus procarbazine, and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin and leucovorin), Thomase-MORR (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide and leucovorin plus standard MORR), ProMACE-CytaBOM (prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate and leucovorin) and the MASSORA-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, fixed-dose prednisone, bleomycin and leucovorin). Cm. Shipp et al. (1997) to determine the standard doses and regimens. CHOP also combined with bleomycin, methotrexate, procarbazine, nitrogen mustard, citizenoriented and etoposide. Less widely used drugs for the treatment of NHL include: 2-chloromethoxypropyl (2-CDA), 2'-deoxycoformycin and fludarabine. For patients with NHL of the intermediate and the high degree who fail to achieve remission or who relapse, use the salvage therapy. When therapy "salvation" used drugs such as citizenoriented, cisplatin, etoposide and ifosfamide, which are given separately or in combination. Arnold S. Freedman and Lee M. Nadler, Malignant Lymphomas, in HARRISON′S PRINCIPLES OF INTERNAL MEDICINE 1774-1788. In cases of recurrent aggressive forms of NHL recommended the following protocols IMVP-16 (ifosfamide, methotrexate and etoposide), MIME (methyl-gag, ifosfamide, methotrexate and etoposide), DHAP (dexamethasone, high dose tsitarabina and cisplatin), ESHAP (etoposide, methylpredisolone, HD-cytarabine, cisplatin), SERRE(B) (cyclophosphamide, etoposide, procarbazine, prednisone, and bleomycin), and CAMP (lomustin, mitoxantrone, cytarabine and. prednisone) using batching schemes and applications described in Shipp et al., (1997).

Recommended the following protocols for NHL in children on the basis of histology and stage:

Table 3
HistologyProtocol
Lymphoblastic
Stage 1CHOP
Stage 2The OMRS
Stage 3ARO
Stage 4LSA2L2, NHL-BFM 86
CHOP
The OMRS
NHL-BFM 86, St. Jude Total In,
LMB 89
Stage 4/B-ALLLMB89
Both
Stage 1CHOP
Stage 2The OMRS
Stage 3APO
Stage 4NHL-BFM 86, HAZOR

APO = doxorubicin, prednisone, and vincristine; list = cyclophosphamide, oncovin, methotrexate and prednisone. Cm. .J.Weinstein et al., "Leukemias and Lymphomas of Childhood," IN CANCER: PRINCIPLES AND PRACTICE OF ONCOLOGY, vol.2, 2145-2165 (DeVita et al., eds., 5thed. 1997) and cited in this work the links all work included in this description by reference.

Anti-CD40L antibodies and antagonists can be used in combination with any of chemotherapeutics and/or radiation therapy, currently used for the treatment of BH or diagnosed subtypes of NHL. Anti-CD20 antibodies can also be added to the mixture used therapeutic agents. A number of chemotherapeutic agents used in combination with anti-CD40L-antibody or CD40L antagonists may vary subject to the sludge which may be entered in accordance with the what is known in this field. See, for example, Bruce A Chabner et al., Antineoplastic Agents, in GOODMAN AND GILMAN′S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS 1233-1287 (Joel G. Hardman et al., eds., 9thed. 1996).

Leukemia and other malignant tumors.

Consider therapy and methods of treatment according to the present invention can also be used to treat b-cell leukemias, including ALL-L3 (leukemia type of Burkitt's lymphoma and chronic lymphocytic leukemia (CLL), and monocytic leukemia and other malignant tumors that Express CD40.

Treatment of ALL involves the use of vincristine and prednisone. With this treatment you can also add anthracycline, cyclophosphamide, L-asparaginase. Other methods of induction therapy includes a combination of four drugs (vincristine, prednisone, anthracycline and cyclophosphamide or asparginase) or five drugs (vincristine, prednisone, anthracycline, cyclophosphamide and asparginase). To explore additional methods of treatment and doses, see D. A. Scheinberg et al., "Acute Leukemias." IN CANCER: PRINCIPLES AND PRACTICE OF ONCOLOGY, vol. 2, 2193-2321 (DeVita et al., eds., 5thed. 1997).

Treatment of CLL includes chemotherapeutic combinations IDA, CVP, CHOP, COP and CAP (cyclophosphamide, doxorubicin and prednisone). Treatment of patients with refractory CLL involves the use of purine analogues (for example, monophosphate fludarabine, 2-is logitechvideotray and pentostatin). Cm. A. C. Deisseroth et al., "Chronic Leukemias," IN CANCER: PRINCIPLES AND PRACTICE OF ONCOLOGY, vol. 2, 2193-2321 (DeVita et al., eds., 5thed. 1997).

Anti-CD20 and anti-CD40-ahtiala.

This invention also encompasses the combination of anti-CD40L antibodies such as IDEC-131, anti-CD20-antibody or therapeutically effective fragments and/or anti-CD40-antibodies or therapeutically effective fragments. Preferred anti-CD20-antibody is RITUXAN®and B1 (see U.S. patent No. 5843398). To explore the description, receipt and use of anti-CD40L (also known as anti-Dr), see jointly assigned application for patent U.S. registration No. 08/554840, filed November 7, 1995, 08/925339, filed September 8, 1997, 09/069871, filed on April 30, 1998, and 09/332595 filed June 14, 1999, and U.S. patent No. 5747037. Preferred anti-CD40 antibodies and obtaining them are described in U.S. patents№5874085; 5874082; 5801227; 5667165; 5674492 and 5667165, all of which are included in this description by reference in full. All discussions relating to anti-CD20 antibodies described herein are also applicable to anti-CD40-antibodies.

Because disorders of b-cells in peripheral blood by definition may require having access to blood for treatment, the preferred route of administration of immunologically active chimeric anti-CD20 antibodies and radioactively labelled anti-CD20 antibodies are the two who is injecting; in the sense used here, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration. Of them most preferably intravenous.

Immunologically active chimeric anti-CD20 antibodies and radioactively labelled anti-CD20 antibodies will usually be prepared using standard methods with pharmaceutically acceptable buffer, for example, sterile saline, sterile buffered water, propylene glycol, combinations of the above, etc. Methods of preparation of parenteral input means described in the PHARMACEUTICAL CARRIERS AND FORMULATIONS, Martin, Remington′s Pharmaceutical Sciences, 15thEd. (Mack Pub. Co., Easton, Pa. 1975), which is incorporated in this description by reference, or as described above.

The specific therapeutically effective amount of immunologically active chimeric anti-CD20 antibodies used to obtain a unique therapeutic action in any given patient can be determined by standard methods, well known to specialists in this field. Effective dose (i.e. therapeutically effective amount) of immunologically active chimeric anti-CD20 antibodies are in the range from about 0.001 to 30 mg/kg body weight, more preferably from about 0.01 to 25 mg/kg body weight and the most is her preferably from about 0.4 to 20.0 mg/kg of body weight. At other doses is also saved vitality. Factors affecting dose include, but are not limited to, the severity of the disease; previous treatment approaches; the General health of the patient; the patient's age; others present illness, etc. the person skilled in the art will easily cope with the assessment of the individual patient and determining the appropriate dose that falls within the specified limits, or if necessary, beyond the extent required. Introduction immunologically active chimeric anti-CD20-angiel or other CD20 antibodies (e.g., RITUXAN®or B1) in the specified dose limits can be done as a single treatment or over a series of treatments. As for the chimeric antibody, it is preferable that such introduction was made during a series of treatments. Specified preferred approach is dictated by the method of treatment associated with the disease. In fact, despite the fact that a single dose of benefits and it can be used effectively for the treatment/management of the disease, the preferred course of treatment can be carried out in several stages; most preferably in the range of about 0.4 and 20 mg/kg of body weight immunologically active chimeric anti-CD20 antibodies or other anti-CD20 antibody is administered to the patient once in the components for approximately 2 to 10 weeks, most preferably for about 4 weeks.

With regard to the application of radioactively labeled anti-CD20 antibodies and/or anti-CD40 antibodies, preferably the antibody is a (1) non-chimeric antibodies or (2) chimeric humanized antibodies with delegated domains, or (3) human antibodies. The specified preference dictated a much shorter half-life of circulating antibodies with delegated domains, or mouse antibodies compared to the full chimeric or humanitarianism antibodies (i.e. with a longer half-life of circulating radionuclide is present in the patient for longer periods of time). However, radioactively labeled chimeric antibodies can be used for treatment values with lower doses in millicurie (mcure")used in connection with its chimeric antibody against the antibody. This plan of action helps to reduce the toxicity in the bone marrow to an acceptable level, while maintaining therapeutic efficacy. Specific radioactively labeled chimeric form of the anti-CD20 can be obtained, as described in U.S. patent No. 5776456 and 5843439.

Effective single treatment dosages (i.e. therapeutically effective amount) labeled with yttrium-90 anti-CD20 antibodies are within primer is from 5 to 120 mcure, more preferably about 10 to 40 mcure for mouse antibodies and from about 30 to 100 mcure for antibodies with delegated domain. Effective single treatment dose labeled with iodine-131 anti-CD20 antibodies, not requiring removal of bone marrow, are within about 5 to 70 mcure, more preferably from about 5 to 40 mcure. Effective single ablative dose (i.e. they may require autologous bone marrow transplantation) labeled with iodine-131 anti-CD20 antibodies are in the range of about 30 to 600 mcure, more preferably from about 50 to less than 500 mcure. As for the chimeric anti-CD20 antibodies, due to the longer half-life from the circulation compared with murine antibodies, an effective single therapeutic doses labeled with iodine-131 chimeric anti-CD20 antibodies, not requiring removal of bone marrow, are within about 5 to 40 mcure, more preferably less than about 30 mcure. Criteria for imaging, for example, for the label indium-111, typically comprise less than about 5 mcure. Additional discussion of the production and use of radioactively labeled anti-CD20 antibodies can be found in U.S. patent No. 5843398 and 5843439, and both patents hereby incorporated by reference in full.

Radioactive labeled antibody.

In respect of the application radioactiveman antibodies (for example, specific for CD40, CD40L and/or CD20) is preferred nahimana antibody; the specified preference is dictated by the significantly longer half-life period of circulation of chimeric antibodies compared with murine antibodies (i.e. with a longer half-life of circulating radionuclide is present in the patient for longer periods of time). However, radioactively labeled chimeric antibodies can be used for treatment values with lower doses in millicurie (mcure")used in connection with chimeric antibody, compared to the murine antibody. This plan of action helps to reduce the toxicity in the bone marrow to an acceptable level, while maintaining therapeutic efficacy.

Many radionuclides suitable for this invention, and specialists in this field can easily determine which radionuclide is most suitable in different occasions. For example, iodine-131 (131I) is a well-known radionuclide used for aiming at a target of immunotherapy. However, the clinical usefulness of131I may be limited by several factors, including the period of the physical half-life of eight days; dihalogenoalkane iodirovannoi antibodies in the blood, and tumors; the characteristics of the emission (n is an example, large gamma component), which may be suboptimal for the localized deposition of dose in the tumor. With the advent of chelating agents the best quality possible protein binding groups, chelating metals, has increased the potential application of other radionuclides, such as indium-131 (131In) and yttrium-90 (90Y).90Y has several advantages when used in radioimmunotherapeutic applications: half-life90Y is 64 hours, long enough to allow the antibody to accumulate in tumors, and in contrast to, for example, from131I90Y is a pure beta emitter of high energy emission, not accompanied by gamma rays when it decays, with limits light penetration from 100 to 1000 cell diameters. In addition, the minimum number of penetrating radiation allows outpatient introduction labeled90Y antibodies. In addition, the internalization of labeled antibodies is not required for cell death and local emission of ionizing radiation must be lethal to the surrounding tumor cells without antigen target.

One not related to therapy restrictions90Y based on the absence of significant gamma radiation, complicating the visualization. To avoid the specified item is oblea, you can use diagnostic "imaging" radionuclide such as indium-111 (111In) to determine the location and relative size of the tumor before the introduction of therapeutic doses labeled90Y anti-CD20. Indium-111 is particularly preferred as a diagnostic radionuclide, because at doses in the range of about from 1 to 10 mcure it can be inserted without risk, without apparent toxicity; and data visualization, as a rule, give the forecast distribution antibodies labeled90Y. In most cases, when research for visualization using 5 mcure labeled111In antibodies, as indicated dose of both secure and has a high efficiency when rendering compared with lower doses, with optimal visualization takes place in the period from three to six days after administration of the antibody. See, for example, Murray, J. of Nuc. Med. 26: 3328 (1985) and Carraguillo et al., J. Of Nuc. Med. 26: 67 (1985).

Effective single treatment dosages (i.e. therapeutically effective amount) of labeled90Y antibodies (eg, anti-CD40L, anti-CD20 anti-CD-40 antibodies) are in the range of about 5 to 75 mcure, more preferably about 10 to 40 mcure. Effective single treatment dose of labeled131I antibodies, not requiring removal of bone marrow, located within the Ah from about 5 to 70 mcure, more preferably from about 5 to 40 mcure. Effective single ablative dose (i.e. they may require autologous bone marrow transplantation) labeled131I antibody are in the range of about 30 to 600 mcure, more preferably from about 50 to less than 500 mcure. As for chimeric antibodies, due to the longer half-life from the circulation compared to the murine antibody effective single therapeutic doses labeled with iodine-131 (131I) a chimeric antibody that does not require removal of bone marrow, are within about 5 to 40 mcure, more preferably less than about 30 mcure. Criteria for imaging, for example, for the taglllIn typically comprise less than about 5 mcure.

As for radioactively labeled antibodies for therapy, the dosing can also be carried out using a single therapeutic treatment or multiple treatments. Because of the presence of radioactive component preferably before processing to "collect" peripheral stem cells ("PSC") or bone marrow ("VM") in those patients where it is assumed potentially unavoidable toxic effect on the bone marrow caused by radiation. VM and/or PSC collected using standard methods and then purified and freeze is possible for re-infusion. Additionally, most preferably before treatment was conducted diagnostic dosimetric study of a patient using diagnostic labeled antibodies (e.g., using111In), the purpose of which is to obtain assurance that therapeutically labeled antibody (e.g., using90Y) does not become too "concentrated" in any normal organ or tissue.

Additional radioisotopes which can be used includel23I125I131In32R64Cu67Cu211At,177Lu,90Y186Re,212Pb212Bi47Sc,105Rh,109Pd.153Sm188Re,199Au,211At and213Bi, the Number of delivered radiation will be partially dependent on half-life and type of corpuscular radiation.

In the experiments described below used the following materials and methods. The examples below do not limit the invention described or claimed, but only represent variants of the claimed invention.

EXAMPLES

Example 1

The properties of cells In the lymphoma cells DHL-4

The idea that anti-CD40L-antibody can block mediated by CD40L-CD40 survival of malignant b cells in chemotherapy-induced toxicity/apoptosis, about Eraly in vitro, using IDEC-131 and cell line B-lymphoma, DHL-4 (Roos et al., Leuk. Res. 10: 195-202 (1986))exposed to adriamycin (ADM). IDEC-131 is a humanized version of the murine monoclonal antibodies against CD40L person, 24-31.

First determined the minimum concentration of ADM, cytotoxic against cells DHL-4, exposing cells DHL-4 for 4 hours effects of various concentrations of ADM. Cytotoxicity towards cells DHL-4 after 5 days of cultivation was measured through an analysis based on the recovery of the dye Alamar Blue (see Gazzano-three-bet et al., J. Immunol. Meth. 202: 163-171 (1997)). Briefly, 1×105cells DHL-4 in the growth medium (RMPI-1640 plus 10% fetal calf serum) were incubated with different concentrations of ADM (1×10-6M to 1×10-8M) in test tubes for cell cultures at 37°C for 4 hours. After incubation, the cells were washed again suspended in the growth medium at a concentration of 1×105cells/ml, and 200 μl of cell suspension was added into each well of 96-well plate with flat bottom. The plates were incubated at 37°and tested for cytotoxicity at different time points. During the last 18 hours of incubation, each well was added 50 μl of the redox dye Alamar Blue (Biosource International, No. in catalogue DAL 1100). After incubation tablets cooled by incubation at room Tempe is the atur for 10 minutes on the shaker and determined the intracellular recovery of the dye. Fluorescence was recorded using a 96-well fluorimeter when the wavelength of excitation of 530 nm and emission 590 nm. The results were expressed in relative fluorescent units (RFU). The percentage of cytotoxicity was calculated as follows: [1-(average RFU of the test specimen ÷ average RFU control cells)] × 100%.

Built titration curve of cytotoxicity ADM and chose the minimum concentration of a medicinal product to ensure cytotoxicity in further analyses.

The results are depicted in figure 1, show cytotoxicity against cells DHL-4, cultured for 5 days after exposure to ADM (2×10-7M and 4×10-8M ADM) for 4 hours before cultivation. Cells were washed once after exposure and were cultured in the growth medium for 5 days, cytotoxicity was determined by analysis of the absorption of the dye Alamar Blue as described above. In addition, characterized cells DHL-4 membrane expression of the selected CD molecules using flow cytometry. Discovered that the cells of DHL-4 Express molecules CD19, CD20, CD40, but not detected in the expression of CD40L.

Example 2

Anti-CD40L-antibody cancels CD40L mediated resistance cells In the lymphoma to death under the action of adriamycin

On figa shows the effect of anti-CD40L antibody (IDEC-131) mediated the th CD40L-CD40 resistance cells DHL-4 cell death, induced ADM. Cells DHL-4 (0,5×106cells/ml) were incubated in the presence of 10 μg/ml of soluble CD40L (sCD40L, P. A.Brams, E.A.Padlan, .Hariharan, .Slater, J.Leonard, R.Noelle, and R.Newman, "A humanized anti-human CD154 monoclonal antibody blocks CD154-CD40 mediated human cell activation" (manuscript submitted for publication)) for 1 hour at 37°C. After incubation for 1 hour was added in small concentrations of ADM (2×10-7M 4×10-8M) and incubated for another 4 hours in the presence or in the absence of CD40L (10 µg/ml). After exposure to ADM cells were washed and resuspendable in the growth medium at a concentration of 0.5×106cells/ml, and 100 μl of cell suspension was added into each well of 96-well plate with flat bottom in repeats with sCD40L or without him. sCD40L (10 μg/ml) was added to the cultures, which are continuously exposed to sCD40L during processing, ADM, and to the cultures, which were not sCD40L during the exposure with ADM. In addition, cultures were added IDEC-131 at a concentration of 10 μg/ml, in order to determine its effect on cell DHL-4, incubated with sCD40L and ADM. After 5 days cytotoxicity was measured by analyzing the absorption of the dye Alamar Blue as described.

Data show that SCD40L prolonged the survival of cells DHL-4 after processing ADM, while, as expected, increased cytotoxicity was observed compared to cells that were exposed to the effect of ADM in the absence of SCD40. Furthermore, addition of anti-CD40L antibody (IDEC-131) was cancelled CD40L mediated survival of cells, leading to increased cellular cytotoxicity (figa).

Adding only one IDEC-131 had no action on the cells of DHL-4 treated sCD40L, and this suggests that the antibody does not in itself have any direct or any abscopal cytotoxic activity against cells DHL-4 (pigv). Cells DHL-4, pre-incubated with sCD40L or without, were cultured in the presence of different concentrations of IDEC-131, RITUXAN®anti-CD20 antibodies CE 9.1 and anti-CD4 antibodies (Anderson et al., Clin. Immunol. and Immunopathol. 84: 73-84 (1997)). After 5 days was determined by a cytotoxicity/cell proliferation DHL-4 with analysis on the basis of Alamar Blue as described above. On FIGU shows the no action IDEC-131 on the proliferation or cytotoxicity of cells DHL-4, while RITUXAN®as expected, inhibited cell proliferation and induced cytotoxicity. Observed no effect in cells DHL-4, cultured with anti-CD4 antibodies.

Example 3

Signal transmission CD40L-CD40 prevents apoptosis of cells In lymphomas induced by anti-CD20-antibody RITUXAN®

The effect mediated by CD40L-CD40 signal induced on anti-CD20-antibody apoptosis of cells In the lymphoma was determined using the in vitro system, which includes the cell is DHL-4 and the surface cross-linking RITUXAN®. Cells DHL-4 (from 0.5 to 1×106cells/ml) were cultured with sCD40L (10 μg/ml) at 37°C. After culturing overnight, the cells were collected and incubated with 10 μg/ml RITUXAN®or control antibodies (CE 9.1; anti-CD4 antibody) in the presence or without sCD40L (10 μg/ml) on ice. After 1-hour incubation, the cells were centrifuged to remove unbound antibody, resuspendable at a concentration of 1×106cells/ml in the growth medium (5% FCS-RPMI) and were cultured in test tubes for tissue culture. Antibodies associated with the surface of the cell, cross-linked by injection of F(ab′)2-fragments of antibodies goat, specific against Ig-Fcγ man at a concentration of 15 μg/ml, and cultures were incubated at 37°until analysis for apoptosis. Apoptosis was detected using analysis kasaei-3 according to the method of flow cytometry. Cultured cells were collected after 4 and 24 hours, washed and fixed with 4°using Cytofix (Set Cytofix/CytopermTM, Phanningen katal. No. 2075 KK). After fixation for 20 min cells were washed and added to 15 µl of affinity purified PE-conjugated polyclonal rabbit antibodies against caspase-3 (Pharmingen, No. in catalogue 67345) and 50 μl Cytoperm (Pharmingen; no directory 2075KK). Cells were incubated on ice in the dark for 30 minutes After which incubatee cells were washed once and resuspendable in Cytoperm. Data flow cytometry was received on FACScan and analyzed using a computer program WinList from Verity Software House.

Table 4 shows the resistance induced RITUXAN® apoptosis in lympany cells DHL-4 under the influence of sCD40L. In these studies, activation of caspase-3 was used as a replacement token, as a preliminary study the authors showed a good correlation between the analysis of caspase-3 and Tunel analysis. Cross-linking RITUXAN®on the surface of cells DHL-4 in the presence of sCD40L reduced the levels of apoptosis, whereas in cells not exposed to sCD40L occurred apoptosis. When compared to cultures incubated in the presence of the antibodies of the same isotype control antibodies (SE), apoptosis of cells occurred. Thus, the data suggest that sCD40L-induced signal transmission path CD40 may lead to the development of indirect RITUXAN®cell death In lymphoma.

Table 4
Conditions of cultivation% Apoptosis (MIF)(a)
4 hours24 hours
Cells DHL-4, exposed with sCD40L
Only cells 3,35 (17,42)4,94 (7,62)
Cells + RITUXAN1,97 (1,97)4,54 (6,54)
Cells + RITUXAN + F(ab′)2antibodies against human IgG21,17 (17,39)9,62 (13,44)
Cells + CE 9.12,31 (13,25)4,15 (7,85)
Cells + SE + F(ab′)2antibodies against human IgG2,09 (22,14)4,14 (to 9.57)
Cells + F(ab′)2antibodies against human IgG1,93 (12,57)5,13 (8,02)
Cells DHL-4, are not exposed with sCD40L
Only cells4,36 (14,34)5,08 (17,62)
Cells + RITUXAN5,67 (10,66)1,08 (17,92)
Cells + RITUXAN + F(ab′)2antibodies against human IgG74,82 (22,80)30.63 per (26,84)
Cells + SE5,99 (14,00)3,05 (18,24)
Cells + SE + F(ab′)2antibodies against human IgG5,96 (12,11)2,24 (18,19)
Cells + F(ab′)2antibodies against human IgG6,09 (12,27)1,85 (17,27)
(a)The percentage of positive cells with active caspase-3 and the average intensity of their fluorescence is orescence in logarithmic scale.

Example 4

The influence of IDEC-131 on the survival cell chronic lymphocytic leukemia (CLL)

To determine the effect of IDEC-131 on the growth and survival of cells of B-CLL in vitro, cells of B-CLL were cultured in vitro with IDEC-131 or without him in the presence of CD40L. From the blood of patients with CLL were isolated mononuclear cells of peripheral blood (RVMS) using centrifugation in density gradient ficol-hipac. Survival was determined by the method of exclusion staining dye Trifanova blue, and it was >98%. Analyses based on flow cytometry showed that >70% of the lymphocytes were CD19+/CD20+.

Cell CLL (RVMS) were cultured in the growth medium CLL (for example, the medium RPMI-1640 with 5% FCS or 2% autologous plasma donor with the addition of 2 mm L-glutamine and 100 units/ml penicillin-streptomycin). In addition, for some experiments, purified b cells CD19+using CD19 beads+Dynabeads according to the manufacturer's instructions (Dynal, katal. No. 111.03/111.04), and were cultured as described above. The CLL cells or treated cells of B-CLL, cultured in the growth medium, typically underwent spontaneous apoptotic cell death. However, culturing these cells in the presence of sCD40L extended their survival in cultures. Table 5 shows cell survival of B-CLL CD19+grown in outstay or in the absence of sCD40L (5 μg/ml) at different time points, and shown longer survival of CLL cells. Cells of B-CLL patient No. 1, cultured with sCD40L, had a survival rate of > 60% for more than 2 weeks, while cells grown in the absence of sCD40L, had a survival rate of less than 10%.

Table 5
Survival of b-cells in the presence of SCD40L
Sample B-CLLTime (hours)% Survival(a)
(-)CD40L(+)CD40L
Patient No. 10

48

96

144
≥90

88

46

30
≥90

90

77

72
Patient No. 20

48

96

144
≥90

40

31

17
≥90

72

65

51
(a)equal to the survival rate, defined by the elimination method using dye Trypanosoma blue.

Figure 3 shows the effect of IDEC-131 on the growth and survival of cells of B-CLL after 7 days of cultivation. Purified cells from B-CLL from a patient with CLL (2×106cells/ml) was divided into two tubes for cultivation. Cells in one test tube was mixed with sCD40L (5 μg/ml) in an equal volume of medium growth, whereas the other is a second test tube was incubated with an equal volume of medium growth as a control. After incubation for 1 hour at 37°With cells, gently mixed, and 100 μl of medium with the cell suspension was added into each well of 96-well plate with flat bottom in repeats in the presence of different concentrations of IDEC-131 (10 μg/ml to 0.3 μg/ml) or without it. After seven days was determined survival/cell death in culture, using analysis based on the Alamar Blue as described above. The results showed the survival of cells in cultures with sCD40L. Adding to the culture of IDEC-131 resulted in increased cell death, which testified to the switching survival of cells to cell death or sensitization of cells to death. In addition, RITUXAN®entered in the same concentration as IDEC-131, acted on cell death and less at a lower level than IDEC-131 (pigv).

Example 5

Mediated by CD40L-CD40 increasing regulation of molecules HLA-DR in B-CLL

To determine violated the path of signal transduction via CD40L-CD40, CLL cells from patients with CLL were cultured (5×105cells/ml) in the presence and without 5 g/ml CD40L at 37°C. After 48 hours and 144 hours was determined the expression of class II molecules, HLA-DR on the cell CD19+flow cytometry using standard methods. Briefly, cultured cells were collected at different time points and analyzed for surface expression of molecules, COI is lsua antibodies associated with either fluorescein (FITC)or phycoerythrin (PE) for single or double staining using flow cytometer FACScan (Becton-Dickinson). To dye for flow cytometry 1×106cells in test tubes for cultivation were incubated with the appropriate following antibodies: anti-CD45-FITC to skip the signal of lymphocyte population on a scatter plot; anti-CD19-PE(Phanningen, cat. No. 30655) or anti-CD20-FITC antibodies (Phanningen; cat. No. 33264)to determine b-cells CD19+and/or CD20+; anti-CD3-FITC antibodies (Phanningen; cat. No. 30104)to drop the signal T-cells; anti-CD19-RPE anti-HLA-DR-FITC antibodies (Phanningen; cat. No. 32384)to determine the expression of class II molecules on cells CD19+. Cells were once washed by centrifugation (200×g for 6 min), 2 ml of cold PBS, and incubated with the antibody for 30 min on ice, after which cells were washed once, fixed in 0.5% paraformaldehyde and stored at 4°C until analysis. Data flow cytometry was received on FACsan and analyzed using a computer program WinList software (Verity Software House). The instrument was configured to autoproducido signal to provide a survey of the quadrants containing cells that were subjected to a single colour or RPE, or FITC, not stained or tugged double staining. Figure 4 p is shown the comparison of the expression of HLA-DR cells CD19 +-CLL, cultivated with sCD40L, and in those cells that were not cultured with sCD40L. Revealed a high level of expression of HLA-DR on B cells-CLL, cultured in the presence of sCD40L (table 6).

Table 6
Mediated by CD40L-CD40 increasing regulation of molecules HLA-DR in B-CLL
SampleTimeHLA-DR+(a)
% positiveMFI
Control48 hours8192
144 hour881655
Cells + sCD40L48 hours88101
144 hour952943
(a)B-cells CD19+that are positive in relation molecules HLA-DR, and the average intensity of fluorescence (MIF).

Example 6

Processing IDEC-131 and RITUXAN®

For the treatment of malignant tumors CD404the subject of intravenous (IV) impose IDEC-131 at a concentration of about 10 to 50 mg/ml in buffer of composition: 10 mm Na-citrate, 150 mm Nal, of 0.02% Polysorbate 80, at pH 6.5. IDEC-131 is administered before, after or together with RITUXAN®. The dose RITUXAN® are within the prima is but from 3 to 10 mg/kg weight of a subject.

Example 7

Processing IDEC-131 and RITUXAN®

For the treatment of malignant tumors CD40+sensitive CHOP (for example, Hodgkin's disease, non-Hodgkin lymphoma and chronic lymphocytic leukemia, as well as rescue therapy in malignant tumors, in which cells are cells CD40+), IDEC-131 is administered by infusion in doses of prisoners from about 3 to 10 mg per kg of body weight of the patient immediately before the CHOP cycle. Introduction IDEC-131 will be repeated before each cycle of CHOP just for 4 to 8 cycles.

Example 8

The introduction of anti-CD40L in combination with RITUXAN®for the treatment of b-cell limfoma the subject

Combination therapy is particularly useful as salvage therapy or for treatment of recurrent or aggressive forms of malignant tumors CD40+(for example, Hodgkin's disease, non-Hodgkin's lymphoma and CLL). When IDEC-131 is administered in combination with CHOP and RITUXAN®, IDEC-131 is administered as discussed above in example 6, then the scheme-specific introduction of CHOP-IDEC-131, described in example 7.

All discussed above references are therefore incorporated by reference in full.

1. A method of treating malignant tumors CD40+that includes the introduction of a therapeutically effective amount of the antibody or antibody fragment, catalystbed with CD40L and which inhibits interaction of CD40/CD40L or transmission of the signal through CD40.

2. The method according to claim 1, where the malignant tumor CD40+is b-cell lymphoma or b-cell leukemia.

3. The method according to claim 2, where b-cell lymphoma is a Hodgkin's disease (HD) or nahodkinskuju lymphoma (NHL).

4. The method according to claim 3, where the NHL is the NHL low-grade, intermediate grade or high grade.

5. The method according to claim 3, where the NHL is selected from the group subtypes, consisting of small cell lymphocytic, follicular predominantly small cells with cleaved nuclei, follicular from a mixture of small cells with cleaved nuclei and large cell, follicular and preferably both, diffuse small cells with cleaved nuclei, diffuse from a mixture of small and large cell, diffuse both, both immunoblastic, lymphoblastic, small cells with unsplit cores type Burkitt and non-Burkitt's lymphoma, lymphoma, AIDS-related angioimmunoblastic lymphadenopathy, lymphoma mantle cell and monocytoid b-cell lymphoma.

6. The method according to claim 2, where b-cell leukemia is a chronic b-cell leukemia, acute lymphoblastic leukemia b-cell lines or chronic lymphocytic leukemia b-cell line.

7. The method according to claim 2, where the antibody or antibody fragment that binds to CD40L, is IDEC-131, 3E, N, N, 4D9-8 AND 4D9-9, 24-31, 24-43, 89-76 or 89-79.

8. The method according to claim 7, where the antibody or antibody fragment is chimeric, bispecific, human or humanized.

9. The method according to claim 2, where the antibody fragment is Fab, Fab', scFv, or F(ab')2.

10. The method according to claim 2, additionally comprising introducing a therapeutically effective amount of a second antibody or its fragment, where the second antibody is an anti-D20-antibody or anti-CD40-antibody, chemotherapeutic agents, combination chemotherapy and/or radiation therapy.

11. The method according to claim 10, where radiation therapy is an external treatment with radiation or radioactively labeled antibody.

12. The method according to claim 11, where radioactively labeled antibody is radioactively labeled antibody IDEC-131, rituximab or B1, or fragments thereof.

13. The method according to item 12, where radioactively labeled antibody radioactively mark123I125I131I111In131In32R64Cu67Cu211At,177Lu,90Y186Re,212Pb212Bi47Sc,105Rh,109Pd153Sm188Re,199Au,211At and213Bi.

14. The method according to claim 10, where the chemotherapeutic treatment for BH is any one or more of the following: an alkylating agent, a Vinca alkaloid, procarbazine, who ethotrexate or prednisone.

15. The method according to claim 10, where the chemotherapeutic agent for the treatment of NHL is any one or more of the following: an alkylating agent, cyclophosphamide, chlorambucil, 2-CDA, 2'-deoxycoformycin, fludarabine, citizenoriented, cisplatin, etoposide or ifosfamide.

16. The method according to claim 10, where the combination of chemotherapeutic agents for the treatment of BH is MORR, ABVD, ChlVPP, CABS, MOPP plus ABVD, MORR plus ABV, BCVPP, VABCD, ABDIC, CBVD, PCVP, SER, EVA, MOPLACE, MIME, MINE, SEM, MTX-CHOP, EVAP or EPOCH.

17. The method according to claim 10, where the combination of chemotherapeutic agents for the treatment of NHL is a CVP, CHOP, C-MOPP, CAP-BOP, m-BACOD, ProMACE-MOPP, ProMACE-CytaBOM, MACOP-B, IMVP-16, MIME, DHAP, ESHAP, CEPP(B) or CAMP.

18. The method according to claim 10, where the chemotherapeutic agent for the treatment of b-cell leukemia is any one or more of the following tools: anthracycline, cyclophosphamide, L-asparaginase and purine analog.

19. The method according to claim 10, where the combination of chemotherapeutic agents for the treatment of b-cell leukemia is a vincristine, prednisone, anthracycline and cyclophosphamide or asparginase; vincristine, prednisone, anthracycline, cyclophosphamide and asparginase; CHOP; CMP; CVP; COP or CAP.

20. The method according to claim 10, where the second antibody is an anti-D20-antibody.

21. The method according to claim 20, where anti-D20-antibody is rituximab or its fragment, or B1 or fragment.

22. The method of treatment is a malignant tumor CD40 +that includes the stage of introduction of anti-D40L-antibody or its fragment, which blocks the interaction of CD40-CD40L or inhibits the transmission of the signal through CD40; and the introduction of anti-D20-antibody or its fragment.

23. The method according to item 22, where the malignant tumor CD40+is b-cell lymphoma or b-cell leukemia.

24. Composition for treatment of malignant tumors CD40+including CD40L antagonist and at least one of the following components: (a) a chemotherapeutic agent or combination of chemotherapeutic agents, (b) radioactively labeled antibody, (C) anti-D20-antibody or its fragment, and (d) anti-CD40-antibody or its fragment.

25. The composition according to paragraph 24, where radioactively labeled antibody radioactively mark123I125I131I111In131In32R64Cu67Cu211At,177Lu,90Y186Re,212Pb212Bi47Sc,105Rh,109Pd153Sm188Re,199Au,211At and213Bi.

26. The composition according to paragraph 24, where a malignant tumor CD40+is a b-cell leukemia or b-cell lymphoma.

27. The composition according to p, where b-cell lymphoma is a BH or NHL.

28. The composition according to item 27, where the NHL is the NHL low-grade, intermediate grade or high grade.

29. To notice on item 27, where the NHL is selected from the group consisting of the following subtypes: small cell lymphocytic, follicular predominantly small cells with cleaved nuclei, follicular from a mixture of small cells with cleaved nuclei and large cell, follicular and preferably both, diffuse small cells with cleaved nuclei, diffuse from a mixture of small and large cell, diffuse both, both immunoblastic, lymphoblastic, small cells with unsplit cores type Burkitt and non-Burkitt's lymphoma, lymphoma associated with AIDS angioimmunoblastic lymphadenopathy, lymphoma mantle cell and monocytoid b-cell lymphoma.

30. The composition according to p, where b-cell leukemia is a chronic b-cell leukemia, acute lymphoblastic leukemia b-cell lines or chronic lymphocytic leukemia b-cell line.

31. The composition according to paragraph 24, where CD40L antagonist is an anti-D40L-antibody or its fragment.

32. The composition according to p, where anti-D40L-antibody is IDEC-131 or its fragment.

33. The composition according to p, where the fragment of the anti-D40L is a Fab, Fab', scFv, or F(ab')2.

34. The composition according to paragraph 24, where anti-D20-antibody is rituximab or its fragment, or B1 or fragment.

35. The composition according to item 27, where the chemotherapeutic treatment for BH what is-is for one or more of the following: an alkylating agent, the Vinca alkaloid, procarbazine, methotrexate or prednisone.

36. The composition according to item 27, where the chemotherapeutic agent for the treatment of NHL is any one or more of the following: an alkylating agent, cyclophosphamide, chlorambucil, 2-CDA, 2'-deoxycoformycin, fludarabine, citizenoriented, cisplatin, Etoposid or ifosfamide.

37. The composition according to item 27, where the combination of chemotherapeutic agents for the treatment of BH is MORR, ABVD, ChlVPP, CABS, MOPP plus ABVD, MORR plus ABV, BCVPP, VABCD, ABDIC, CBVD, PCVP, SER, EVA, MOPLACE, MIME, MINE, SEM, MTX-CHOP, EVAP or EPOCH.

38. The composition according to p, where the combination of chemotherapeutic agents for the treatment of NHL is a CVP, CHOP, C-MOPP, CAP-BOP, m-BACOD, ProMACE-MOPP, ProMACE-CytaBOM, MACOP-B, IMVP-16, MIME, DHAP, ESHAP, CEPP(B) or CAMP.

39. The composition according to p, where chemotherapeutic agent for the treatment of b-cell leukemia is anthracycline, cyclophosphamide, L-asparaginase, a purine analog.

40. The composition according to item 27, where the combination of chemotherapeutic agents for the treatment of b-cell leukemia is a vincristine, prednisone, anthracycline and cyclophosphamide or asparginase; vincristine, prednisone, anthracycline, cyclophosphamide and asparginase; CHOP; CMP; CVP; COP or CAP.

41. Composition for treatment of malignant tumors CD40+containing (i) anti-D40L-antibody or fragment indicated antibodies, prepared for primeneniyu combination with any one or more of the following components: (ii) radioactively labeled antibody which binds CD40L or CD20, (iii) anti-D20-antibody or its fragment, or (iv) a chemotherapeutic agent or combination of chemotherapeutic agents.

42. Composition for treatment of malignant tumors CD40+in paragraph 41, where a malignancy is a b-cell lymphoma or b-cell leukemia.

43. The composition according to § 42, where b-cell lymphoma is a Hodgkin's disease or NHL.

44. The composition according to paragraph 41, where radioactively labeled antibody is radioactively labeled antibody IDEC-131, RITUXAN®or B1.

45. The composition according to item 44, where radioactively labeled antibody radioactively mark123I125I131I115In131In32P,64Cu67Cu211At,177Lu,90Y186Re,212Pb212Bi47Sc,105Rh,109Pd153Sm188Re,199Au,211At and213Bi.

46. The composition according to item 43, where the NHL is the NHL low-grade, intermediate grade or high grade.

47. The composition according to item 43, where the NHL is selected from the group consisting of the following NHL subtypes: small cell lymphocytic, follicular predominantly small cells with cleaved nuclei, follicular from a mixture of small cells with cleaved nuclei and large cell, follicular and preferably both, diffuse from Elka cells with cleaved nuclei, diffuse from a mixture of small and large cell, diffuse both, both immunoblastic, lymphoblastic, small cells with unsplit cores type Burkitt and non-Burkitt's lymphoma, lymphoma associated with AIDS angioimmunoblastic lymphadenopathy, lymphoma mantle cell and monocytoid b-cell lymphoma.

48. The composition according to paragraph 41, where anti-D40L-antibody is IDEC-131 or its fragment.

49. The composition according to paragraph 41, where anti-D20-antibody is rituximab or its fragment, or B1 or fragment.

50. The composition according to § 42, where the chemotherapeutic treatment for BH is any one or more of the following: an alkylating agent, a Vinca alkaloid, procarbazine, methotrexate or prednisone.

51. The composition according to item 43, where the chemotherapeutic agent for the treatment of NHL is any one or more of the following: an alkylating agent, cyclophosphamide, chlorambucil, 2-CDA, 2'-deoxycoformycin, fludarabine, citizenoriented, cisplatin, etoposide or ifosfamide.

52. The composition according to item 43, where the combination of chemotherapeutic agents for the treatment of BH is MORR, ABVD, ChlVPP, CABS, MOPP plus ABVD, MORR plus ABV, BCVPP, VABCD, ABDIC, CBVD, PCVP, SER, EVA, MOPLACE, MIME, MINE, SEM, MTX-CHOP, EVAP or EPOCH.

53. The composition according to item 43, where the combination of chemotherapeutic agents for the treatment of NHL is a CVP, CHO, C-MOPP, CAP-BOP, m-BACOD, ProMACE-MORR, ProMACE-CytaBOM, MACOP-B, IMVP-16, MIME, DHAP, ESHAP, CEPP(B) or CAMP.

54. The composition according to § 42, where the chemotherapeutic agent for the treatment of b-cell leukemia is anthracycline, cyclophosphamide, L-asparaginase, a purine analog.

55. The composition according to § 42, where the combination of chemotherapeutic agents for the treatment of b-cell leukemia is a vincristine, prednisone, anthracycline and cyclophosphamide or asparginase; vincristine, prednisone, anthracycline, cyclophosphamide and asparginase; CHOP; CMP; CVP; COP or CAP.

56. A method of treating malignant tumors CD40+that includes the introduction of CD40L antagonist and at least one of the following effects: (a) the introduction of chemotherapeutic agents or combinations of chemotherapeutic agents, (b) radiation therapy (C) the introduction of anti-D20-antibody or its fragment, and (d) the introduction of anti-CD40 antibody or its fragment.

57. The method according to p where radiation therapy is an external radiation treatment or handling of radioactively labeled antibody.

58. The method according to § 57, where radioactively labeled antibody radioactively mark123I125I131I111In131In32P,64Cu67Cu211At,177Lu,90Y186Re,212Pb212Bi47Sc,105Rh,109Pd153Sm188Re,199Au,211At and213Bi.

59. The method according to p, where a malignant tumor CD40+is a b-cell leukemia or b-cell lymphoma.

60. The method according to p, where b-cell lymphoma is a BH or NHL.

61. The method according to p, where the NHL is the NHL low-grade, intermediate grade or high grade.

62. The method according to p, where the NHL is selected from the group consisting of the following subtypes: small cell lymphocytic, follicular predominantly small cells with cleaved nuclei, follicular from a mixture of small cells with cleaved nuclei and large cell, follicular and preferably both, diffuse small cells with cleaved nuclei, diffuse from a mixture of small and large cell, diffuse both, both immunoblastic, lymphoblastic, small cells with unsplit cores type Burkitt and non-Burkitt's lymphoma, lymphoma associated with AIDS angioimmunoblastic lymphadenopathy, lymphoma mantle cell and monocytoid b-cell lymphoma.

63. The method according to p, where b-cell leukemia is a chronic b-cell leukemia, acute lymphoblastic leukemia b-cell lines or chronic lymphocytic leukemia b-cell line.

64. The method according to p where CD40L antagonist is an anti-D40L-antibody or its fragment.

65. The method according to p, where anti-D40L-anti-Christ. elom is IDEC-131 or its fragment.

66. The method according to p, where a fragment of an anti-CD40L is a Fab, Fab', scFv, or F(ab')2.

67. The method according to p, where anti-D20-antibody is rituximab or its fragment or B1 or fragment.

68. The method according to p, where chemotherapeutic treatment for BH is any one or more of the following: an alkylating agent, a Vinca alkaloid, procarbazine, methotrexate or prednisone.

69. The method according to p, where chemotherapeutic agent for the treatment of NHL is any one or more of the following: an alkylating agent, cyclophosphamide, chlorambucil, 2-CDA, 2'-deoxycoformycin, fludarabine, citizenoriented, cisplatin, etoposide or ifosfamide.

70. The method according to p, where the combination of chemotherapeutic agents for the treatment of BH is MORR, ABVD, ChlVPP, CABS, MOPP plus ABVD, MORR plus ABV, BCVPP, VABCD, ABDIC, CBVD, PCVP, SER, EVA, MOPLACE, MIME, MINE, SEM, MTX-CHOP, EVAP or EPOCH.

71. The method according to p, where the combination of chemotherapeutic agents for the treatment of NHL is a CVP, CHOP, C-MOPP, CAP-BOP, m-BACOD, ProMACE-MOPP, ProMACE-CytaBOM, MACOP-B, IMVP-16, MIME, DHAP, ESHAP, CEPP(B) or CAMP.

72. The method according to p, where chemotherapeutic agent for the treatment of b-cell leukemia is anthracycline, cyclophosphamide, L-asparaginase and purine analog.

73. The method according to p, where the combination of chemotherapeutic agents for the treatment of b-cell leukemia represents incretin, prednisone, anthracycline and cyclophosphamide or asparginase; vincristine, prednisone, anthracycline, cyclophosphamide and asparginase; CHOP; CMP; CVP; COP or CAP.

74. The use of anti-D40L-antibody or its fragment for the manufacture of a medicinal product for use in the treatment of malignant tumors CD40+in combination (i) with radioactively labeled antibody that binds CD40L or CD20, (ii) with anti-D40L-antibody or its fragment, or (iii) a chemotherapeutic agent or combination of chemotherapeutic agents.

75. Use p, where malignant tumor CD40+is b-cell lymphoma or b-cell leukemia.

76. The application of item 75, where b-cell lymphoma is a Hodgkin's disease (HD) or nahodkinskuju lymphoma (NHL).

77. Use p where radioactively labeled antibody is radioactive labeled antibody IDEC-131, rituximab or B1.

78. Use p where radioactively labeled antibody radioactively mark123I125I131I115In131In32P,64Cu67Cu211At,177Lu,90Y186Re,212Pb212Bi47Sc,105Rh,109Pd153Sm188Re,199Au,211At and213Bi.

79. Use p, where the NHL is low-grade, intermediate-grade or high-grade malignant the property.

80. Use p, where the NHL is selected from the group consisting of the following subtypes: small cell lymphocytic, follicular predominantly small cells with cleaved nuclei, follicular from a mixture of small cells with cleaved nuclei and large cell, follicular and preferably both, diffuse small cells with cleaved nuclei, diffuse from a mixture of small and large cell, diffuse both, both immunoblastic, lymphoblastic, small cells with unsplit cores type Burkitt and non-Burkitt's lymphoma, lymphoma associated with AIDS angioimmunoblastic lymphadenopathy, lymphoma mantle cell and monocytoid b-cell lymphoma.

81. Use p, where anti-D40L-antibody is IDEC-131 or its fragment.

82. Use p, where anti-D20-antibody is rituximab or its fragment, or B1 or fragment.

83. Use p, where chemotherapeutic treatment for BH is any one or more of the following: an alkylating agent, a Vinca alkaloid, procarbazine, methotrexate or prednisone.

84. Use p, where chemotherapeutic agent for the treatment of NHL is any one or more of the following: an alkylating agent, cyclophosphamide, chlorambucil, 2-CDA, 2'-deoxycoformycin, fludarabine, C is tainabilit, cisplatin, etoposide or ifosfamide.

85. Use p, where the combination of chemotherapeutic agents for the treatment of BH is MORR, ABVD, ChlVPP, CABS, MOPP plus ABVD, MORR plus ABV, BCVPP, VABCD, ABDIC, CBVD, PCVP, SER, EVA, MOPLACE, MIME, MINE, SEM, MTX-CHOP, EVAP or EPOCH.

86. Use p, where the combination of chemotherapeutic agents for the treatment of NHL is a CVP, CHOP, C-MOPP, CAP-BOP, m-BACOD, ProMACE-MOPP, ProMACE-CytaBOM, MACOP-B, IMVP-16, MIME, DHAP, ESHAP, CEPP(B) or CAMP.

87. The application of item 75, where the chemotherapeutic agent for the treatment of b-cell leukemia is anthracycline, cyclophosphamide, L-asparaginase and purine analog.

88. The application of item 75, where the combination of chemotherapeutic agents for the treatment of b-cell leukemia is a vincristine, prednisone, anthracycline and cyclophosphamide or asparginase; vincristine, prednisone, anthracycline, cyclophosphamide and asparginase; CHOP; CMP; CVP; COP or CAP.



 

Same patents:

FIELD: organic chemistry, chemical technology, medicine, pharmacy.

SUBSTANCE: invention relates to compounds of the formula (I)

or their pharmaceutically acceptable salts or esters hydrolyzing in vivo and possessing activity inhibiting the cellular cycle and selective with respect to CDK-2, CDK-4 and CDK-6. Compounds can be used in cancer treatment. In the formula (I) R1 represents halogen atom, amino-group, (C1-C)-alkyl, (C1-C6)-alkoxy-group; p = 0-4 wherein values R1 can be similar or different; R2 represents sulfamoyl or group Ra-Rb-; q = 0-2 wherein values R2 can be similar or different and wherein p + q = 0-5; R3 represents halogen atom or cyano-group; n = 0-2 wherein values R3 can be similar or different; R4 represents hydrogen atom, (C1-C6)-alkyl, (C2-C6)-alkenyl, (C2-C6)-alkynyl, (C3-C8)-cycloalkyl, phenyl or heterocyclic group bound with carbon atom wherein R4 can be optionally substituted at carbon atom with one or some groups Rd; R5 and R6 are chosen independently from hydrogen, halogen atom, (C1-C)-alkyl, (C2-C6)-alkenyl or (C3-C8)-cycloalkyl wherein R5 and R6 can be substituted at carbon atom independently of one another with one or some groups Re; Ra is chosen from (C1-C6)-alkyl, (C2-C6)-alkenyl, (C2-C6)-alkynyl, (C3-C8)-cycloalkyl, (C3-C8)-cycloalkyl-(C1-C6)-alkyl, phenyl, heterocyclic group, phenyl-(C1-C)-alkyl or (heterocyclic group)-(C1-C6)-alkyl wherein Ra can be substituted optionally at carbon atom with one or some groups Rg and wherein if indicated heterocyclic group comprises residue -NH- then its nitrogen atom can be optionally substituted with group chosen from the group Rh; Rb represents -N(Rm)C(O)-, -C(O)N(Rm)-, -S(O)r-, -OC(O)N(Rm)SO2-, -SO2N(Rm)- or -N(Rm)SO2- wherein Rm represents hydrogen atom or (C1-C6)-alkyl, and r = 1-2. Also, invention relates to methods for synthesis of these compounds, a pharmaceutical composition, method for inhibition and using these compounds.

EFFECT: improved preparing method, valuable medicinal properties of compounds and pharmaceutical compositions.

24 cl, 3 sch, 166 ex

FIELD: organic chemistry, vitamins, medicine, pharmacy.

SUBSTANCE: invention relates to a new compound of the formula (I): wherein X means hydrogen atom or hydroxy group; R1 and R2 that can be similar or different mean hydrogen atom, (C1-C4)-alkyl; R3 means hydrogen atom, methyl group, fluorine or chlorine atom. Also, invention relates to its esters able to hydrolysis in vivo in combination with pharmaceutically acceptable acids. Also, invention relates to a pharmaceutical composition eliciting the inhibitory activity with respect to proliferation and promoting differentiation of cells and comprising the effective dose of compound of the formula (I) in common with pharmaceutically acceptable carriers and/or excipients. Also, invention relates to applying compound of the formula (I) for preparing a medicine used in treatment and prophylaxis of disease characterizing by abnormal differentiation of cells and/or proliferation of cells.

EFFECT: valuable medicinal properties of compounds.

13 cl, 3 sch, 3 tbl, 6 ex

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to new inhibitors of farnesyltransferase of the formula (I):

wherein R1 means hydrogen atom (H), group of the formula R5C(O)- wherein R5 means phenyl, pyridyl or N-methylpiperidine; R2 means hydrogen atom (H), isopropyl, cyclopentyl or N-methyltetrahydropyridyl; R3 means hydrogen atom (H), halogen atom; R4 means hydrogen atom (H), halogen atom; L means -CH2-Z- wherein Z means NH; Y means sulfur atom (S), S(O) or S(O)2; or its salt. Compounds of the formula (I) inhibit activity of enzyme, farnesyl(protein)transferase, that allows their using in pharmaceutical composition in cancer treatment.

EFFECT: valuable medicinal properties of inhibitors.

18 cl, 3 tbl, 3 sch, 6 ex

FIELD: medicine, oncohematology.

SUBSTANCE: the present innovation deals with treating elderly patients with chronic lympholeukosis accompanied with cardiovascular failure. The method deals with applying chemopreparations and cytoprotector. Moreover, 1 wk before the onset of chemotherapeutic therapy one should prescribe preductal at the dosage of 105 mg daily. At this background one should sample blood out of elbow vein at the volume of 200 ml into a vial with glugicir to centrifuge it, isolate plasma, divide into two portions, add into the 1st vial - cyclophosphan 600-800 mg/sq. m, vincristin 1.4 mg/sq. m, into the 2nd vial - adriamycin 50 mg/sq. m to be incubated for 30 min at 37 C and intravenously injected by drops for patients. Simultaneously, the intake of prednisolone should be prescribed at the dosage of 60 mg/sq. m since the 1st d and during the next 5 d and preductal at the dosage of 105 mg daily during a week, and then 2 wk more at the dosage of 60 mg daily. All the procedures should be repeated in above-mentioned sequence 4-6 times. The method enables to decrease toxic manifestations of chemotherapy while applying adequate dosages of cytostatics, anthracycline antibiotics, among them, at no great manifestations of their toxicity due to preductal's cardioprotective action.

EFFECT: higher efficiency of therapy.

1 ex, 5 tbl

FIELD: medicine, oncology.

SUBSTANCE: invention relates to a method for treatment of chronic lympholeukosis. Method involves intravenous drop and jet administration of antitumor chemopreparations and carrying out the autochemotherapy. At the 1-st and 8-th day of treatment cyclophosphan in the dose 750 mg/m2, vincristine in the dose 1.4 mg/m2 and doxorubicin in the dose 30 mg/m2 incubated with 200 ml of autoblood are administrated to patients. From the 1-st to 14-th day of treatment prednisolone is used every day in the therapeutic dose. The treatment course is repeated in 30-35 days depending on blood indices and patient state. The total treatment of courses is 4-5. Method provides reducing cardiotoxicity of doxorubicin and cumulative toxicity of chemopreparations that allows carrying out administration of antitumor chemopreparations in the full volume to patients of elderly age groups.

EFFECT: improved method for treatment.

1 ex

The invention relates to derivatives of 5-imino-13-deoxy of anthracycline formula

where R1, R2and R3are H, HE or och3group and R4represents the following groups:

The proposed pharmaceutical composition having antitumor activity, containing at least one derivative of 5-imino-13-deoxy of anthracycline

The invention relates to veterinary

The invention relates to medicine and relates to antibodies with reduced total positive charge

The invention relates to medicine, in particular to Oncology and immunology, and for the treatment of b-cell lymphoma

The invention relates to medicine, specifically to the production of anti-allergic drugs on the basis of human immunoglobulin and biologically active substances

FIELD: immunology, medicine.

SUBSTANCE: claimed recombinant antibody (Ab) has at least constant regions in heavy and light chains representing human Ab regions. Said At inhibits bonding of integrine recognizing RGD and SVVYGLR sequences to integrine or fragment thereof. Also disclosed are nucleotide sequences (NS) encoding heavy and light chains of recombinant Ab as well as expression vectors containing respective NS. Described are host cell for Ab production, transformed with two vectors for expression of Ab heavy and light chains and method for abovementioned host cell application to produce recombinant Ab. Ab of present invention is useful in diagnosis and treatment of autoimmune diseases, rheumatism and rheumatoid arthritis.

EFFECT: therapeutic methods of increased efficiency.

45 cl, 14 tbl, 28 ex

FIELD: immunobiotechnology.

SUBSTANCE: method for isolating antibodies involves a procedure of adsorption chromatography using water-insoluble uncharged adsorbent (variant 1), or a procedure for desalting antibodies from egg yolk aqueous extract resulting to specific precipitation of IgY antibodies (variant 2), or using a successive procedure of adsorption chromatography (by variant 1) and desalting procedure (variant 2) in combination (variant 3). Invention provides carrying out the selective purification of antibodies IgY (▵Fc) and IgY from lipids under conditions of their increased content in egg yolk of Anseres order birds. Invention can be used for quantitative and qualitative analysis or preparing pharmaceutical compositions directed to etiological factor representing the interest.

EFFECT: improved preparing method.

23 cl, 4 dwg, 2 tbl, 6 ex

FIELD: medicine, biopharmacology, immunology.

SUBSTANCE: invention relates to development of agent possessing immunoregulatory property, in particular, ability to induce suppressive activity of mononuclear cells and secretion of cytokines by these cells. Proposed agent comprises trophoblastic β-1-glycoprotein and immunoglobulin (Ig) of G, A or M class. According to the second invention the agent comprising trophoblastic β-1-glycoprotein and Ig is used in treatment of autoimmune diseases and this agent is administrated by parenteral route. Invention provides the accelerated clinical effect in the autoimmune disease exacerbation stage.

EFFECT: valuable medicinal properties of agent.

9 cl, 30 tbl, 38 ex

FIELD: immunology, biotechnology.

SUBSTANCE: invention describes murine antibody and its humanized variant (CDP870) showing specificity to human tumor necrosis factor-alpha. Amino acid sequence is given in the description. Also, invention describes compounds showing affinity with respect to human tumor necrosis factor-alpha based on humanized antibody wherein lysylmaleimide group bound covalently with one or some methoxypoly(ethylene glycol) molecules by lysyl residue is joined to one of cysteine residues by C-end of heavy chain of the humanized antibody. Invention discloses DNA sequences encoding antibodies showing specificity to human tumor necrosis factor-alpha and variants if expression vectors involving indicated DNAs. Also, invention describes variants of a method for preparing a host-cell using expression vectors and variants of a method for preparing antibodies based on prepared host-cells. Invention discloses therapeutic compositions used in treatment of pathology mediated by tumor necrosis factor-alpha based on antibodies. Invention provides providing antibodies showing high affinity: 0.85 x 10-10 M for murine antibodies and 0.5 x 10-10 M for its humanized variant and low immunogenicity for human for humanized antibodies. Part of patients with improved ACR20 in administration of 5 and 20 mg/kg of CDP870 is 75% and 75% in 8 weeks, respectively. Half-life value of CDP870 in plasma is 14 days.

EFFECT: valuable biological and medicinal properties of antibodies.

58 cl, 24 dwg, 6 tbl, 1 ex

FIELD: medicine, immunology.

SUBSTANCE: invention relates to antibodies raised against myelin-associated glycoprotein (MAG). Invention involves humanized antibody or its functional fragment that bind with MAG (myelin-associated glycoprotein) and a pharmaceutical composition containing thereof, and to using such antibodies in treatment and/or prophylaxis of neurological diseases/disturbances being insult especially. The advantage of invention involves enhancement of the neuroprotective effect in acute phase of disease.

EFFECT: valuable medicinal properties of antibodies.

6 cl, 6 dwg, 4 ex

FIELD: medicine, immunology, pharmacy.

SUBSTANCE: invention relates to antibody, drug based on thereof used in treatment or diagnosis of infection caused by microorganism expressing of the protein GrfA homolog. Also, invention discloses a method for preparing a drug said and a pharmaceutical package used in treatment of the diseases said. The described antibody shows the established amino acid sequence represented as the sequence SEQ ID NO:2 given in the description, and antibody or its antigen-binding fragment that binds specifically with epitope allowing by peptide with amino acid sequence given in SEQ ID NO:3 and disclosed in the description. Using the invention provides enhancing the therapeutic effectiveness of a glycopeptide antibiotic as compared with using a single antibiotic.

EFFECT: enhanced effectiveness of infection.

22 cl, 1 dwg, 10 tbl

FIELD: biochemical engineering; medicine.

SUBSTANCE: method involves producing immunoglobulin preparation by concentrating III Kohn spirit method fractions by means of ultrafiltration under рН 3.5-5.0 condition, washing from ethanol and carrying out final solution concentration process. The subsequent restoration hydrate immunoglobulin envelopes is carried out by means of molecular filtration method by creating concentration gradient by introducing low-molecular substance in concentration of 0.1-2.5% by weight into immunoglobulin solution, with water of рН 3.8-4.5 being used, cleared from dialyzing solution. Then stabilizing agent is added and рН value is fixed at 3.5-5.0. Sterilizing filtration is carried out and the produced preparation is dried by subliming when needed at the final stage of refinement process. The so produced immunoglobulin preparation for making intravenous injections has solution transparency index less than 0.01, and contains monomer immunoglobulin IgG more than 97% under full absence of polymeric forms, proteins 4.5-11% and sodium chloride no more than 0.3%.

EFFECT: reduced risk of side reactions when administering the immunoglobulin preparation; mechanically aided and automated process of preparation production; prolonged storage period.

5 cl, 1 dwg, 2 tbl

FIELD: medicine, immunology, biotechnology.

SUBSTANCE: invention describes antibody raised against osteopontin that inhibits binding integrin and osteopontin or its fragment wherein integrin recognizes a sequence RGD or sequences SVVYGLR, SVAYGLR, SLAYGLR, SVAYGLK and SVAYRLK of osteopontin. Antibody is prepared in using peptide as an antigen comprising amino acid sequence RGDSVVYGLR or relates sequence RGDSVVYGLR. Also, invention proposes a medicinal agent for treatment of autoimmune disease and a method for treatment of autoimmune diseases based on indicated antibody wherein autoimmune disease is chosen from rheumatism, rheumatic arthritis and osteoarthritis. Invention describes using antibody for producing a medicinal preparation used in treatment of autoimmune disease chosen from rheumatism, rheumatic arthritis and osteoarthritis. Using the invention improved arthritic coefficient, delays development of arthritis and reduces legs edema in experimental arthritis in mice and can be used in medicine.

EFFECT: valuable medicinal properties of antibody.

25 cl, 16 dwg, 13 tbl, 20 ex

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