JP2000509015A - Use of antibodies against CD48 for the treatment of T and B cell lymphomas and leukemias - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for treating T-cell or B-cell lymphoma or leukemia. The method comprises administering to the subject an antibody of the isotype IgG that renders it CD48.

Description

DETAILED DESCRIPTION OF THE INVENTION               For the treatment of T cell and B cell lymphomas and leukemias Use of antibodies against CD48 Technical field   The present invention relates to a method for treating T cell and B cell lymphomas and leukemias. The method Uses an antibody that targets CD48. Background of the Invention   Over the past decade, significant progress has been made in leukemia and lymphoma chemotherapy. Was. High rates of remission have been achieved with combination chemotherapy and bone marrow transplantation (BMT) You. However, many of these remissions are not long-lasting and are diagnosed as hematological cancers. Most patients who are rejected eventually die from the disease. Therefore non-Hodgkin Long-term survival rates for patients with lymphoma and adult acute chronic leukemia are still low. Current, For acute lymphoblastic leukemia (ALL) and moderate or advanced non-Hodgkin's lymphoma Only 40% of patients achieve long-term survival.   At the same time as these advances in chemotherapy, Kohler and Milstein A unique report has been published describing the production of a monoclonal antibody (MoAb). M This is because oAbs can bind to antigens expressed on the surface of malignant hematopoietic cells. Can be used in serum therapy to specifically target and destroy such cells. There was an optimistic outlook to try. In addition, MoAb therapy is Providing an alveolar toxic mechanism could avoid tumor cell resistance And it was. However, the immunogenicity of rodent immunoglobulins, Modulation of antigen, nonspecific uptake of antigen by poor phagocytes, low binding of some antibodies Numerous major issues, such as the problem of affinity, antigens circulating in plasma in certain cases, etc. Obstacles have limited the potential of this method. About some of these issues However, even more difficult is that most MoAbs are Activates the human complement cascade or binds to cytotoxicity after binding to target surface antigen Kill cells by promoting antibody-dependent cellular cytotoxicity mediated by T lymphocytes Lack of ability. Lack of this cytolytic effector function in vitro How Intravenous injection of antibodies in patients with leukemia and lymphoma has generally poor results This is reflected in the fact that they have not been obtained (11).   CD48, a 47 kd glycophosphatidylinositol binding glycoprotein, With some features suggesting that it can be a good target for immunotherapy I have. CD48 is widely expressed on lymphoid malignancies but not on other tissues Not (2,3,4). Both normal and malignant T cells and B cells express CD48 However, most CD34 positive cells do not express CD48. CD48 is a T cell and B cell It also exists at a high level on the surface. Biological function of CD48 in humans still unclear Not sure. CD48 is a high affinity ligand for CD2 in mice However, it is a low affinity ligand for human CD2 (6, 7).   Anti-CD48 antibodies have been disclosed (3), one such antibody being used in anti-tumor therapy. (13). In this study, the highest IgM anti-CD48 antibody was tested. Up to 50 mg has been injected into four patients with chronic lymphocytic leukemia. However, temporary circulation A decrease in peripheral lymphocytes is only observed, and the underlying disease It had no effect on progress. These results indicate that anti-CD48 antibody is a strong antitumor It suggests no ulcer effect.   We now have the appropriate isotype for the CD48 antigen in animal models. Antibodies were found to have strong anti-tumor effects. This anti-tumor effect is Treatment of mice bearing tumor cells with an anti-CD48 antibody, It resulted in longer survival (healing) than those treated with the control antibody. Summary of the Invention   Accordingly, a first aspect of the present invention is directed to T-cell or B-cell lymphoma or leukemia. A method of treatment, wherein the method requires a class IgG antibody against CD48. Administration to the subject (patient) to be administered.   The IgG isotypes are mouse IgG2a, human IgG1, human IgG2 and It can be an isotype selected from rat IgG2b. The subject is a human In some cases, the preferred isotype is human IgG1 or IgG2. Target is Ma If it is a mouse, the preferred isotype is mouse IgG2a. The target is Is In some cases, the preferred isotype is rat IgG2b.   The term “antibody” as used herein has a binding domain with the required specificity. Any specific binding substance. That is, the term refers to natural or synthetic, Antibody fragments, including any polypeptide comprising an immunoglobulin binding domain; Derivatives, functional equivalents and homologs of antibodies are included. Therefore, another port A chimeric moiety containing an immunoglobulin domain or equivalent fused to a polypeptide Including children.   In a preferred embodiment, the antibody is a recently cloned and sequenced HuLy m-3 antigen (9).   We have developed a chimeric anti-CD48 in which the Fc portion has been replaced by a human Fc region. The antibodies were found to exhibit improved characteristics over the mouse anti-CD48 antibody. these Are characterized by higher effector effects, longer serum half-life, and more There is low immunogenicity etc.   Thus, in another preferred embodiment, the antibody against CD48 is chimeric (i.e., (Humanized) antibodies. In this chimeric antibody, at least a part of the Fc portion is a human Fc. It can be a mouse antibody replaced by the c portion.   The efficacy of the anti-CD48 antibody may be, for example, radioisotope, cytokine, protein toxin It can be understood that the conjugate can be enhanced by conjugation with an amino acid, an anticancer agent, etc. . Anticancer drugs include doxorubicin, cisplatin, taxol, interferon, Pseudomonas exotoxin A, fumagillin, AGM-1470, tamoxifen, Nitrosoureas such as ACNU, BCNU, CCNU and PCNU, diazicons, Decarbazine, Hydrea, Semustine, Matsulon, Teniposide, and Terrazan You can choose from   The method of the first aspect of the present invention provides a method for treating non-Hodgkin's lymphoma or lymphoid leukemia. Can be used for treatment.   One advantage of the present invention is that CD48 is only present in less than 5% of CD34-positive progenitor cells. It is in our discovery that it is expressed. That is, anti-CD48 antibody Targets papillary leukemia and lymphoma cells but retains most of the CD34 + progenitor cells To compensate for the lack of cell types. Detailed description of the invention   In order that the present invention can be more clearly understood, preferred embodiments thereof will be described with reference to the following examples. It will be described in the light of the above. The figures in the examples are as follows.   Figure 1: Internalization of anti-CD48 antibody HuLy-m3.   Figures 2 (a) and 2 (b): Survival after CD48 treatment on SCID / Raji (Kaplan Meier curve). For SCID mice, (a) 1x10⁶Raji cells or (b) 5 × 10^FourInject Raji cells Fired. A group of 5 mice received 200 ug mice on days 0, 2, and 4 after Raji cell injection Anti-CD48 antibodies, HuLym3 or isotype control antibodies were injected.   FIG. 2 (c): Effect of different anti-CD48 antibody doses. 1x10 for SCID mice^FiveRaji Cells were injected and a group of 5 mice were given 200 Tg on days 0, 2, and 4 after Raji cell injection. Isotype control, 200Tg or 20Tg IgG2a anti-CD48 antibody HuLym3 Fired.   FIG. 2 (d): Effect of different anti-CD48 antibodies. 5x10 for SCID mice^FiveRaji cells Injected, a group of 5 mice received 200 Tg of Ig on days 0, 2, and 4 after Raji cell injection. G2a isotype control, 200Tg IgG2a anti-CD48 antibody HuLym3 or 200Tg Of IgM anti-CD48 antibody.   Figure 3: Human PBMC effector cells using Raji cell line as target cells The ADCC updates of the chimeric HuLym3, mouse HuLym3 and rat Campath antibodies used Surname. Results shown are 1 ug / ml antibody concentration and 25: 1 effector to target ratio. Is about. Materials and methods Cell line   The HuLym3 hybridoma cell line is a Dr. Mauro S Sandrin, The Austin Research  A gift from the Institute, Melbourne (17), including 10% fetal bovine serum (FBS). Or RPMI16 containing 10% bovine IgG-free serum (Starrate), 2 mM glutamine Cultured at 37 ° C in a 37 ° C incubator in 40 ° C. Cell lines COS1, CHO- K1, Raji, Daudi, MOLT-4, U-937 and CCRF-CEM were obtained from ATCC . COS And CHO cells were incubated at 37 ° C, 5% in 1: 1 DMEM / F12 (CSL) containing 10% FBS. CO_TwoAnd cultured. Raji, Daudi, MOLT-4, U-937 and CCRF-CEM are R PMI 1640, 2 mM glutamine, 10% FCS at 37 ° C. and 5% CO_TwoAnd cultured. Production and purification of antibodies   Antibodies (IgG2a) were cultured in HuLy-M3 hybridoma-conditioned medium. Or from ascites produced by nude mice or BalbC / CBA hybrids Was purified using Protein A affinity chromatography (Pharmacia). Was. Antibody purity was confirmed by 10% SDS-PAGE, and activity was And confirmed by flow cytometry. Antibody for animal experiments Further purification by on-exchange chromatography and gel filtration. IgG2a eye Sotype control antibody (anti-human TSH) was obtained from Bioquest and repurified as described above. Was. Protein concentration was measured by absorbance at 280 nm (18). Rat campath -1 antibody was a gift from Bob Hale, MRC, Cambridge, UK. IgM The anti-CD48 antibody WM63 was obtained from Ken Bradstock, Department of Haematology, Westmead H Obtained from ospital. CD34 / CD48 expression by flow cytometric analysis of human mononuclear cells   Normal individuals or St. Vincent's Hospital, Sydney, Australia CCL and Human peripheral blood (10 ml from patients, 50 ml from normal individuals) was obtained from NHL patients, and To prevent coagulation and to collect lymphocytes and monocytes (PBMC) on a Ficoll gradient. Prepared. Viable cells were counted by trypan blue exclusion and 1 × 10 5 in PBS.⁶Fine Resuspended at a final concentration of cells / ml. For flow cytometry analysis, anti-CD34- FITC, anti-CD45-PerCP and biotinylated anti-CD48 or isotype pairs 5.0x10⁶Incubate with the cells for 30 minutes at 4 ° C and add 2 ml of wash medium (PBS / 1% BSA), then add streptavidin-PE and place at 4 ° C for 30 minutes Was. Wash cells twice, 1% sucrose and 0.5% paraformaldehyde in PBS Immobilize in 500 μl of fixation buffer containing PBS / 1% BSA was added and stored at 4 ° C. Coulte within 24 hours of sample preparation r Epi Fluorescence was measured with a cs flow cytometer. Measurement of the number of CD48 molecules per cell   Prepare human peripheral blood mononuclear cells as described above,⁶resuspended at a final concentration of / ml did. Use Simply Cellular Microbeads and follow the manufacturer's instructions to The number of binding sites was determined. Antibody internalization   Testing for internalization of the CD48 monoclonal antibody is performed by flow cytometry. It was performed by tree (19) and observation with a confocal microscope. Human peripheral blood mononuclear cells as described above And isolated. 5-10x10 for flow cytometry⁶Cells with excess anti CD48 or control antibody (10-20ug / 0.5-1.0x10⁶Cells) and phosphate buffered saline ( (PBS) for 60 minutes at 4 ° C. After washing, cells were washed in complete medium (RPMI / 10% FCS) and resuspend in 5% CO_TwoIncubate at 37 ° C for a certain period in an incubator. Incubated. After a suitable incubation time, the FITC-conjugated anti-mouse Alternatively, an anti-human IgG1 antibody was added and the mixture was kept at 4 ° C. for 30 minutes. Twice with cold PBS / 1% BSA After washing, cells were washed with 0.5% paraformaldehyde and 1% sucrose in PBS at room temperature for 1 hour. Fix for 5 minutes, resuspend in PBS / 1% BSA and analyze by flow cytometry. The analysis was performed.   For confocal microscopy, prepare human PBMC and test monoclonal antibody And at 4 ° C. and / or 37 ° C. as described above. After washing, suspend cells Duplicate turbidity and add 4% paraformaldehyde in PBS (cell surface staining) or Is 4% paraformaldehyde / 0.1% Triton X-100 (intracellular and surface staining) in PBS Color) for 30 minutes at room temperature. After washing with PBS / 1% BSA, the cells were Incubated with C-conjugated anti-mouse or anti-human IgG1 antibody for 30 minutes at 4 ° C. So After washing the cells with PBS, the pellet was washed with PPD (90% (v / v)) as an anti-fading agent. Lycerol, 1 mg / ml r-phenylenediamine in 10% (v / v) PBS, pH 8.0) Mix, place on glass slides and cover with circular coverslips. Ab Inter To investigate the narration, Salastro 2000 CLSM (Molecular Dynamics, S unnyval e, CA, USA), using a flat achromatic 60x / 1.40 NA oil immersion lens and Confocal laser scanning microscopy (CLSM) went. Excitation at 488 nm using a 50 mm fixed pinhole, 510 nm Light section through FITC-labeled cells with a liter and 510 nm barrier filter ( (Usually at 0.3 mm intervals). Image processing is Silicon Graphics Personal Iris 4D 35 Performed using a workstation. Cell viability is due to trypan blue exclusion Examined. Treatment of SCID mice with Raji lymphoma by HLM3   Six to eight week old female SCID mice (CB17) were transferred to the Animal Resources Center, West. Obtained commercially from ern Australia. Mice are free of specific pathogens (SPF ) Reared in C1 Lab maintained as equipment. RPMI 16 in 5 mice per group Raji cells in 40 (200 Tl) were injected intravenously on day 0. Then 200 Tl R Antibodies in PMI 1640 were injected i.v. on days 0, 2, and 4. Observe mice daily and weigh Was measured and sacrificed at the onset of hind limb paralysis. Blood and tissue for further analysis A sample was taken. Immediately analyze bone marrow collected from one femur and blood, The collected tissues were frozen in liquid nitrogen or fixed in formalin / PBS. Bone Marrow and blood analyzed by flow cytometry to determine human cell percentage did. Anti-mouse CD45-PE (PharMingen) and anti-human CD45-FITC (Becton Di mouse and human by flow cytometry as described above using Leukocytes were detected. Recovered tissue for histological staining and immunohistochemical analysis And sectioned. Histological staining was performed using hematoxylin and eosin staining. Was. For immunohistochemical analysis, block endogenous peroxidase. Sections are incubated with normal rabbit serum, then have anti-CD20 (Dako) Or HuLy-m3 for 2 hours at 37 ° C. After washing, cut the sections into rabbit anti-mouse Followed by Ig-HRP (Silenus) and counterstained with hematoxylin and Scott Blue Colored. After washing again, it was dehydrated and mounted in Eukitt. Cloning of HLM3 variable region gene   Isolate total RNA from HuLym3 hybridoma (19) and use Ig-Primer Kit (Navagen) Used for the synthesis of first-strand cDNA using their heavy or light chain 3'-primers. . For PCR amplification of the variable heavy and light chain regions, various 5'-primers (Ig-Prime Ki t, Novagen) for 35 cycles of PCR in separate tubes containing . The VH and VL PCR products were electroporated into the T-vector (20). And transformed into E. coli (DH5a). VH and VL PCR products For all of the above, use the T7 Sequencing kit (Pharmacia Biotech) DNA sequencing was performed for both word and reverse directions. About each PCR product Sequence at least two clones to minimize PCR errors Was. The amino acid sequences of the heavy and light chains are determined by the 477A Protein Sequencer & 120A Analyzer (Applied Biosystems) for N-terminal amino acid sequencing of another alkylated chain Decided. Construction of chimeric HLM3   Expression vectors HCMV-Crl and HCMV-Ck (21) were obtained from Mary Bendig (Medica l Donation from Research Council, Laboratory of Molecular Biology, UK) there were. Insert VH and VL inserts into vector by Hind III and Bam HI digestion Removed. Greg Winter (Medical Research Council; Laboratory of Molecular)  Biology, UK) from the M13-VkPCR1 vector to Hind III And digestion with Apa I yielded the leader sequence. VH and VL are re- Amplify to introduce a 5 'Apa LI site in both variable regions, a 3' Bam HI site and Bgl II sites were introduced into VH and VL, respectively. Hind III-Apa LI Leader sequence and Apa LI-Bam HI VH sequence or Apa LI-B The glIIVL sequence was ligated to the HindIII-BamHI digested HCM by ternary ligation. Ligated to V vector. Electroporation using Gene Pulser (BioRad) Ligation product (2.2 kv, 250 uF capacitance) into E. coli DH5a Changed. Expression in COS1 and CHO-K1 cells   Chimeric antibodies were expressed in COS and CHO-K1 cells. Gene Pulser device (27 DNA by electroporation using 0 Volt, 250 mF, BioRad). The cells were introduced into COSI and CHO-K1 cells. Transformants were cultured under G418 (4 mg / ml) selection. Using a sandwich ELISA specific for cultured and assembled human antibodies High yield clones were screened.   Protein A Sepharose CL-4B (Pharmac) eluted with 100 mM glycine (pH 3.0) ia) or POROS 50A affinity chromatography (Perfusion Chromat Purification of chimeric HLM3 from cell culture supernatant using For dialysis, dialysis was performed against PBS (pH 7.4). Stock solution concentration from 1mg / ml high. ELISA for expression of assembled chimeric antibodies   Use sandwich ELISA to measure the concentration of assembled human antibodies did. Goat anti-human IgG Fc-specific antibody (Jackson ImmunoResearch Laboratorie s, Inc., USA) on Nunc Maxisorb plates at 2 ug / ml at 4 ° C overnight. I let it. Anti-human κ light chain-tracking biotin (The Binding Site Ltd., England) (0.5ug / ml), then alkaline phosphatase in carbonate buffer Conjugated egg white-avidin (Jackson) (0.5 ug / ml) and 1 mg / ml phosphatase substrate (p-d Trophenyl phosphate disodium, Sigma) without washing between each step. Rolls were sequentially added to the plate. With ELISA Reader (Dynateck MR7000, Baxter) The absorbance at OD 405 nm was read. Human IgG (Jackson ImmunoResearch Labor atories, Inc., USA) were used as standards (0.25-1000 ng / ml). All antibodies Incubation was for at least 1 hour at 37 ° C. ADCC assay   ADC using Cytotoxicity Detection Kit (LDH) (Boehringer Mannheim) C was measured. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and Used as target cells. The human Raji cell line was used as a source of target cells. Campath1 (CDw52) monoclonal antibody (rat IgG2b) used as a positive control did. Mouse IgG2a (anti-TNP, Pharmagen) and human IgG1 (The Binding Si te) Antibodies were used as HuLym3 and chimeric HuLym3 isotype controls, respectively. P BMC was placed in assay medium (RPMI 1640 with 1% BSA) at 1 × 10⁷resuspend with lml It became cloudy. Raji cells are 1x10 in assay medium^Five/ ml final concentration, and Incubated for 30 minutes at room temperature with different concentrations (0.01-10 ug / ml). F Vector and Ab-labeled target antibody (1 × 10^Five) In various proportions at 37 ° C, 5% CO_Twoso Incubated for 4 hours. Released lactate dehydrogenase (LDH) activity Sex was measured using a cytotoxicity detection kit according to the manufacturer's instructions. Three times Calculate the average absorbance of performed experiments and correct for background (assay medium only) And substituted into the following equation.   Low control: target cells in assay medium   High control: target cells and 2% Triton X-100   E + T-Ab: target cells labeled with effector and Ab   E + T: effector and target cells only result Characterization of CD48 antigen Measurement of CD34 + / CD48 + cells   The percentage of CD34 + cells expressing CD48 was determined by comparing CD34, CD45 and CD4 Measured by trilabeled flow cytometry using an antibody to 8. CD34 + Cells were first selected and then examined for CD48 + / CD45 + cells. Two healthy donna -And two patients with CLL and NHL were examined for PBMC. Healthy individuals and CLL And less than 5% of CD34-positive cells in patients with NHL express CD48. Was. Approximately 4% (± 1%) levels of CD34 + cells where all of the samples examined are CD48 + Had. Number of anti-CD48 binding sites per cell   The number of anti-CD48 binding sites per cell is determined by Quantum Simply Cellular Microbeads And using flow cytometry. Healthy individuals (2) and CLL patients (2) Has a similar number of binding sites, with approximately 40,000 ± 10,000 binding sites per cell Was. B-lymphoma cell line Raji has higher levels than patients or normal individuals It expressed CD48 and had about 200,000 binding sites / cell. Internalization of CD48 monoclonal antibody   Antibody internalization involves the following steps: i) Loss of surface-labeled antibody And ii) the location of the labeled antibody using confocal microscopy (surface Or intracellular). Internals easily into cells OKT3, an anti-CD3 antibody that is known to be (22) The results are shown in FIG. FIG. 1 shows the labeling with OKT3 at 37 ° C. It shows a rapid loss of signal from the incubated cells, and after about 30 minutes It was half of the original value. In contrast, cells labeled with anti-CD48 antibody No signal loss was noted shortly after. Healthy individuals, CLL patients and leukemia cells All experiments performed on PBMCs isolated from the system gave similar results. Establishment of B cell lymphoma model in SCID mice   To investigate whether antibodies against CD48 can mediate antitumor effects in vivo Thus, a model of B cell lymphoma was established in SCID mice. Human Raji B All untreated SCID mice injected intravenously with a cell lymphoma cell line developed hind limb paralysis Awake. The onset of paralysis was reproducible and depended on the dose of cells injected. 1x10⁶ Or 5x10^FourSCID mice injected with 19 cells Or paralysis 34 ± 3 days later. Mice were sacrificed at the onset of paralysis. mouse Autopsy of various organs such as liver, kidney, ovary, spleen, lung, and lymph nodes Sporadic tumors were seen. Large neoplastic infiltrates often have distinct white It formed a knot nodule. Distinct nodules of tumor cells by immunohistochemical analysis Was confirmed. These cells are stimulated by antibodies specific for human CD20 and CD48. Stained positive. Bone marrow from femurs of sacrificed mice contains 20-60% human cells However, no human cells were detected in peripheral blood. Inhibition of tumor cell growth in SCID mice by anti-CD48 antibody   A group of 5 SCID mice injected with Raji cells on day 0 was challenged with 200 ug mouse anti- Treated on days 0, 2, and 4 with CD48 antibody or isotype control antibody. Result is These are shown in FIGS. 2 (a) and (b). 1x10⁶Mice injected with Raji cells anti-CD48 antibody Treatment increased the time to hind limb paralysis by 40%. 5x10^FourCells of cells Four of the five mice injected with the dose and treated with the same dose survived long term. Eye Those treated with the sotype control antibody developed hind limb paralysis about 32 days later. Anti-CD48 anti The effect of body dose on the survival of mice was also examined. FIG. 2c shows the antibody dose of 20 Tg Show that 60% of mice can extend the time to hind limb paralysis. Figure 2d shows the anti-tumor effect of anti-CD48 antibodies of different isotypes. IgM anti Body did not prolong mouse survival relative to control antibody, whereas IgG2a antibody was prominent It showed a survival effect. These results indicate that the mouse anti-CD48 mouse antibody HuLym3 It shows that an antitumor effect can be produced in vivo. Cloning of HLM3 variable region gene   For construction of mouse / human chimeric antibodies based on potent in vivo antitumor activity The variable region of the anti-CD48 antibody was cloned. Degenerate primers (Ig-Primer Kit, No (vagen, USA). These primers , Multiple PCR products were generated. Two differences about Vh Product and three individual products for Vl. Predicted amino acid distribution The correct Vl product was determined by comparing the sequence to the determined N-terminal amino acid sequence. And confirmed by searching the database. Vh products are based on Kabat database Source search, which indicated that one product contained a 50 bp deletion. Was. No amino acid sequence was obtained from the heavy chain, which is probably blocked at the N-terminus. It seems to have been done. Construction of chimeric HLM3 and expression in CHO cells   Chimeric antibody expression vector using anti-CD48 Vh and Vl gene segments -, HCMV.Vh and HCMV. Vl was constructed. Plus Kmid DNA To COS cells for stable expression and to CHOK1 cells for stable expression. Transfected. Chimera using sandwich ELISA after 418 selection The CHOK1 clone expressing the highest level of antibody was picked. Structure of chimeric antibody Construction was confirmed by SDS-PAGE and Western blot (23). Chimeric antibody Is a mouse cell line examined in various cell lines and 4PBMC samples. Has almost the same binding characteristics as mouse (23), and was examined by competitive ELISA (23). Have the same relative affinity as that of the other. ADCC of mHLM3 and cHLM3   The chimeric antibody is an ADCC using human PMBC as effector cells. Significant lysis could occur in the assay. 6: 1, 12: 1, 25: 1, 50: 1 and Antibody concentration range from 0.01 to 10 ug / ml in a ratio of 100: 1 effector to target cells Was tested. Rat Campath-1 antibody (IgG2b) was also used in some assays. Used. Chimeric HuLym3 is 2-6 times higher than mouse HuLym3 and 2-3 times higher than Campath1 A specific dissolution of efficiency occurred. Specific lysis levels in chimeric HuLym3 are 0.1-5 ug / ml The same is true for body concentration and the ratio of effector to target from 12: 1 to 50: 1, Chimeric HuLym3 usually produces specific lysis greater than 60%. Typical ADCC assay The results are shown in Table 1 and FIG. The maximum specific cell lysis observed has been described so far. (24) Dependent on the source of effector cells. Consideration   CD48 is the majority of lymphocytes, monocytes, and lymphocytic leukemia and lymphoma cells (2,3). CD48 for the treatment of lymphoma and leukemia Some properties of CD48 were further characterized in order to evaluate its targeting ability . CD34 represents an early marker in hematopoietic cell development (25). White Cures hematologic or lymphoma cells but not CD34 + cells It is considered to be advantageous as a therapeutic agent. We found that CD48 was less than 5% of CD34 + cells Antibodies to CD48 target most CD34 + cells Not shown. Therefore, if all CD48 + cells were removed by antibody treatment, Proliferating remaining CD34 + progenitor cells and reconstituting the number of their depleted cell types Will be able to.   Cross-linking with a bivalent antibody patches and caps the surface antigen and ultimately Causes turnover. The rate at which this process occurs depends on the antigen . Certain lymphoid differentiation antigens, such as CD3 and CD7, , But in others such as CD45 and CAMPATH-1 The writing process can take several hours. Anti-CD48 antibody at least 24 hours without adjustment Remains on the surface of the cell for a while. The stability of the anti-CD48 antibody on the cell surface Plays an important role in the observed cytotoxic effects mediated by this antibody It seems to be.   Expression level of CD48 on the surface of PBMC from healthy donors and CLL patient donors There does not appear to be a significant difference in the bells. Antibodies at approximately 40,000 sites / cell on both cell surfaces Was combined. CD48 is induced to high levels of expression on EBV-infected B cells (26). This is the fraction of the human Raji cell line that is EBV + and expresses about 200,000 sites / cell. It was confirmed by analysis.   IgM anti-CD48 antibodies have been used in phase I preliminary clinical trials (13). Four people CLL patients treated with up to 64 mg of antibody for 6 days and significantly but temporarily A significant decrease in the number of circulating leukocytes was observed. IgM anti-CD48 antibody is potent in vitro Was able to activate complement, which is a major cause of only a temporary loss of circulating tumor cells. The same as the IgM Campath antibody that was able to cause complement activation. Campath-1 Additional clinical trials with different isotypes of antibodies have shown that It has been suggested to be important for potent antitumor effects in o (14,15). mouse It has been shown that IgG2a and human IgG1 antibodies mediate ADCC and antitumor activity (27).   We show that anti-CD48 antibodies can mediate potent in vivo antitumor effects, ICD injected with Raji cells causes hind limb paralysis about 34 days after treatment with a control antibody It has been shown that mice can survive for long periods. The manifestation of the disease in this model is Other CID cells using human B cell lines (28), for example the Daudi cell line (29) Similar to the mouse model. However, the time to hind limb paralysis depends on the Raji cell line. Shorter than other models. For example, 1x10⁶IV Daudi cells SCID mice develop hind limb paralysis about 34 days later,⁶Injection of Raji cells The shot causes hind limb paralysis about 20 days later. The Raji cell line forms disease faster You. So we have the same cell dose of 1x10 as reported in other models.⁶ Administration of Raji cells and the untreated animals reported in the Daudi SCID model Low cell dose to allow mice to survive for approximately one month, with a survival time similar to that of Report the results of the quantity. At higher doses, treatment with anti-CD48 antibody Increased the time to hind limb paralysis by 40%. In lower cell dose experiments, anti-CD48 The majority of mice show long-term survival after 32 days of treatment with the antibody and untreated animals Hind limb paralysis occurred after the mice survived at least ten times longer. We have antibodies Is also active at much lower doses.   The therapeutic effect of anti-CD48 antibody in SCID mice was It appears that the isotype control antibody has no therapeutic effect and is therefore antigen-specific. You. The cytotoxic effect of anti-CD48 antibody is due to its inability to modulate antigen, mouse Several features such as effective interaction of mouse IgG2a by effector function Seems to depend. Since SICD mice lack significant T and B cell responses, Possible effector response is direct poisoning of monoclonal antibodies against tumor cells Or neutrophils, macrophages, or natural killer cells Assembling other cells having such cytotoxic activity. Anti-CD48 antibody Does not have direct cytotoxicity to Raji cells (unpublished results), The most likely hypothesis of the antitumor effect observed to aggregate effector cells It is clear.   We also want to produce antibodies in which the Fc portion has been replaced by a human Fc region. Some issues with using murine monoclonal antibodies for therapy Tried to solve. Chimeric antibodies are higher than the original mouse antibodies Effector function, longer serum half-life, lower immunogenicity (14,15,16) It is expected to have improved features for floor use. Human IgG1 constant Region-based chimeric antibodies often use human effector cells ADCC assay showed higher in vitro cell killing activity (14,15) . Chimeric antibodies generally have a half-life that is at least 5 times longer in humans than mouse antibodies. (16) and is 75% human, but the immune response to the chimeric antibody is Often much lower than their counterparts (16).   In vitro and in vivo by replacing mouse Fc with human Fc region New or enhanced effector functions. This has been clearly demonstrated in vitro, using human effector cells to produce chimeric antibodies. Mediates significantly enhanced CD48 + Raji cell lysis compared to that of mice I was able to. Similar results have been obtained for several chimeric antibodies (30). The mouse IgG2a isotype of this antibody is We believe that it is likely to be most effective at mediating Antibodies were tested in SCID mice.   That is, we have shown that antibodies against CD48 have potent antitumor activity in SCID mice. That can mediate the effect, and that the chimera of this antibody is human PBMC It has been shown that stronger ADCC activity can be mediated by the human cells. This These properties indicate that anti-CD48 antibodies are useful in many diseases, including lymphocytic leukemia, lymphoma, etc. Suggests that it may be useful in treatment.   Those skilled in the art will appreciate that they do not depart from the broadly described concept and scope of the invention. Many variations and / or modifications of the invention described in the specific embodiments are possible. Will understand. Therefore, the embodiments shown here are in every sense It is illustrative and not restrictive. References 1. Longo DL: Non-Hodgkin's lymphoma. Current Opin ion in Hematolagy 1: 295, 1994 2. Henniker AJ, Bradstock KF, Grimsley P, Atkinson MK: Human lymph A novel non-linear antigen on a sphere: characterization with two CD-48 monoclonal antibodies (A n ovel non-lineage antigen on humaan leucocytes: characterization with two CD-48 monoclonal antibodies.) Disease Markers 8: 179, 1990 3. Vaughan HA, Thompson CH, Sparrow RL, McKenzie IFC: HuLy-m3-hi HuLy-m3-a human leukocyte antigen. Transplantation 36:44 6, 1983 4, Hadam MR: N10 Cluster report: CD48, NK- and non-linear antigen (CD48, NK-and non-lineage antigens.) Fourth international workshop and confer ence on human leukocyte differentiation antigens P661, 1989 5. Kato K, Koyanagi M, Okada H, Takanashi T, Wong YW, Williams AF , Okumura K, Yagita H: CD48 is a counter receptor for mouse CD2 and activates T cells (CD48 is a counter-receptor for mouse CD2 and is involved  in T cell activation.) J Exp Med 176: 1241, 1992 6: Arulanandam ARN, Moingeon P, Concino MF, Recny MA, Kato K, Yag ita H, Koyasu S, Reinherz EL: Soluble multimeric recombinant CD2 protein Identifies CD48 as a low affinity ligand for human CD2: human and Of CD2 ligand during the evolution of mice and mice (A soluble multimeric recombinan t CD2 protein identifies CD48 as a low affinity ligand for human C D2: d ivergence of CD2 ligands during the evolution of humans and mlce.) J Exp Med 177: 1439, 1993 7. Sandrin MS, Mouhtouris E, Vaughan HA, Warren HS, Parish CR: C D48 is a low affinity ligand for human CD2 (CD48 is a low aff inity ligand for human CD2.) J Immunol 151; 4606,1993 8. Korinek V, Stefanova I, Angelisova P, Hilgert I, Horejsi V: human Leukocyte antigen CD48 (MEM-102) is closely related to activation marker Blast-1 (The human leucocyte antigen CD48 (MEM-102) is closely related to the activation marker Blast-1.) Immunogenetics 33: 108, 1991 9. Vaughau HA, Henning MM, Purcell DFJ, McKenzie IFC, Sandrin M S: Isolation of cDNA clone of CD48 (The isolation of cDNA clones for CD48. Immunogenetics 33: 113, 1991 Ten. Stauton DE and Lawson DAEMBO (1987) EMBO J. 6.3695-370 1 11． Grossbard ML, Press OW, Appelbaum FR, Bernstein ID, Nadler LM: Treatment of leukemia and lymphoma with monoclonal antibodies (Monoclonal antibo dy-based therapies of leukemia and lymphoma.) Blood 80: 863, 1992 12． Maloney DG et al., Chimeric anti-Cd20 monoc in patients with recurrent B-cell lymphoma Phase using increasing single dose injection of lonal antibody (IDEC-C2B8) Phase I clinical trail using escalating single dose infusion  of chimeric anti-Cd20 monoclonal antibody (IDEC-C2B8) in patient s with recurrent B-cell lymphoma.) Blood, 84, 2457-2466. 13. Greenaway S, Henniker AJ, Walsh M, Bradstock KF: Chronic lymphoid white Preliminary Advent of Two Mouse Monoclonal Antibodies That Fix Human Complement in Hematological Patients Floor test (A pilot clinical trlal of two murine monoclonal antibodies fixin g human complement in patients with chronic lymphatic leukemia.) Leukemi a and Lymphoma 13: 323, 1994 14, Martin JS, Dyer G et al. (1989) In vivo in patients with lymphoid malignancies Effect of Campath-1 antibody; Effect of antibody isotype (Effect of Campath-1 antib odies in-vivo in patients with lymphoid malignancies; Influences of antib ody isotope) Blood, 73, 1431-1439. 15． Bruggemann M, Williams GT, et al. (1987) matched sets of chimeric antibodies. Comparison of the effector functions of the human immunoglobulins used (Comparison of the ef fector functions of human immunogloblins using a matched set of chimeric  antibodies) J. Exp. Med., 166, 1351-1361. 16． Shin SU (1991) chimeric antibody: in drug delivery and immunotherapy Potential applications (Chimeric antibody: Potential applications for drug deli very and immunotherapy) Biotherapy. 3, 43-53. 17． Vaughan HA, Thompson CH, Sparrow RL, McKenzie IFC: HuLy-m3- Human Leukocyte Antigen (HuLy-m3-a human leukocyte antigen.) Transplantation 36: 446, 1983 18． Harlow E and Lane D Antibody: Laboratory Manual (Antibodies: A laborator y manual.) Cold Spring Harbor Laboratory, USA. 1988. 19. Chomczynski P and Sacchi N, acid guanidinium thiocyanate-pheno Single-step method of RNA isolation by extraction with toluene-chloroform RNA isolation by acid guanidinium thiocyanate-phenol-chlorofolm extra cti on) Anal. Biochem. 1987; 162: 156-159 20. Marchuk D et al., Construction of T Vector, Direct Cloning of Unmodified PCR Products A rapid and genera system for the rapid and genera l system for direct cloning of unmodified PCR products) Nucleic Acid Research. (1991) 19, 1154. twenty one. Maeda H, Matsushita S, Eda Y, Kimachi K, Tokiyoshi S, Bendig M M: Construction of a new type of human antibody having HIV neutralizing activity (Construction of reshaped) human antibodies with HIV-neutralizing activity.) Hum Antibod Hybr idomas 2: 124, 1991 twenty two. Telerman A, Amson RB. Romasco F, Wybran J, Galand P, Mosselman s R: Internalization of human T lymphocyte receptor (Internalization of hu man T lymphocyte receptors.) Eur J Immunol 17: 991, 1987 twenty three. Sun HP, Smith GM, manuscript in preparation twenty four. Greenwood J and Clark M. Antibodies for prophylactic and therapeutic uses in humans Of recombinant human IgG subclass antibodies in antibody production of molecules Effector functions of matched sets of recombinant huma n IgG subclass antibodies in Antibody Engineering of antibody molecul es for prophylactic and therapeutic applications in man) (1993) Ed by Cl ark M. Academic Press. twenty five. Huang S and Terstappen LWMM. Hematopoietic microrings from a single human bone stem cell Formation of harmatopoietic microenvironment and h aematopietic stem cells from single human bone stem cells.) Nature (1992 ) 360, 745-749. 26 Thorley-Lawson, DA, Schooley RT, Bhan AK and Nadler LM (1982 ) Cell, 30, 415-425. 27. Herlyn, D and Koprowski H (1982) IgG2a monoclonal antibody Inhibits human tumor growth through interaction with effector cells (IgG2a monoclo nal antibodies inhibit human tumour growth through interaction with effe ct or cells.) J Biol Chem 79, 4761-4765. 28. Kawata A, Yoshida M et al. (1994) Novel SCID in human B leukemia / lymphoma Establishment of mouse and nude mouse models and immunotoxins and monoclonal antibodies Effective Treatment of Tumors: Anti-tumor Efficacy of Monoclonal Antibodies in SCID Mice and Significant differences between nude mouse models (Establishment of New SCID and nude mouse models od human B leukemia / lymphoma and effective therap y of the tumours with immunotoxin and monoclonal antibody: Marked differe nce between the SCID and nude mouse models in the antitumour efficac y of monoclonal antibody.) Cancer Research. 54, 2688-2694. 29. Gretie GA et al. Show the dissemination of human B-cell tumors (Daudi) in SCID mice. Disseminated or localized growth of a human B cell tu mour (Daudi) in SCID mice.) Int. J. Cancer (1990) 45, 481-485. 30. Mount PF, Sutton VR et al. (1994) chimeric anti-colon cancer antibody c30.6 is scid / scid Inhibits the growth of human colorectal cancer xenografts in mice (Chimeric anti-colo n cancer antibody c30.6 inhibits the growth of human colorectal cancer x enografts in scid / scid mice.) Cancer Rasearch, 54, 6160-6166.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/30 C12P 21/08 16/46 A61K 43/00 C12N 15/09 37/02 C12P 21/08 C12N 15/00 A (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AU, CA , JP, US (72) Inventor Sung, Haiping Australia 2010 Victoria Street, Darlinghurst, New South Wales 384

Claims (1)

[Claims] 1. A method for treating T-cell or B-cell lymphoma or leukemia, wherein the method is CD Prior to administering a class IgG antibody to 48 to a subject in need thereof. Notation. 2. The IgG isotypes are mouse IgG2a, human IgG1, human IgG2 and 2. The method according to claim 1, wherein the method is selected from the set IgG2b. 3. The method according to claim 1 or 2, wherein the antibody is against HuLym-3 antigen. . 4. The antibody is a radioisotope, cytokine, protein toxin or anticancer agent The method according to any one of claims 1 to 3, which is conjugated to a compound selected from the group consisting of: 5. T-cell or B-cell lymphoma or leukemia is non-Hodgkin's lymphoma or Is a lymphoid leukemia. 6. The method according to any one of claims 1 to 5, wherein the antibody is a humanized antibody. 7. The humanized antibody, wherein the Fc region or a part thereof is a human Fc region or a part thereof 7. The method of claim 6, which is a mouse antibody that has been replaced by: 8. The subject is a human and the isotype is human IgG1 or human IgG2. The method according to claim 1.