JP2000516594A - Improved treatment of immune cell-mediated systemic diseases - Google Patents

Abstract

  (57) [Summary] Improved methods of treating immune cell mediated systemic diseases, particularly T and B cell mediated diseases, are provided by increasing the systemic exposure, or bioavailability, of the therapeutic protein. Such therapeutic proteins are selected from the group consisting of monoclonal antibodies, soluble receptors, and soluble ligands that bind to antigens expressed on the surface of immune cells.

Description

DETAILED DESCRIPTION OF THE INVENTION                 Improved treatment of immune cell-mediated systemic diseases                                Field of the invention   The present invention generally relates to monoclonal antibodies, routes of administration, and immune cell mediated diseases. Related to the therapeutic field.                                Background of the Invention   Recently, in vivo applications such as clinical tests or various pregnancy tests, diagnosis of sepsis, Therapeutic applications, such as the treatment of organ transplants, autoimmune diseases, restenosis, certain types of cancer, and There are many prophylactic uses, for example, monoclonal antibodies as antiviral agents. Scripture Typically, such antibodies are administered either intravenously (iv) or subcutaneously (sc). Although given, other routes of administration are also possible, for example, intramuscular (im) and intranasal. In general, iv administration requires catheterization for home administration, and Hospital administration requires medical care and, in more extreme situations, hospitalization Is required, sc administration is preferred over iv administration. In addition, administered iv Therapeutic agents take longer to administer when compared to sc administration, and as a result, This is an expensive treatment. However, sc administration is not without its drawbacks. example For example, there are physical limits on the maximum dose that can be administered at the site of injection.   Large polypeptides, e.g., antibodies, when administered subcutaneously, It has also been observed that it is absorbed into the lymphatic system and subsequently travels into the bloodstream [Weinst ein et al., Science,222: 423-426 (1983); Weinstein et al., Cancer Invest.,Three: 85-95 (1 985); Supersaxo et al., Pharm Res.,7: 167-169 (1990)]. Not located in the lymphatic system Systemicity of sc-administered antibodies with respect to antigen targets such as respiratory syncytial virus Exposure is comparable to that given iv [Davis et al., Drug Met. Disp.,twenty three: 1028-10 36 (1995)].   However, Applicants have determined that the antibody can be used to control immune cells, such as T (or B) cells. When targeting or binding antigens expressed on the surface, the extent of absorption (e.g., That is, systemic exposure) is due to the binding of antibodies to such antigens in the lymphatic system. Restricted, That is, they have found that the antibody is prevented from entering the bloodstream. Accordingly Therefore, antibodies are effective in treating systemic immune cell-mediated diseases such as rheumatoid arthritis. It is desirable to reach the final site of action in quantity. In the case of the subcutaneous route, the antibody It is important that they do not invade and remain in the lymph nodes or other areas of the lymph system . Thus, treating systemic immune cell mediated (eg, T or B-cell mediated) diseases Requires the efficient administration of antibodies and other therapeutic proteins. Exist. The methods described herein will become apparent to those of skill in the art upon reading this specification. It will be.                                Summary of the Invention   The present invention relates to therapeutic proteins that bind to selected antigens on the surface of immune cells. Provide improved treatment of immune cell-mediated diseases by increasing systemic exposure To offer. Systemic exposure to such therapeutic proteins is first achieved at saturating levels. Providing (or administering) a therapeutic protein, followed by such a therapeutic protein Quality is increased by subjecting the substance to a second dose administered subcutaneously, thereby increasing the systemicity of the second dose. Exposure is at least greater than an equivalent subcutaneous dose given without saturating dose benefit. 50% larger. Preferably, systemic exposure of such therapeutic proteins is low. It increases at least two-fold, more preferably at least four-fold.   In a preferred embodiment, the immune cells are T (cell) lymphocytes.   In another preferred embodiment, the immune cells are B (cell) lymphocytes.                             Brief description of drawings   Figure 1 shows [^ThreeH] CE9.1 was transferred to CD4 + (〇) and CD4- (□) In iv after administration of iv (0.4 mg / kg) to mice Shows serum radioactivity. Open symbols indicate total radioactivity, solid symbols indicate cephalo Represents radioactivity associated with sucrose-conjugated soluble CD4. 24 and 48 hours [two animals Mouse used (average% for 24 and 48 hours)] Two male mice were employed.   FIG. 2 shows that [[spleen (O) and thymus (□)]^ThreeH] CE9.1 was converted to CD4 + (Solid) and CD4- (open) transgenic mice were administered iv (0 4 mg / kg), shows the total radioactivity in% dose. CD4 + Mau In Spleen radioactivity approaches 20% of the dose in 2 hours, while CD4-mice No absorption was observed in the spleen or in the thymus of CD4 + or CD4- mice .   FIG. 3 shows that [[] in the liver (○), kidney (□) and lung (肺)^ThreeH] CE9.1 To CD4 + (black) and CD4- (open) transgenic mice The figure shows the total radioactivity expressed as a dose% after v administration (0.4 mg / kg). Spleen CD4 receptor-mediated absorption in liver, kidney and lung, according to Not significant.   FIG. 4 shows that [Spleen (A) and liver (B)^ThreeH] CE9.1 was converted to CD4 + Or administered radiation after iv administration to CD4-transgenic mice Shows the dose dependence of% activity. ○ indicates a dose of 0.4 mg / kg, while □ indicates a dose of 10 mg / kg. Represents a dose of 0 mg / kg. Black symbols indicate data from knockout (CD4-). And open symbols represent data from CD4 + transgenic mice. You. As dose increases, liver radioactivity increases proportionately and spleen radioactivity increases Did not increase. Radiolabeling properties in the spleen of CD4 + mice at high doses are low. Similar to the characteristics of CD4-mice at the dose.   FIG.^ThreeH] CE9.1 was transferred to CD4 + (＋) and CD4- (□) Blood (%) after sc administration (0.4 mg / kg) to male mice Shows serum radioactivity. Open symbols represent total radioactivity, solid symbols are Sepharose 9 shows radioactivity associated with S-conjugated soluble CD4. One experiment at each time point The thing was adopted. Biologically active anti-CD4 mAb has been Residues in plasma but active after sc administration in animals with human receptors No mAb was observed (plasma CE9.1 concentration was much more unquantifiable , LLQ = 10 ng / ml).                             Detailed description of the invention   The present invention relates to the use of immune system mediated diseases Therapeutics using therapeutic proteins that recognize antigens expressed on You. Applicants have identified that immune cell antigens can be used to saturate therapeutic proteins such as monoclonal antibodies. Lonal antibodies, or other binding proteins, such as soluble receptors (or Soluble Riga And then subcutaneous administration of the therapeutic protein Results from systemic exposure compared to that observed from one equal sc dose alone , Preferably increased by at least a factor of two.                                   Definition   An “immune cell” is a cell that is important for the immune response in an individual and generally Refers to cells found in the lymphatic system, and especially in the lymph nodes. Such cells , T cells (or T lymphocytes), B cells (or B lymphocytes), macrophages And dendritic cells.   "Saturation" refers to the complete binding of selected immune cell antigens in the lymphatic system , Upon subsequent administration of the therapeutic protein, The amount of therapeutic protein required to prevent identifiable binding of U. The amount of therapeutic protein required depends on the immune cell antigens present in the lymphatic system. Amount, affinity of the therapeutic protein for such antigens, and therapeutic tamper It depends on the half-life of the protein in vivo. One skilled in the art will recognize the You can check the amount. For example, for a human anti-CD4 monoclonal antibody, Saturation amounts are typically in the range of 0.5 to 5 mg / kg.   “No identifiable binding” means subcutaneous dose and saturated dose after saturated dose. Plasma AUC (sub-plasma concentration vs. time curve) Area) is at least doubled.   “Selected T (or B) cell antigen” is expressed on T or B lymphocytes Cell surface protein. Such proteins are typically receptors Or a receptor ligand (also called a counter receptor) And is involved in T (or B) cell mediated diseases. Such antigens are Not specified, but CD3, CD4, CD8, CD11, CD18, CD20, CD 23, CD28, gp39 (CD40 ligand, CD40 counter receptor Or T-BAM), CD40, CD80, CD86 and CD family Includes other elements.   The “therapeutic protein” can be a monoclonal antibody or selected Exemption It can be another protein that binds to the epidemic cell "target" antigen. Such a protein Quality can compete with the natural ligand of said antigen, or otherwise It suppresses the interaction between immune cells, for example, the interaction between T cells and B cells. For treatment Proteins can be monoclonal antibodies or soluble receptors or soluble ligands Other binding proteins, such as obtain. Soluble receptors (or counter receptors) are transmembrane And / or proteins in which the cytoplasmic region is deleted. Yes, if desired Soluble receptors (or counter receptors) are fused with other proteins, Desired properties can be enhanced or created. For example, soluble The puter (or counter receptor) is fused to an immunoglobulin Fc region, The circulating half-life in vivo can be increased.   “Systemic exposure to therapeutic proteins” is the cure in the bloodstream as measured by AUC. Level of therapeutic protein (bioavailability).                                   Characteristic   The present invention relates to the surface of immune cells in systemic immune cell mediated diseases, such as autoimmune diseases. Improved therapeutics using therapeutic proteins that recognize antigens expressed above Related. This improvement is due to the systemic exposure of therapeutic proteins that bind to immune cell antigens. Is highly dependent on the route of administration and the presence and distribution of such antigens in the lymphatic system. It is based on the observation that it exists. Applicants may be given a primary or initial dose administered subcutaneously. If such therapeutic proteins are not administered at very high doses, An unexpected finding was that it was not detected in the circulation (bloodstream), which was probably To prevent antigens and therapeutic protein complexes in the blood system from entering the bloodstream Seem.   By administering a therapeutic protein according to the present invention, subcutaneous dosing systemically Sexual exposure (ie, plasma AUS) increases dramatically. That is, this systemic exposure May be increased by a factor of two or more (100% or more). Preferably, Systemic exposure from subcutaneous dosing increases by a factor of four or more. Better Preferably, this increases by a factor of 10 or more. In this way, Treatment of plague cell-mediated diseases involves increasing systemic exposure to therapeutic proteins Better.   Preferably, the therapeutic protein is a monoclonal antibody, but is not limited to this. Not done. More preferably, the antibody is a human monoclonal antibody [eg, WO94 / 0 6448 “Human Neutralizing Monoclonal Antibodies to RSV”; Barbas et al.Method sa Companion to Methods in Enzymol.,2 (2): 119-124 (1991) and Burton et al.P roc.Nat'l.Acad.Sci ,88: 10134-10137 (1991)] or modified immunity Encoded by a globulin coding region and expressed in a selected host cell The modified antibody is a modified protein. Such a modified antibody may be a treated antibody (Eg, a chimeric or humanized antibody) or the entire immunoglobulin constant region or Is a partially missing antibody fragment, eg, Fv, Fab, F (ab)_TwoSuch as Either.   For repeated or prolonged dosing, monoclonal antibodies are human or Preferably, it is any of the processed antibodies. Such a treated antibody is A portion of the light and / or heavy chain variable region of the selected acceptor antibody is One or more donors having specificity for a selected epitope of a cell antigen -A full-length synthetic antibody (eg, antibody fragment Chimeric or humanized antibodies). For example, like this Such a molecule is a humanized heavy chain (or a complex thereof) associated with an unmodified light chain (or chimeric light chain). Converse). The processed antibodies will also have donor antibody binding specificity. Antibody light and / or heavy variable region framework for retaining It is characterized by a change in the nucleic acid sequence encoding the region. These antibodies are One or more (preferably all) of the CDRs from the antibody are described herein. Replacement with CDRs from different donor antibodies. For the construction of processed antibodies Techniques are known in the art [eg, Queen et al.,Proc . Natl Acad Sci USA,86 : 10029-10032 (1989); Hodgson,Bio / Technology,9: 421 (1991)] .   Preferably, the monoclonal antibody administered according to the present invention is selected T (or B) cell antigen. One preferred embodiment is that of humans on T lymphocytes. Antibodies against the CD4 receptor protein (antigen). Such antibodies are Can be chimeric, human or humanized to minimize antigenicity. Illustrative purpose For example, a human anti-CD4 antibody is a primatized® antibody, eg, CE9 .1 [for example WO93 / 02108 describing a primate-human chimeric antibody, i.e. primatized (R) Newman et al., Bio / Technology, 10, 1455-1460 (1990), which described CE9.1. You. Other preferred antigens are CD3, CD8, CDI1, CD18, CD20, C D28, gp39 (also known as CD40 ligand), CD40, CD80 and And CD86.                                 Administration route   Generally, subcutaneous administration is more desirable to physicians and patients than intravenous administration. sc Administration can be completed in minutes, rather than hours, for the iv infusion. In addition, iv infusion can be performed either at (i) hospital; (ii) clinic; or (iii) at home. Is administered using a catheter, but sc administration is practical without catheterization It can be done anywhere.   Sc administration also typically involves pain and pain often associated with intramuscular (im) injections. It is also advantageous in avoiding scars. This can occur daily or at a higher frequency (e.g., (E.g., weekly, twice weekly). Can be sc administration, compared to iv administration, It is also believed that a longer circulating half-life can be achieved in this regard. For example, the soluble form The CD4 receptor protein sCD4 [or sT4;Nature,331: 82-84 (1988)] is about 6 minutes when administered iv. Is about 1 hour when sc is administered.   A disadvantage of subcutaneous dosing is the amount of therapeutic protein that can be administered. sand In other words, administration of a therapeutic protein by the sc administration route is due to It is not always feasible due to limitations on the total amount of protein. This, in turn, It is determined by the amount of solution that can be administered to the patient and the solubility of the therapeutic. As a general rule Thus, the maximum volume of solution that can be administered is about 2 ml. Volume should not exceed 1.5ml Preferably, it does not exceed 1.3 ml.   Another disadvantage of subcutaneous dosing is that some therapeutic proteins are not stable in the lymph. is there. That is, in some cases, degradation may occur after subcutaneous administration.   However, saturating amounts of immune cell antigens such as T (or B) cell antigens K Conjugation to therapeutic proteins such as nal antibodies allows for subsequent subcutaneous administration. Achieves an increase in systemic exposure of the therapeutic protein, preferably the increase is It is at least twice as compared to that observed from equivalent (primary) sc dosing. all For somatic diseases, such as rheumatoid arthritis, a significant amount of It is advantageous to enter the systemic circulation without loss (and degradation). The present invention Will help facilitate this event, thus making the subcutaneous route of administration more clinically viable. It is the law.   Thus, the advantages of the present invention provide a comparable therapeutic use without chronic iv administration. The ability to obtain systemic exposure of the protein. Furthermore, the present invention relates to the treatment A simpler method for chronic administration of protein (eg, mAb) And thus allow for more practical treatments. Another feature is the initial administration By carrying out, the immune cell antigens of the lymphatic system And the systemic exposure is greater for subsequent doses, Allows for less drug administration or longer than without Dosing can be done at intervals, resulting in fewer dosings and overall therapeutic Use of Parkin can be reduced.   Preferably, the subcutaneous injection site is used for therapeutic treatment before entering the thoracic or right lymphatic vessels. The protein is selected to minimize the number of lymph glands or nodes accessible. (The thoracic and right lymph vessels carry lymph fluid into the blood, the systemic circulation The main pipe. ) For example, injections into the upper arm generally target the superficial axillary node . The lymph effluent from these nodes then enters the thoracic or right lymphatic vessels. chain Subcutaneous injections into the supraoscapular and suprascapular areas will result in lymph from these areas Bypassing the nodes and moving only into the clavicle or scapula on the way into the blood, It is even more advantageous.   Other potentially advantageous injection sites are the abdominal wall and upper thigh. Injection into the abdominal wall At this time, monoclonal antibodies are supplied to the superficial lumbar gland, and the derived phosphate from these nodes is The liquid is led into the thoracic duct. By subcutaneous injection into the upper thigh, the surface groin is targeted Become. The lymph drainage from these nodes is ultimately received by the lumbar vertebrae, To the chest tube Flows.   The saturating dose of the invention is such that the drug to be administered, typically an antibiotic, is Load administered iv to rapidly raise antibiotic levels to status levels Note that it is not equal to In the context of the present invention, the reaction rate is non-linear In form, the saturating amount is administered for a completely different purpose. That is, The saturating dose is the final dose of action due to the second administration of the therapeutic protein administered subcutaneously. Reach the site (for example, in the case of rheumatoid arthritis, an enlarged joint) It is intended to bind to endogenous target antigens in the lymphatic system to achieve a major therapeutic effect. Figure.                                   Symptoms   The present invention relates to immune cell mediated conditions, such as conditions mediated by T (or B) cells. To provide effective treatments. Such symptoms include lymphomas (T and B cells ), Various leukemias, infectious diseases (eg AIDS), transplantation, autoimmunity and inflammation Symptomatic diseases. Therapeutic tampering as a means to induce immunosuppression The quality of transplantation is high, such as transplant rejection (eg, heart, lung, kidney, cornea, bone marrow, skin, etc.) Therapeutic or prophylactic use of autoimmune or inflammatory diseases (eg rheumatoid arthritis) H, psoriasis, lupus erythematosus, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, reed Moto thyroiditis, myasthenia gravis, type 1 diabetes, uveitis, nephrotic syndrome, Reflux obstructive airway disease, enteritis, for the treatment or prevention of topical dermatitis And allergies (eg, asthma, childhood steatorrhea, proctitis, eosinophilic gastroenteritis, obesity Cytopathy, Crohn's disease, ulcerative colitis, etc.) and even food allergies (eg (Eg, migraine, rhinitis, and eczema).   Preferably, the condition to be treated is rheumatoid arthritis, asthma and / or psoriasis It is. Another preferred embodiment is for therapeutic or prophylactic use in transplant organ rejection. It is a means for inducing immunosuppression as in the process.                                   dosage   The amount and frequency of sc administration of the therapeutic protein will depend on the immune cell target antigen (eg, CD4 receptor), the level of such targets in patients, half the therapeutic protein Therapeutic proteins required for effective treatment such that a reduction in life and improvement in symptoms are observed Quality whole body Depends on sexual exposure. Such parameters can be determined by one skilled in the art. For example, Effective doses for treating rheumatoid arthritis include tender joint count (TJC), swelling Means well known to those skilled in the art, such as the evaluation of the number of articulated joints (SJC) [ie, Ritchie Articul ar Index, Ritchie et al.QJ . Med.,37.: 393-406 (1968)] ACR (American College of Rheumatology) response index, and / or Or early morning stiffness is a rare but effective means of evaluating effective treatment.   As an example of a dosing regimen, a saturating amount of 80-280 mg of anti-CD4 antibody in the first week Iv in an amount in the range of Alternatively, anti-CD4 antibody can be administered twice a week (Ie, 40, 80, 100, 120 or 140 mg twice weekly). So Subsequent doses are then administered subcutaneously. Such subcutaneous administration is performed for about 3 weeks (for example, , Table 5). If symptoms are observed as needed with repeated dosing (ie , For arthritis, or at predetermined intervals (eg, 3, 6, or 9). Months). Preferably, the dosage is 80-12 on a twice weekly basis. 0 mg. However, those skilled in the art will recognize that more frequent dosing (eg, daily, daily) ) Or less frequent dosing (eg, weekly) may also be appropriate. I can solve it. The amount of therapeutic protein administered can be adjusted as appropriate.   In addition, other therapeutic compositions may be co-administered with the therapeutic proteins of the present invention. For example, as a treatment for rheumatoid arthritis, DMARD (disease-modifying anti-rheumatic drug) May be administered simultaneously. DMARDs are well known in the art and include methotrexate ( mtx), azathioprine, penicillamine, hydroxychloroquine, IM goal Do, oral gold, sulfasalazine, cyclosporine and chlorambushi Includes                                Pharmaceutical composition   The therapeutic protein of the present invention can be used as an active ingredient in a pharmaceutically acceptable carrier. Such a protein is prepared as a pharmaceutical composition containing an effective amount. The preventive measures of the present invention Or a therapeutic agent, such as water containing a therapeutic protein, e.g., a monoclonal antibody. Aqueous suspension or solution, preferably buffered to physiological pH and immediately It is preferably in a form that can be cut. Compositions for parenteral administration usually comprise antibodies or Contains a solution of the cocktail in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Do it. varied Aqueous carriers such as 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulates. These solutions are traditional and Sterilization may be achieved by well-known sterilization techniques (eg, filtration). The composition comprises a pH adjuster And pharmaceutically acceptable auxiliaries necessary to approximate physiological conditions such as buffers May be contained. The concentration of the antibodies of the invention in such pharmaceutical preparations can vary widely. That is, from less than 0.5% to usually about 1% or at least about 1% to 15% Or up to 20% by weight, depending on the particular mode of administration chosen, the fluid volume , Viscosity and the like.   Nonionic surfactants suitable for use in the present invention are preferably human Erythrocyte lysis to a significant extent at the appropriate concentration with little toxicity to Does not cause blood. Suitable nonionic surfactants include, but are not limited to, poly Sorbate 20 (monolaurate), polysorbate 60 (monostearate) And polysorbates such as polysorbate 80 (monooleate) Loxyethylene sorbitan). Preferred nonionic surfactants are Resolvate 80. Polysorbate 80 is generally available from Tween® It is sold under 80 trade names. The non-ionic surfactant is preferably about 0.5. It is made into a pharmaceutical composition in an amount of from 0.01% to about 0.6%, preferably about 0.02%. Book Sugars useful in the pharmaceutical compositions of the invention serve as swelling and tonicity adjusting agents. Suitable Suitable sugars are mannitol, sucrose, trehalose and sorbitol. Which sugars. The preferred sugar is sucrose. The sugar is about 3 in the pharmaceutical composition % To about 8% w / w.   In addition to monoclonal antibodies and buffers, the pharmaceutical compositions of the invention may optionally be Other agents suitable for parenteral administration, such as bacteriostats, tonicity adjusting agents, and anti-cooling agents May be contained. Suitable bacteriostatic agents are benzyl alcohol and methyl and Includes ropirparaben.   Hydrophilic polymer cooling agents such as hydroxyalkyl cellulose, gelatin, Acacia gum, polyvinylpyrrolidone (for example, having a molecular weight of 10,000 to 600 00) and polyalkylene glycols such as polyethylene Clycols (eg, , Molecular weight 4000 to 40000) is included in the pharmaceutical composition of the present invention. like this The use of new drugs should be used in lyophilization and in reconstitution after lyophilization. Increase qualitative (ie, minimize loss of activity and proteolysis) .   The stability of the pharmaceutical composition of the invention increases at low temperatures. Therefore, they are preferably A temperature in the range of -70 ° C to 15 ° C, preferably about -40 ° C to about 8 ° C, and more Preferably, it is stored at about 4 ° C to about 8 ° C. The lyophilized composition is preferably Administered within 8 hours after reconstitution, preferably kept at 4 ° C to 8 ° C after reconstitution.   The pharmaceutical composition of the present invention provides an effective amount to a patient in need thereof, particularly a human patient. Pharmaceutical dosage units prepared to be administered intravenously, subcutaneously, or intramuscularly I.e. in a sterile container such as an ampoule, syringe, vial, bottle or bag Can be packaged. The exact concentration of the mAb in the pharmaceutical dosage unit as well as the exact Dosage volume will depend on factors such as the condition being treated, the severity of the condition and the weight of the patient. You. Optimization of a given dose of the pharmaceutical composition of the present invention is based on standard pharmaceutical and medical practice. Can be performed in accordance with Monoclonal antibody concentration in each drug dosage unit Can exceed 80 mg / ml. Preferably, the concentration of the monoclonal antibody is about It ranges from 40 to about 80 mg / ml.   Patients typically receive a dose of 1.0 to 4.0 mg / kg / week of mAb. You. This treatment is given for about 4 weeks, then if repeated dosing is necessary [ie If the person shows symptoms again (extended for rheumatoid arthritis) or At regular intervals, for example, at 3, 6 or 9 month intervals]. Intramuscular administration I.e., as an initial or saturating dose), the pharmaceutical composition of the present invention Is administered by injection into the large muscles of. If the total volume of the dose exceeds about 5 ml, The dose may be divided and injected in two or more places.   In a preferred embodiment of the present invention, sucrose in sterile water or 6% sterile water The pharmaceutical composition is stored in lyophilized form for a final reconstitution solution, such as. Lyophilized Pharmaceutical compositions are prepared by lyophilizing a pharmaceutical composition in aqueous form using conventional techniques. Prepare from The lyophilized pharmaceutical composition is preferably a single unit for ease of administration. It is stored in single dosage units, but may be stored in larger amounts in lyophilized form. Freeze drying Dried composition Items should be reconstituted in sterile vials or other containers and finally or immediately parenterally administered. Can be stored until given. In preparing pharmaceutical preparations for lyophilization, the aqueous solution It may be more dilute than the original pharmaceutical composition. For example, having a mAb of 40 mg / ml 1 ml of the aqueous solution was lyophilized and then reconstituted with 0.5 ml of sterile water, A solution with a g / ml mAb is obtained.   In another preferred embodiment of the invention, the invention relates to one or more freeze-dried Sterile container for pharmaceutical composition in form and one or more separate sterile containers for reconstitution solution A kit comprising: The reconstitution solution can be further added to a multi-compartment container, such as for And may be contained in different compartments of a two-compartment syringe designed for administration. This Lyophilized mAbs and syringes in a syringe or other two-compartment container such as The solution can be broken, for example, by crushing a syringe or container. As a result, the mAb and the reconstitution solution are separated by a membrane wall in which they are mixed. Single key The amount of reconstitution solution, mAb and amount of reconstitution solution in the Final reconstitution with a mAb of about 40 to about 80 mg / ml at the selected pH Select so that the product is obtained. The preferred solution for reconstitution is sterile water. Return The parent solution may further contain bacteriostats or other substances suitable for parenteral administration. .                            T and B cell antigens   In addition to the CD4 receptor protein, expressed on T cells and for therapeutic purposes Molecules that can also be used as "targets" and are administered according to the present invention [Will iams et al.Annu . Rev. Immunol.,6: 381-405 (1988)] There are other elements of the family. Such molecules include, but are not limited to, CD3 CD8 (CD4 and CD8), CD11 / CD18 (eg, Xie et al.,J . Im munol .,155: 3619-28 (1995)), CD28 (Aruffo et al.,Proc.Natl.Acad.Sci.,84: 8573-7 (1987)), low levels on CTLA-4, activated CD8 + and CD4 + T cells. A homologue of CD28 transiently expressed at scepter density (Brunet et al.Nature,32 8 : 267-270 (1987)), and gp39 (CD40 ligand, T-BAM, and [Also known as TRAP] [Noelle et al.Proc.Natl.Acad.Sci.,89:6550-6554 ( 1992), Foy et al.J . Exp.Med.,178: 1567-1575 (1993), Banchereau et al.,Annu . Rev . Immunol .,12: 881-922 (1994)]. The therapeutic protein can be a monoclonal antibody, Or possibly soluble ligands (ie, cytoplasmic and transmembrane Region, optionally fused to a constant region of an immunoglobulin, such as an Fc region. Natural ligand).   Other suitable "target" molecules include CD28, CTLA-4, and others found on B cells. And a ligand or counter receptor for gp39. like this The ligand or counter receptor is CD86 (also known as B7.1) ( Linsley et al.Proc . Natl. Acad. Sci.,87 :5031-5035 (1990), Freeman et al.,J . Ex p. Med .,174: 625-631 (1991)), also known as CD86 (B7.2 and B70). (Freeman et al.,J.Exp.Med.,178: 2185-2192 (1993), Freeman et al.,Science,262: 909-911 (1993)) and CD40 (Stamenkovic et al.,EMBO J.,8: 1403 (1989)) Include. With respect to antibodies that bind to CD40, such antibodies are non- It must be proliferative or non-irritating [see, for example, WO 94/01547 (Cetus Oncol ogy) and WO 95/09653 (Immunex)]. Other suitable on human B cells One "target" molecule is the CD20 antigen.   The following examples illustrate various aspects of the invention and do not limit the scope. it is conceivable that. Reagents and materials were obtained from commercial sources unless otherwise noted. Was.                                  Example   Materials and methods   Chemical substances   Cynomolgus monkey / human chimeric antibody (mAb) that binds to human T cell receptor CD4 CE 9.1 [Newman et al., Bio / Technology 10, 1455-1460 (1992)] was used in the following experiments. Was. Other monoclonal antibodies to human CD4 can be used as well Is understood. CE9.1 (unlabeled-reference standard) in 5 mg / ml solution or stable Supplied as a lyophilic substance with a sex-enhancing excipient (50 mg / ml on reconstitution) . Soluble CD4 (also called sCD4, sT4, Deen et al.,Nature,331: 82-84 (19 88)) was obtained as the lyophilic substance (10 mg / ml for reconstitution). CE 9.1 and sCD 4 is a recombinant protein expressed in Chinese hamster ovary cells. Protein A Sepharose was purchased from Sigma (St. Louis, MO). Horstra Dish peroxidase conjugated mouse anti-human IgG1 mAb (clone HP 6069) is Zyme Laboratories Inc. (San Fransisco, CA) . All other chemicals were reagent grade or better.   [^ThreeH] CE9.1   CE9.1 is basically described by Davis et al. (Drug Metab. Dispos. 20, 695-705 (1992)). ) In Chinese hamster ovary cells as described in Metabolic labeling with primed leucine. Antibody is concentrated from the culture supernatant and protein A chromatographic Purified by luffy (total protein recovery about 70%). 400μCi or more Purified metabolic label [^ThreeH] CE9.1 was converted to 5 mCi [^ThreeH] Leucine (Dupont / NEN , 144 Ci / mmol). The specific radioactivity is about 1 μCi / μg (20 00 DPM / ng). Pure[^ThreeH] CE9.1 is typically greater than 99% Of precipitating trichloroacetamide, 98% of the radiolabel was antigen-bound (Sepharos e-sT4 binding, see below). Radiochemical purity (Evaluated by SDS-PAGE) was typically greater than 90%.   Animal breeding   Male CD4 + and CD4-transgenic mice (weight about 26-4 3g) was used in this experiment and the Regents of the University of California (Kil leen et al., EMBO J12, 1547-1553 (1993)). Animals laying wood chips Group (up to 5 animals) in a controlled cage (25 (± 2 ° C .; 50 ± 10% relative humidity) with a 12 hour light / dark cycle. Diet (Purina Certified rodent chow, Purina Mills, Inc., St. Louis, MO) And filtered tap water was made available ad libitum.   [^ThreeH] iv or sc administration of CE9.1   Experimental animals are given iv dosing by injection into the tail vein or injection under the skin on the back Sc administration. The drug solution was about 0.1 for the 0.4 mg / kg dose group. mg / ml [^ThreeH] CE was 9.1 and 100 μCi / ml. 100mg / kg group For iv group only, the drug solution is about 14 mg / ml CE9.1 , 9 μCi / ml. 100 μl (low dose) or 200 μl 1 (high dose) of the target drug was administered. Vehicle is phosphate-glycine buffer or This was a mixture of PBS. Precise dosing volume weighs syringe before and after dosing And measured. The exact administered total radioactivity and dose of CE 9.1 is Measured by analysis of solution volume (oxidation / scintillation measurement and ELI below) SA).   At selected times after dosing, experimental animals were sacrificed (combined sampling, n = 1 / Time points other than described). In low-dose experiments, CD4 + iv and sc groups And 10, 30, 60, 120, 240, 480 minutes and 24 hours and 48 Time display time was adopted. 10,120 for CD4-iv and sc groups Display times of 480 minutes and 24 and 48 hours were employed. 0.4mg / kg   Additional 72 hours, 1 and 2 weeks for sc group (CD4 + and CD4-) Additional indicated sacrifice times were used. In high dose experiments, iv group (CD4 + only) With respect to 10, 30, 60, 120, 240, 480 minutes and 24 hours and A display time of 48 hours was employed. sc group (CD4 +, 100mg / kg) Thus, display times of 7, 24, 48, 72 hours and one week were employed.   After sacrifice, blood (about 1 ml) was removed by vena cava and cardiac puncture and 100 μl Aggregation was prevented by the addition of 1 129 mM trisodium citrate. Ants of whole blood Place coat on combusto-pad for oxidation and centrifuge remaining blood The sera was collected. Place an aliquot of plasma on combusto-pad for oxidation and allow remaining Plasma and blood cell pellets are frozen on dry ice and Stored for analysis. Liver, kidney, lung, spleen and thymus (or high dose iv In the experiments, only the liver and spleen) were removed and total radioactivity was measured. Tissues can be dissolved in ethanolic KOH or combus for direct oxidation. Placed on to-cone. The debris is dissolved in ethanolic KOH and the remaining total radiation Activity was evaluated.   SDS-PAGE   The plasma and blood radiochemical properties of the selected samples were determined using non-reduced SDS-PA It was evaluated by GE. For analysis of plasma proteins, 8% (w / v) polyacrylic acid was used. Le Degradation was performed using an amide gel. 16% (w / v) polyacrylic protein Degradation was performed using luamide gel. Frozen cells ( (No plasma) was thawed at room temperature and PBS was added (body equivalent to plasma volume drawn) product). Next, the cells were resuspended and diluted in SDS loading buffer. Typically, 1 0-20 μl of plasma or suspended blood cells were loaded directly on the gel lane for analysis . After electrophoresis, the gel was stained with Coomassie Brilliant Blue and dried, Sliced and radiolabeled properties evaluated (recovery> 80%). Radiolabel and all Macroscopic observation of protein (coomassie) properties, and known swimming of CE9.1 Assess radiolabel incorporation into endogenous proteins using mobility and molecular weight standards (Davis et al., Supra).   Sepharose-sT4 extraction   Characterize pure radiolabel and radiochemistry in plasma samples (ex vivo) Antigen binding activity was assessed with Sepharose conjugated soluble CD4 (sT4) . Plasma samples containing the radiolabel (typically <100 μl) were separated on Sepharose -Added to sT4 in PBS slurry (typically 100% 1: 1 resin in PBS) μl). Enhances separation of supernatant and Sepharose and ensures high total protein concentration In some cases, PBS or rat plasma diluted in PBS may be further Was added. After mixing, the supernatant was collected by centrifugation. Total radiolabel extracted (% ), The radioactivity in an aliquot of the supernatant is compared to the radioactivity in an appropriate aliquot of the original solution. It was determined by comparing to the amount of activity. All Sepharose-sT4 binding experiments It is considered that there is a large excess of resin compared to the CE9.1 equivalents present. available.   To evaluate the administered radiation dose, the radioactivity (%) per plasma volume is Literature values for mouse weight and plasma volume [50 ml / kg, Davies et al., Ph. arm. Res, 10: 1093-1095 (1993)] to evaluate total radioactivity in whole plasma. To calculate total radioactivity bound to Sepharose-sT4, total plasma radioactivity was calculated. Is multiplied by the fraction-sT4 binding already described.   ELISA   Sera samples were analyzed for antigen (sCD4) and C against anti-human IgG1 mAb. E Analysis was performed for CE 9.1 concentration using an ELISA based on 9.1 simultaneous binding. In analysis, microtiter with soluble CD4 (sT4) bound to CE9.1 -Captured from plasma in plate. The sT4 / CE9.1 composite is then Mouse anti-human IgG specific for CH2 region directly conjugated with tissue peroxidase 1 mAb (Hamilton et al., J. Immunol. Methods 158, 107-122 (1993)). I did The standard curve is 10% (v / v) with citrated rat plasma or PBS. 2.) 2 to 50 ng / ml CE9.1 in citrated rat plasma (obviously For analysis of samples above the standard curve range. sT4 / anti-γ1 E The LLQ (lower limit of quantitation) for LISA was measured in neat mouse plasma (50 μl). 10 ng / ml. Concentration in the range of 10 to 100000 ng / ml As described above, the with-run coefficient is in the range of 5.6 to 7.5%, while the average accuracy is 88. To 110%.   Oxidation of tritiated proteins for radioactivity measurements   To determine the radioactivity in a sample containing tritium, Was applied on the Combusto-pad in the Combusto-cone. 0.25 mL before oxidation Of Conbusto-Aid was added to the sample. Oxidation is Packard Nodel C306 Tri-Card Proceeded in the Sample Oxidizer. 15-18 mL of water labeled with tritium Collected in ase S and mixed. 5 mm for polyacrylamide gel slice × 20 mm sections were cut into two or three and placed directly into Combusto-Cone. Organization Liquid aliquot of a sample dissolved in ethanolic-KOH Or tissues were placed in Combusto-Cone and oxidized directly. Combustion efficiency (Conventionally> 98%) with flammable and non-flammable standards (Packard Spec-Chec Tritium s). The evaluation was made by comparison of the radioactivity in tandard). Scintillation county The sample was cooled to 4 ° C. overnight before aging. Scintillation counting Perform measurement with a Beckman LS3800 or 5801 scintillation spectrophotometer to improve measurement efficiency. Measured by external standard method using sealed quench standard.   Pharmacokinetics   Plasma CE 9.1 concentration-time data were analyzed using the unfractionated method (M. Gibaldi and D. Perrier: "Pharmacokinetics," 2nd ed. Marcel Dekker, Inc., New York, 1982) Analysis was performed using in-houses of twarepackage. AUCO-t (composite ELISA data Logarithmic trapezoidal formula for decreasing plasma concentration and increasing plasma concentration Was evaluated using the linear trapezoidal rule. Half-life of loss of radioactivity from the spleen ( 0.4 mg / kg iv, CD4 + mice) log converted dose (%) vs. time data Calculated from linear regression. Similarly, sc into CD4 + transgenic mice Reported half-life from total plasma radioactivity data after dosing (0.4 mg / kg) Log converted dose (%) vs. time data (24 h to 2 weeks) obtained from linear regression .   Intravenous administration   Has hCD4 T cell receptor (CD4 +) or CD4 receptor Once in transgenic mice (CD4-, knockout) without any Amounts of metabolically radiolabeled [^ThreeH] CE9.1 (0.4 mg / kg) was administered iv. , Whole blood and plasma radioactivity, radioactivity in plasma capable of antigen binding, and liver, kidney Sacrifices were made to evaluate total radioactivity in the glands, lungs, spleen, and thymus. Plasma In addition, we analyzed for CE9.1 using the sT4 / anti-γ1 ELISA. Another In one study, CD4 + mice received a single dose of [^ThreeH] CE9.1 (100 mg / kg) was administered iv and whole blood and plasma radioactivity, plasma CE 9.1 and spleen and And total radioactivity in the liver were evaluated.   0.4 mg / kg [^ThreeH] Administration of iv to CD knockout mice of CE9.1 As a result, the administered radiochemical dose of the active antibody in plasma at 48 hours <ELISA According to the method, a level of about 1.5 μg / ml, which was higher than 19% of FIG. 1) was maintained. all An apparent difference between plasma radioactivity and plasma radioactivity bound to Sepharose conjugate antigen There was no difference. In addition, the ratio of blood to plasma by 48 hours exceeds 0.68. Foremost, this indicates little blood cell radiolabel binding (Table 2).   In contrast, the administered radioactivity in plasma was CD4 + transgenic mouse. [^ThreeH] less than 20% by 2 hours after CE9.1 administration (FIG. 1). dosage Four hours later, less than 5% of the plasma radioactivity bound to the Sepharose-conjugated antigen. plasma The CE9.1 concentration was determined 4 hours after iv administration (LLQ 10 ng / ml) by ELISA In A, quantitative It was impossible.   2 and 3 show [^ThreeH] CD4 + and knockout transgenes of CE9.1 Figure 5 shows total tissue radioactivity versus time after iv administration to enic mice. CD4 + mouse Up to about 18% of the dose is recovered in the spleen 2 hours after administration to the active antibody. Despite higher blood levels, the dose of Only 0.6% was recovered (FIG. 2). Absorption by the spleen of CD4 + mice Occurred in a time frame similar to the loss from the serum compartment. CD4 + mouse spleen Loss of radiolabel from the gut was characterized by a half-life of about 10 hours. 0.5 dose Less than% was recovered in the thymus of CD4 + mice. Total radioactivity only in liver It resembled that of the spleen (up to about 13%, FIG. 3). But in the liver, A quantity of radiolabel was recovered from the knockout (9.4%), indicating that the liver had [^ThreeH] C Plays an important but probably not antigen-specific role in the predisposition to E9.1. And Radioactivity in the thymus, kidney and lung is CD4 + or CD4-real Less than 5.4% of the radioactivity administered was less than It remained in the remains of the gut, thymus, liver, kidney and lung.   High dose of 100 mg / kg [^ThreeH] CE9.1 dose was clearly 0.4 mg / The CD4 receptor dependence of the molecular predisposition observed in kg was satisfied. Smell of plasma At 100 mg / kg in D4 + mice based on ELISA data Average residence time of CE9.1 was about 1 day, while 0.4 mg / kg The retention time was less than one hour. Table 1 shows the dose normalized AUC (area under the curve) (100 mg / kg, CD4 + mice) in CD4-animals (0.4 mg / kg) It was within factor 2 of what was observed. Of drug rate in knockout The linearity assumption is: 1) The drug rate is linear in animals without CD4 receptor binding And 2) uncorrelated IgG1 specificity for exogenous antigens Of pharmacokinetics (1-200 mg / kg in monkeys, Davis et al., Drug Metab . Dispos. 23, 1028-1036 (1995)).   0.4 mg / kg [^ThreeH] Plasma release only 4 hours after iv administration of CE9.1 Most of the radioactivity was incorporated into endogenous plasma proteins. In contrast, 100 mg / Kg [^ThreeH] 24 hours after iv administration of CE9.1, plasma radiolabel was mainly Body (80-90%). 100 mg / kg iv administration to CD4 + mice The ratio of blood to plasma (B / P) is shown in Table 2. CE9.1 was converted to CD4- High doses of iv at any time, as did low doses of iv in mice. They were temporarily allocated for up to 24 hours after giving. At these points, the radiolabel is essentially It was unchanged CE 9.1.   5A and 5B show 100 mg / kg in spleen and liver, respectively. of[^ThreeH] CE9.1 (CD4 + mice) administered radioactivity (iv) after iv administration ). For comparison, data at 0.4 mg / kg is also shown (CD4 + and CD4-mouse). Generally, the spleen of CD4 + mice receiving high doses of iv Radiochemical properties similar to those observed at low doses in CD4-mice Was. Apparent time dependence of spleen absorption at high doses in CD4 + mice Was not evident, and the total administered radioactivity averaged 0.5%. At low doses, radioactivity Sex reached a maximum of 18% in 2 hours. In contrast, the administered radioactivity in the liver Gender (%) characteristics were: CD4 + mice at 100 mg / kg, 0.4 mg / kg As in CD4 + mice and CD4- mice at 0.4 mg / kg No apparent dose or hCD4 receptor-dependence was observed.   Subcutaneous administration   CD4 + and CD4-transgenic mice received 0.4 mg / kg [^Three H] CE9.1 administered sc, whole blood and plasma radioactivity, in plasma capable of antigen binding Radioactivity in the liver, kidney, lung, spleen, and thymus For sacrifice at selected time points. Plasma also contains sT4 (soluble T4 or Was also determined for CE9.1 concentration using soluble CD4) / anti-γ1 ELISA. Was analyzed. In another experiment, CD4 + mice received 100 mg / ml of unlabeled CE9.1 once. kg was administered sc to evaluate plasma anti-hCD4 mAb concentration.   Consistent with extravascular systemic exposure of IgG in the absence of endogenous antigen (Davis et al. , Drug Metab. Dispos. 23, 1028-1036 (1995)), [^ThreeH] CE9.1 CD4 Knock Administration of sc to kuout mice resulted in plasma active antibody levels of 8 hours to 1 hour. weekly It was maintained at a level above 10% of the dose (> 1 μg / m L, FIG. 5). Sepharose after total plasma radioactivity and iv administration to CD4-mice No apparent difference was seen between plasma radioactivity binding to S-conjugated antigen. further The ratio of blood to plasma by 2 weeks did not exceed 0.66, which is significantly The absence of high blood cell radiolabel binding (Table 2).   In contrast, the total dose (%) in plasma is [^ThreeH] CD 9.1 transfection of CE9.1 It was at most 1% from 10 minutes to 2 weeks after sc administration to the nick mice. plasma Radiolabel did not show quantifiable antigen binding activity (FIG. 5). Similarly, plasma C E9.1 concentrations were not quantifiable by ELISA over the entire time period (LL Q 10 ng / ml). Total plasma radioactivity, however, is maximal 1% dose in 24 hours And the apparent half-life decreased in about 4 days.   Table 1 shows that [CD4 + and knockout transgenic mice^ThreeH] Relative blood after iv or sc administration of CE 9.1 (0.4 and 100 mg / kg) Shows serum AUC. AUC was determined after iv administration or 0-1 based on 0-48 hour intervals. Calculated after sc administration at 68 hour intervals. Dose increased to 100 mg / kg Thus, CD4 receptor binding is saturated and the resulting AUC is receptor-free Similar to AUC in animals. For the 0.4 mg / kgsc data, Plasma concentrations are practically unquantifiable and the maximum possible AUC is determined by the lower limit of quantification ( 10 ng / ml).                                   Table 1 Human CD4 + transgene compared to 0.4 mg / kg in CD4-mice Normalized Plasma AUC in Nick Mice  For the iv group, AUC was calculated based on the 0-48 hour interval and for the sc group. Thus, the AUC was calculated based on the 0-168 hour interval. 0.4mg / kg   For the CD4 + sc group, the total plasma concentration is not quantifiable and the maximum possible AU C was evaluated based on the lower limit analysis of quantification. The plasma concentration of CE 9.1 is Measured using an ELISA based on D4 binding.   [^ThreeH] 4 hours of sc administration of CE9.1 to CD4 + transgenic mice SDS-PAGE analysis of the plasma afterwards was similar to that after administration of the same amount of iv. The ratio of blood to plasma at 4 hours was 0.68, indicating that most of the radiolabel Indicates that they were in plasma at these time points and were not bound to blood cells. Only Two weeks after administration, the ratio of blood to plasma was 7.5 (Table 2). cell No trace of binding-invariant CE9.1 was observed.   After sc administration, the level of radioactivity in the spleen of CD4 + mice was Also And very low compared to the iv group (≦ 0.5%, CD4 + mouse For a maximum of 18%). This is because the analysis of circulating radiolabels is Biologically active parent could not be detected (based on 0.4 mg / kg iv data). , Which is believed to accumulate in the spleen). Inspected organization Of which CD4 + mouse liver has the highest administered radioactivity of up to 2.4% did. Similar properties of liver radioactivity observed in CD4 + and CD4- mice This indicates that absorption is not CD4-receptor dependent (data Is not shown).   Absorption of antibodies occurs by the lymph (Weinstein et al., Science 222, 423-426 (1983 ); Weinstein et al., Cancer Invest. 3,85-95 (1985); Supersaxo et al., Pharm. Res. 7, 167-169 (1990)), the lymphatic system in CD4 + mice has relatively high CD4 + T Assuming that it contains lymphocytes, after a small dose of sc, [^ThreeH] of CE9.1 Deletion of systemic availability prevents lymphatic CD4 + T from preventing antibodies from entering the circulation Apparently by binding to cells. further,[^ThreeH] Release after sc9.1 administration of CE9.1 The presence of radiolabeled endogenous blood and plasma proteins indicates significant metabolism in the lymphatic system Is consistent with the fact that has occurred.   Following administration of high doses of sc in CD4 + mice, injection sites into the systemic circulation Significant CE9.1 absorption was observed. Table 1 shows that large amounts of sc on CD4 + mice The dose-normalized AUC after administration was 2% of the AUC observed at low doses of CD4-. It was within 0%. Lymph CD4 + T cell binding observed at low doses It was saturated and therefore CE9.1 was efficiently absorbed into the systemic circulation.                                   Table 2 [^ThreeH] IV of CE9.1 to CD4 + and CD4-transgenic mice Or ratio of total radioactivity to blood plasma after sc administration ^aN = 1/24 and 48 h [2 mice used (average shown)] Iv data at the time.   ^bNQ shows no quantification (very low radioactivity in blood and / or plasma) ).   ^cND indicates no data (experimental animals were not sacrificed at specific time points) .   ^dData from one mouse.   Table 3 shows the systemic exposure of the two subcutaneous doses in transgenic mice. You. In the first case, the initial dose is saturated, in the latter the initial dose is not saturated .                                   Table 3 Administration of 2 sc to huCD4 mice with CE9.1-When the first dose is saturated- Allows the second dose to be absorbed into the systemic circulation from the injection site                    Human CD4 transgenic mouse  * Radioactivity migrates as unchanged CE 9.1 by SDS-PAGE.   As a result of administration of the initial saturating dose (100 mg / kg), plasma Increased the radioactivity by a factor of 30.   In summary, the properties of the anti-hCD4 mAb depend on the presence of the human CD4 receptor. And high dependence on distribution. After administration of a small amount of iv, rapid release from plasma Quick [^Three[H] Loss of CE9.1 is associated with accumulation of radioactivity in the spleen. In contrast In CD4-mice, CE9.1 is associated with IgG1 in the absence of endogenous antigen. Had a long plasma half-life as expected for These phenomena are dose dependent Gender. Saturation of CD4 receptor binding after administration of high doses of iv results in CD4 What is observed at low doses and pharmacokinetic rates and minutes in animals without sceptors Cloth matched.   Following administration of a small amount of sc, complete, active CE 9.1 from the injection site to the systemic circulation. No evidence of absorption was observed. However, high systemic exposure can result in the same amount of CD 4- Seen after sc administration to mice, which shows that the CD receptor of CE9.1 Binding suggests that the mAb prevented entry into the systemic circulatory system. These phenomena were also dose-dependent. CD4 receptor binding after high dose sc administration Is observed at low doses in animals without the CD4 receptor. And the drug rates were consistent.   Comparative data for CE9.1 in transgenic mice and humans Intravenous drug rates are qualitatively similar in these species and therefore in mice The findings described herein provide information about the properties of CE9.1 in humans. It indicates that it must be able to be In particular, transgenics In both mice and humans, the pharmacokinetics were non-linear. Intravenous administration As the amount is increased in transgenic mice and humans, plasma concentration versus A greater than dose proportional increase in the area under the time curve is observed. This The non-linearity of the liposome between the inner and outer peripheral blood trafficking and the CE9.1 CD4 It is likely due to the movement of saturable binding CD4 + T cells. CD4 antigen Evidence of CE9.1 in vivo in both transgenic mice and humans Plays an equally important role in showing movement.   In these CD4 transgenic mice, the human CD4 gene is endogenous. Restores normal helper cell function in mice without the sex CD4 gene Has already been demonstrated (Killeen et al., EMBO J. 12, 1547-1553 (1993). )). Furthermore, the immunity of CD4 distribution in CD4 + transgenic mice Histochemical studies have shown that the human CD4 molecule has been reported in T lymphocytes and humans. Expressed on a macrophage cell / dendritic cell line with a similar distribution Proved. These data are summarized and the transgenic mouse model is The data described in the present invention predict the behavior of CE 9.1 in Suggests that it is important to optimize clinical dosing to maximize efficacy Things.   Chronic administration method for anti-CD4 monoclonal antibody   Anti-CD4 monoclonal antibodies are currently being used in T-cell mediated autoimmune diseases such as For the treatment of rheumatoid arthritis and asthma You. Typical dosing regimens are shown in Table 4.   For chronic subcutaneous dosing, suitable dosing regimens include those outlined in Table 5.   The invention has been described with reference to specific embodiments. However, this application is attached by those skilled in the art. These changes can be made without departing from the spirit and scope of the appended claims. It is intended to cover all modifications and substitutions.                                   Table 4 Anti-CD4 monoclonal antibody in rheumatoid arthritis and asthma Effective chronic medication   Intravenous administration only   Method A   Method B                                   Table 5   Method 1 First, intravenous administration to saturate the lymph system, followed by chronic subcutaneous administration^*dosage   Method 2 First, subcutaneous administration to saturate the lymph system, followed by chronic subcutaneous administration   Method 3-Same as Method 2 dosing at 160 mg / dose weekly ^*Subcutaneous dosing interval and dose level are always limited to those adopted after iv administration Not done.   ^**Initial administration may further include im administration.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 37/02 C07K 14/705 C07K 14/705 16/28 16/28 C12P 21/08 C12N 15/02 A61K 37/02 C12P 21/08 C12N 15/00 C (72) Inventor MacDonald, Brian Richard United States 19481 Liberty Lane No. 10, Valley Forge, Pennsylvania

Claims (1)

[Claims]   1. An improved method for treating immune cell mediated diseases, the improvement comprising:   Monoclonal antibodies, soluble receptors and expressed on the surface of immune cells A saturable amount of a therapeutic tan selected from the group consisting of soluble ligands that bind antigen Administering the protein; followed by a second administration of the therapeutic protein. Here, the secondary administration is administered subcutaneously, and the systemic derived from the secondary administration of the therapeutic protein. Sexual exposure is less than systemic exposure from primary subcutaneous administration of an equal amount of therapeutic protein Both methods are 50% larger.   2. 2. The method according to claim 1, wherein the immune cells are T cell lymphocytes.   3. 3. The method according to claim 2, wherein the T-cell antigen is human CD4.   4. 3. The method according to claim 2, wherein the T-cell antigen is human CD28.   5. 3. The method according to claim 2, wherein the T-cell antigen is human CTLA-4.   6. 3. The method according to claim 2, wherein the T-cell antigen is a human CD40 ligand.   7. The method according to claim 1, wherein the monoclonal antibody is a primate-human chimeric antibody. .   8. The method according to claim 7, wherein the chimeric antibody is CE9.1.   9. 2. The method according to claim 1, wherein the monoclonal antibody is a humanized monoclonal antibody. Law.   10. The method according to claim 1, wherein the monoclonal antibody is a human monoclonal antibody. Law.   11. 3. The method according to claim 2, wherein the T-cell mediated disease is rheumatoid arthritis.   12. 3. The method according to claim 2, wherein the T-cell mediated disease is psoriasis.   13. 3. The method according to claim 2, wherein the T-cell mediated disease is asthma.   14． 3. The method of claim 2, wherein the T-cell mediated disease is a graft versus host disease.   15. 2. The method of claim 1, wherein the saturating amount is administered intravenously.   16. 2. The method of claim 1, wherein the saturating amount is administered intramuscularly.   17． Secondary administration of therapeutic protein to upper arm or supraclavicular or scapula region 2. The method of claim 1, wherein the method is administered subcutaneously to a subject.   18. Claims: Secondary administration of therapeutic protein administered subcutaneously to abdominal wall or upper thigh Item 7. The method according to Item 1.   19. The method according to claim 1, wherein the immune cells are B-cell lymphocytes.   20. 20. The method according to claim 19, wherein the B-cell antigen is human CD40.   21. 20. The method according to claim 19, wherein the B-cell antigen is human CD80.   22. 20. The method according to claim 19, wherein the B-cell antigen is human CD86.   23. 20. The method according to claim 19, wherein the B-cell antigen is human CD20.   24. Monoclonal against therapeutic CD80 or CD86 20. The method according to claim 19, which is a monoclonal antibody.   25. The therapeutic protein is a monoclonal antibody against human CD20. 20. The method of claim 19.   26. Non-proliferating monoclonal antibody against therapeutic CD40 for human CD40 20. The method of claim 19, wherein   27. 20. The method according to claim 19, wherein the B-cell mediated disease is B-cell lymphoma.   28. 20. The method according to claim 19, wherein the B-cell mediated disease is rheumatoid arthritis.   29. 20. The method according to claim 19, wherein the B-cell mediated disease is psoriasis.   30. 20. The method according to claim 19, wherein the B-cell mediated disease is asthma.   31. 20. The method of claim 19, wherein the B-cell mediated disease is a graft versus host disease.   32. Systemic exposure from the second dose of therapeutic protein is At least twice as much as systemic exposure from primary equivalent subcutaneous administration of quality 2. The method according to claim 1, wherein the ratio is at least 100%).   33. Systemic exposure from the second dose of therapeutic protein is At least 4 times greater than systemic exposure from the primary equivalent subcutaneous dose of quality The method of claim 1.