JPH07502727A - Prevention of allograft rejection using antibodies against adhesion molecules - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Prevention of allograft rejection using antibodies against adhesion molecules Field of the invention The present invention relates to the field of transplantation, and more particularly to methods for preventing allograft 1"1 rejection.

Background of the invention Organ and tissue transplantation is an important aspect of the treatment of end-stage organ failure and replacement of damaged tissue. Ru. The use of allogeneic or non-autologous transplants is gaining importance in medicine. death However, the use of allogeneic EM fragments often occurs due to antigenic differences between CItN individuals and recipients. is limited by rejection of the graft tissue by the recipient host.

Differences in antigens between individuals of the same species are called "alloantigens." Allograft rejection When the reaction includes alloantigens, they are called "histocompatibility antigens." °゛ The terms “major histocompatibility antigen” and “major histocompatibility complex” (111c) , refers to the product of one tightly connected region of a gene.

Graft extinction is due to the host's immune response to histocompatibility antigens expressed by the graft tissue. This is the result of the answer. Generally, the allograft is infected for a few days to a few weeks, but its inflammation The tumor is infiltrated by lymphocytes and monocytes. The 11 transplanted tissues ended up showing necrosis. In the case of skin grafts, it falls off the skin.However, in the case of vital organs such as the heart, , tissue rejection can ultimately be fatal to the recipient.

Cyclosporin is a cyclic, water-insoluble, highly non-polar compound consisting of 11 amino acids. It is a molecule. Cyclosporine is widely used to maintain the function of various transplanted organs. It is used. Its immunosuppressive effect is 1. It selectively inhibits cell function and causes myelin suppression. Keeping the allograft, or heart graft, alive without restriction (Meyers et al. al., N., Engl., J., Med.

J. J. 1:699 (1984)).

Major drawbacks of conventional immunosuppressants including cyclosporine and steroids One is the universal suppression of host immunity, which can lead to severe opportunistic infections in patients. wake up Antigen-specific immunosuppression to overcome the severe side effects of conventional drugs is strongly desired.

Additionally, cyclosporin 111 use has been associated with infection and liver and kidney toxicity. Therefore, it is limited to some extent. The clinical use of cyclosporine is blood urea nitrogen ( Reversible medication-related increases in serum creatinine (BUN) and serum creatinine levels; Accompanied by inhibition of atinine clearance. Kidney transplant recipients treated with cyclosporine It has been reported that some nephrotoxicity occurs in almost 80%.

Renal l1lI in heart transplant ψ recipients! ! Irreversible cyclosporine-induced deterioration of have been described (~feyers et al., N., Engl, J.

Med, 311:699 (1984)). Cyclospon'J fM Law Possible irreversible histological findings in the kidneys of transplanted patients have also been published. (Mihatsch et al., Transplant, P. Deterioration of function is a major side effect and is a major side effect for patients during transplantation and non-transplantation. This reduces the practical clinical therapeutic efficacy of crossporin therapy. Therefore mammalian recipients, There is a need for improved methods of inducing transplantation A tolerance, especially in humans.

Related technology A review of the adhesion receptors of the immune system is provided by Springer, T.

Δ, 1j was obtained by Na1ure 346:425 (1990).

Springer pi al, , Ann, Rev, Immunol5: 2 23-252 (1ri87) is a cell adhesion receptor of the immune system, LFA-1, CI )2 and LFA-: 3 molecules are discussed. Especially in L　1”Δ-1 Prevention of graft extinction using monoclonal antibodies has been discussed.

For, 1. Describes the in vivo utilization of monoclonal antibodies against FA-1. ing.

Bc　n　j　a　m　　+1 is a monoclonal antibody-fld resistance induction mechanism We are discussing the structure. Especially monochrome against CD4 molecule on T helper cells. Mice given short-term null antibody 41-L1 were given a The patient developed resistance to protein antigens.

Vang and Rock, J, Immunol, -146+3273-32 79 (1γ9-1) has s1g receptor fixed on B lymphocytes. reported that it induces the expression and function of both t-FA-1 and t-FA-1.

Lymphocyte function-f1 associated antigen 1 (lymphocytc runcti.

Discussion regarding n-associated antigen 1) (LFA-1) The theory is Marlin and Springer, Ccli 5539 (198 Seen in 1).

Outline of the invention Compositions and compositions for sustaining the function of transplanted allografts and preventing graft rejection and how to do so. The method includes a composition comprising an inhibitor of more than one adhesion molecule inhibitor. Including the administration of a substance. It is preferred to use inhibitors of adhesion molecules and their counter-receptor molecules. Delicious. Inhibitors include antibodies against adhesion molecules and receptor ligands.

The compositions also have uses in inflammatory reactions and allergic and autoimmune diseases. The present invention is primarily directed to compositions and methods for sustained graft survival in patients. Hidden The present invention therefore provides a method for preventing allograft rejection.

The composition includes an adhesion molecule inhibitor. "Adhesion molecule inhibitors" are T cells and/or B cells means a molecule that inhibits the activation of Such inhibitors may interfere with immune and inflammatory responses. It acts to prevent cell-cell adhesion. Therefore, the term “inhibitor” Inhibits adhesion molecules by binding to adhesion molecules or receptor ligands of adhesion molecules Contains a wide range of molecules that Such inhibitors primarily involve adhesion molecules or their receptors. Antibodies against protein ligands are included. A variety of cell adhesion and recognition molecules are used in this technique. These include leukocyte integrins, which are known in the art. s), e.g. LFA-1 (lymphocyte function-associated antigen-1), Mac-1 (7 cells) Lofiano anti-11-1), VLA-4 (very late antigen-4) (very 1at e antigen-4), CR3 (complement receptor type 3), CR11 (complement receptor type 3), receptor type 4), 1, euM5, etc., but these are limited to munol, 1 8: LO79-1088 (1988); I) avign.

n et al, , Proc, Na11. Acad, Sci　USΔ78:45 35-4539 (1981): Marlin and Springer, Ce l　I-5↓　813-819　(1987); S+:+r, and cited therein. Please refer to the references provided. Cell adhesion molecules are mainly involved in immune and inflammatory responses. contains cell surface glycoproteins that promote cell-cell adhesion. Other adhesive or rese Examples of protein molecules include LFA-3, Ic\Ml, ICΔM-2, and VCAM. -1, EL ΔN4-1, CD-2, 1) I, '30.1) Melon IJ etc. be caught.

The silkworms of the invention contain more than one inhibitor molecule for adhesion molecules or receptors. nothing. The composition comprises an inhibitor of the receptor-ligand pair. (e.g. LFA-1 used in conjunction with an ICAM-1 inhibitor) Inhibitors and VLA-4 inhibitors used with inhibitors of VCAM-1 ).

Inhibitors of the invention prevent adhesion molecules from generating an immune response. Tl1l immunorecognition ■ Facilitates binding of cells to their targets and transmits regulatory signals to T cells Thus, adhesion receptors as well as 1' cell receptors are required. inhibitor The molecule inhibits activation of antigen receptors on T cells or B cells. favorable inhibition Agents include antibodies directed against adhesion receptors or ligands. The term “antibody” The term refers to polyclonal and monoclonal intact molecules, as well as antigen-binding including both Fab, F(ab)2, and Fv. nothing.

Specific antibodies for adhesion molecules and receptor ligands are known in the art. Contains things.

However, once an adhesion or receptor molecule is identified, the production of antibodies that bind to the adhesion molecule Methods are available in the art for. of antibodies or antibody fragments Several methods for production are available in the art. 1 deviation in such a method al, Cold Spring Ha rbor Laboratory.

Co1d Spring 1 Larbor, NY (1988): Cited by Davi. Please see the references used. Describe the general principles of immunology A,,”Monoclonal Δntibody 1’Al5ivier, A msterdam (1984); and EisPh i l ad-@I Includes research by Ph.I.A. (1980).

Both reclonal and monoclonal antibodies are used according to the invention as described. be able to. Further produced in humans or by recombinant or pond methods antibodies that have been human-adapted (i.e. not immunogenic in humans) or their Functional derivatives can be used. Antibodies adapted to humans can be used, for example, for antibody immunization. Substituting a corresponding but non-immunogenic moiety (i.e. chimera) antibody) produced by 7: Tan i guch i, M,, European patent Application No. 171.496+Mar Regarding General Considerations of Human Adapted Chimeric Antibodies Morrison.

986).

As described, antagonizing agents may be used before or after allograft transplantation to prevent rejection. Ru. The method of the present invention can be applied to, but not limited to, the heart, kidney, liver, bone marrow. For use in the case of any allograft of an organ or tissue, including cells, skin, etc. Can be done.

The inhibitor compositions of the present invention can be administered by injection, rapid infusion, nasopharyngeal inhalation (nasopharyngeal), or skin absorption. It can be administered orally or orally. In another case, the composition It can be administered intramuscularly or intravenously. Compositions for parenteral administration include Includes bacterial aqueous or non-aqueous solutions, renal suspensions and emulsions. An example of a non-aqueous solvent is polyp Lopyrene, glycol, polyethylene, glycol, vegetable oil, e.g. Oliveura , and injectable esters such as ethyl oleate. Increases skin permeability However, a bandage or occlusive dressing can be used to enhance absorption.

The inhibitor composition is 111. Can be used alone or in combination with other therapeutic agents Ru. The compositions of the present invention can be used in pharmaceutical preparations, such as in admixture with a pharmaceutically acceptable carrier vehicle. 6th Ed,) according to known h-methods for the preparation of scientifically useful compositions. 0801. △,,cd,,Mack,lCa5ton.

1) Described in A(+980). The production rJi logically γ suitable for n-effective ``Such compositions may be used in order to form , or suitable i! Contains a single effective dose of inhibitor in combination with l1F-vehicle.

Ike's Pharmaceutical JJ method adds polymers that can control the duration of action. 1w of the antibody or antibody fragment I-/therapeutic composition of the present invention using By combining, a modified release preparation can be obtained.

The therapeutic or diagnostic composition of the present invention shall be administered to a person in a therapeutically effective amount. In other words, to maintain the function of the transplanted allograft and prevent graft rejection. Administer in sufficient amount for The effective amount of the composition depends on the body weight, sex, age, and past medical history. It changes according to medical history. Others that affect the effective amount Factors include the severity of the patient's condition, the type of allograft, and the relationship between the specific protein and therapeutic composition. This includes, but is not limited to, the kinetics of interactions between generally group The product is about tu g - about 200 mg of antibody, and more precisely - about 50 u g - about 10 Administered in dosages ranging from 0 mg. animals to further limit specific dosages. A model can be used.

The antibody/inhibitor molecules of the present invention can be used in any biochemistry to prepare injectable pores. It can also be dissolved in physically acceptable liquids. Molecules in normal saline The pharmaceutical composition of the present invention which prepares such pores by dissolving one It is commonly used in the treatment of transplant recipients before and/or after transplantation. transplanted allograft Treatments can be repeated to preserve explant function. Generally the composition is transplanted or immediately after transplant surgery. Duration of treatment depends on patient condition It will change in about a few hours to a few weeks. In other cases, the first few hours, days or One series of treatments every few weeks (can be done once the first treatment or one series of treatments is completed) Once completed, the composition is only administered from time to time. i.e. after the first treatment the composition is It is given only if there are complications or signs of transplant rejection.

Since adhesion molecules are associated with inflammatory responses, the methods of the invention may be used to treat inflammatory responses. It turns out that you can do this. Additionally, the method can be used to treat autoimmune diseases or other T-cell-mediated diseases. It can be used for =l+ in the response.

Although the present invention has been generally described, reference may be made to certain specific embodiments to illustrate the present invention. light will be better understood. Examples are included herein for illustrative purposes. and are not intended to limit the invention unless otherwise specified.

Moaning Recent advances in research on adhesion molecules have shown that 5-433 in the emergence of immune responses. (1990)). T cell immune recognition promotes the binding of r cells to their Adhesion receptors and Tl11 cell receptors are stimulated by delivering regulatory knobnals to cells. Requires scepter. Lymphocyte function-associated antigen 1 (1, FAi) and intercellular adhesion Child 1 (ICAM-1) has such important heterophi ic) the adhesion receptor-ligand pair Rock, J, Imm, uno-1,14 6 (10) + 3273-9 (199])) activation of the antigen receptor on L FA-1 binds to its ligand with higher affinity. Also, LFA-1 and IC AM-] phase interaction is required for optimal T cell function in vitro (Makg Oba Fart Lord” ↓:619-624 (1989)). Therefore, for these antigens Monoclonal antibodies that inhibit NM heart disease are potential agents for the prevention of graft rejection. Using an allograft model, we demonstrated for the first time the strong effects of these antibodies in allografted mice. show.

Monoclonal antibodies used in this study, KBA (IgG2a) (Ni) Mouse CDI J, ((a chain of LFA-1), CD18 (summer, β chain of FA-1) ) and rat immunoglobulin against ICAM-1. produce these antibodies The hybridoma cells were treated with 10% fetal bovine serum and 1% gentamicin. The cells were cultured in RPM11640 supplemented with

Monoclonal antibodies were used in ascites of nude mice injected with these hybridomas. was purified using a protein G affinity column.

rla　l　+)/C(112')　(All animals are Ch　a　r　l　e　s Purchased from Rivcr Resources (13oSton). motion All physical experiments are on Comm1ttee on Re5earch Δnimal Approved by Care Protocol Review Group, Ma Ssachusetts General l1 ospital finger button (I″f ). The heart was infected with C311/l1c (II) using the microsurgery method. 21’) was transplanted heterotopically into a patient (Isobc C1at, Circ ulat'+on (1991, in press)). Heart graft survival The cessation of the beating of the f8 fragment is explained as the completion of rejection, and f = (Iso be ct at, , C1rculation (1991゜in press)).

TL every day for 6 days starting immediately after surgery! Treated by intraperitoneal injection of antibodies manufactured by I. There are 11 positions.

For complete r of 112i fusion, matching 1-1, immunosuppression (without j The allografts were allografted within I (Table 1). YNl, / L7 or K 13Δ at 1 year old jnIT 1.00 II g dose Treated animals compared to control mice, as shown by the persistence of heartbeats in implants 11. Significantly allogeneic transplantation;) survival of γR was sustained. But all these animals i! The allograft was rejected within 50 [ ]. Treated with the same amount of Ml, 8/2 The graft life (T was not prolonged.YNI/1.7 or KI3A In contrast to the results observed in the case of only 50 μg of KnA with 50 μg of All 6 animals treated with YNI7'17 of ttg remain under observation. All (75-1,50 patients) received cardiac allografts. of these allografts. The beating strength and heart rate were the same as in the syngeneic grafts.

Histology performed on C31f/lie recipients of Ba1l)/c cardiac allografts Analyzes showed that mononuclear cell infiltration in grafts treated with the two antibodies compared to untreated grafts. showed a significant decrease. 7 days after transfer without immunosuppressive treatment ['Allografts show extensive infiltration of leukocytes with myocyte necrosis and intratissue hemorrhage. It showed moisture. The results show that 'r'N+/1.7 and K for 60 years starting immediately after transplantation. In sharp contrast to allograft recipients treated with r3A monoclonal antibody.

These animals showed diffuse intratissue leukocyte infiltration and graded rA rejection at transplantation i & 70 days. Absolutely (nighhamet al,, J, l1eart Tra nsplant 9(6):587-593 (1,990)), myosite There was no necrosis. Allografts examined 40.75 and 120 days after transplantation showed fibrosis. areas were scattered and showed no evidence of active rejection.

Cell-mediated cytotoxic activity of recipient visceral cells was examined at 7.40 and 75 days post-transplant. (Table 2) On day 7, allograft mice that did not receive immunosuppressive treatment Their ILV visceral cells harbor donor syngenic MIIC antigens. It exhibited cytotoxic activity against tumor cells. Kr3Δ or Kr3Δ and YNI Allograft mice treated with both 1.7 and 1.7 were normal virgin mice (virgin ) showed no increased cytotoxic activity. Processed with YNI, /1.7 Mice gave intermediate results. Regarding K13A/YNI/l',...7 treated mice These observations were consistent on days 40 and 75.

To further assess the tolerance of these mice, we attempted skin grafting on them. . Four mice with long-term (65-72 days) viable cardiac allografts were given donor Genotype (Balb/c) and third group (th i rdparty) (C57[ 3L/6.112'') body skin was grafted at the same time.

Usually all animals are transplanted i! ! Reject the 311th skin at day 1-14. but They showed that the fl I accepted it. All heart grafts continued to pulsate during observation. The results are these This clearly demonstrates the existence of antigen-specific tolerance in mice.

Examining the expression of LFA-1 and LCΔM-1 on allogeneic transplanted mouse thallus cells Indirect immunofluorescence staining for mAB treatment was performed on the microscopic surface 7 days after implantation with each antibody. It was shown that the original downmodulation occurred.

This down-regulating noyon was detected by heteroreactive cytotoxic T lymphocyte activity on day 7. The study also explains the inability to produce the antigen, and also explains the induction of tolerance to alloantigens.

Expression of CFA-1 and Ic8-1 returns to normal after transplantation, but heterogeneous Reactive cytotoxicity 1゛lymphocyte +11 was still not detected.

The mechanism of this sustained response has not yet been elucidated. L　FΔ-1/ICΔ Because M-1-mediated cell adhesion is an essential part of T cell function (Spri-mediated adhesion It is reasonable to think that '1. Temporary inhibition of this adhesion system and the introduction of large amounts of alloantigen are It appears to promote the induction of responsiveness. The population of CD11a-positive cells remains normal during chronic Evidence that resistance returns to the normal range indicates that resistance is greater than removal of LFA-1 and ICΔN1-1 molecules. This means that it is maintained by some external mechanism.

The most interesting finding in this experiment is that anti-I CAM-1 and anti-CD11a The body appears to act synergistically to induce tolerance.

In vitro experiments showed that each antibody used in this experiment completely inhibited cell-mediated cytotoxicity in vitro. showed that injection of these antibodies individually had only a modest effect on sustained graft survival. It was shown that there is no such thing. Complete acceptance of the graft eliminates two types of antibodies at the same time! ) after was achieved. Although this synergy must be further investigated, C. FA-1 has at least three types of ligands, Fougerolles et a l,, J, Ishixp, Med, 174, 253-267 (1,991,)) This synergism can be partially explained by the fact that Also, ICAM-1 Mac-1 (Diamond et al., cell 65:961-971 (1991)), which mainly contains lq′ pith and natural expressed on killer cells (Kishi et al., Δdv, + mmuno 1.46: 14G-182 (1989)) o Due to this complexity Although the role of these adhesion molecules in rejection has not been determined, interference with cell adhesion is most effective after inhibition of both sides of the adhesion pair.

Whatever the mechanism, these observations suggest that ICAM-1/LFAi in the pathogenesis of rejection. clearly demonstrate the importance of adhesion and provide a rationale for applying this mode of immunosuppression to patients. Suggests.

Table 1. C: HT/I (e) of cardiac allografts (Balb/c) transplanted into mice. Number of days to live. Heterophilic heart transplantation was performed using the microsurgery method (Cosir ni et at, J. Immunol, 144:4604-4612 (1 990)). Recipient mice received 100 tlg of YNI/1.7.100 ug of Either Kr3A or 50 μg YNI/1.7 plus 50 μg KBA each time. Injections were given starting immediately after surgery and continuing until day 5 after transplantation.

Survival times of YNI/1.7 and KBΔ-treated mice were normal (no immunosuppression); significantly longer than YNI/1.7 or KBA treated mice (p<none 6 7. 7.8.8.8.10 8.0±11YNI/1.7 6 Ratio 12. +2.13 ．． 15.23 14.3±4.5KBΔ5 17.20.25.38,47 29.4±12.7M18/2 6 7.8.9.9. IO, IO8, 8±12 '1'N1/1. 7 6 > 70. >70. >70. >70. >70 plus Kr3Δ>70. >70 Table 2. Cytotoxic T-lymph assay. Ba1b/c recipient C3H/lie mice Mouse hearts were sacrificed 7.110 or 75 days after transplantation. They are opened on the day of transplantation. 100 tt g of YNI/1.7, Kr3A or each daily for the first 5 days. They were given 50 ttg of two different antibodies. to 175mM ammonium chloride After further lysing the red blood cells, fresh glenoid cells were washed three times. Labeled with 51 chromium P815 cells were used as target cells (4xlO4/well) and standard 4h cells were used. Mediated lymphocyte lysis assay was performed. Results are expressed as percent lysis. data is the average of three runs, and the natural release is 15-25% of the maximum release in all experiments. %Met. The experiment was repeated with consistent results.

effector/target Treatment Heart graft Post-operative days 5 20 None + 7 63 20.8 YNI/1.7 + 7 5.2 15.4KB-A + 7 territory 1 6. -8 YN1/1.7 + 7 1.2 83 plus KBA YNI/1.7 + 7 2.5 8.3 plus KBΔ YNI/1.7 + 40 1.8 3.6 Brass KBA None -75*2.3G, 5 *Recipient mice were given donor isogenic and group 3 skin injections 8 days prior to the cytotoxicity assay. The skin was transplanted.

Medicine, immunology, vibridoma forensic pharmacology and/or related matters when carrying out the present invention Modifications to the above format that are obvious to those skilled in the art are within the scope of the following claims. shall be included.

All publications and patent applications cited herein are references to the art to which the present invention pertains. Indicates the level of proficiency of an expert in a field. All literature and patent applications , by citing each document and patent application, the matters described therein become the content of this specification. by citation as if it were specifically and individually indicated. The matters constitute the content of this specification.

The foregoing invention has been described in some detail by way of example and embodiments for clarity of understanding. Although certain changes and modifications may be practiced within the scope of the appended claims. It is clear that it can be done.

Copy and translation of amendment) Submission (Article 184-8 of the Patent Law) March 31, 1994

Claims (12)

[Claims] 1. at least one inhibitor is specific for the receptor of the receptor-ligand pair; and at least one inhibitor is specific for the ligand of the receptor-ligand pair. Compositions comprising at least two anti-adhesion molecule antibody inhibitors are administered in therapeutically effective amounts. Preventing allograft rejection or transplanted allografts, including administering to mammals How to maintain the functionality of explants. 2. The anti-adhesion molecule antibody inhibiting agent is LFA-1, ICAM-1, Mac-1, CR. 3, CR4, LcuM5, VCAM-1, Vl, A-4, ELAM-1, CD- 2. The method according to claim 1, characterized in that the method is selected from LFA-2 and LFA-3. 3. The method according to claim 2, wherein the antibody is a monoclonal antibody. Method. 4. A receptor-specific inhibitor of the receptor-ligand pair inhibits LFA-1. 1CAM is an antibody specific for the ligand of the receptor-ligand pair. 2. The method according to claim 1, wherein the antibody is an antibody against -1. 5. A receptor-specific inhibitor of the receptor-ligand pair inhibits VLA-4. antibodies specific for the ligand of the receptor-ligand pair or VCAM 2. The method according to claim 1, wherein the method is an antibody against -1. 6. at least one inhibitor is specific for the receptor of the receptor-ligand pair; and at least one inhibitor is specific for the ligand of the receptor-ligand pair. A medicament for use in therapy comprising at least two anti-adhesion molecule antibody inhibitors chemical composition. 7. The anti-adhesion molecule antibody inhibitors include LFA-1, ICAM-1, Mac-1, CR 3, CR4, LcuM5, VCAM-1, VLA-4, ELAM-1, CD-2 and LFA-3. 8. Claim 7, wherein the antibody is a monoclonal antibody. Composition. 9. Receptor-specific inhibitors of the receptor-ligand pair inhibit LFA-1. ICAM is an antibody specific for the ligand of the receptor-ligand pair. 7. The composition according to claim 6, which is an antibody against -1. 10. A receptor-specific inhibitor of the receptor-ligand pair is directed against VLA-4. VCA is a receptor-ligand pair ligand-specific inhibitor. The composition according to claim 6, which is an antibody against M-1. 11. To prevent allograft rejection or maintain the function of a transplanted allograft A composition according to any one of claims 6-10 for use in. 12. To prevent allograft rejection or maintain the function of a transplanted allograft The anti-adhesive agent according to any one of claims 6 to 10, for the manufacture of a drug for use in Utilization of adhesion molecule antibody inhibitors.