Prevention and Treatment of Renal Allograft Rejection: New Therapeutic Approaches and New Insights Into Established Therapies1

Christopher V. Lu,2 Stanley Carlos Sicher, and Miguel A. Vazquez

“ hemotherapeutic agents are not only CV. Lu, S.C. Sicher, M.A. Vazquez, The University Trans- . e #{149} ends In themselves but also serve as plant Program at Parkland Memorial Hospital, Division tools for unlocking doors and probing Nature’s mys- of Nephrology, Department of Internal Medicine, Uni- terles. . versity of Texas Southwestern Medical School, Dallas, Gertrude B. Elion, lecture upon receiving the Nobel TX Prize for Physiology or Medicine (1) (J. Am, Soc. Nephrol. 1993; 4:1239-1256) There is a dynamic interaction between clinical transplantation and basic . On the one hand, new insights by basic scientists have resulted ABSTRACT in new therapeutic agents that may totally revolu- Renal transplantation is the preferred treatment mo- tionize therapy in the next decade. On the other hand, dality for patients with ESRD who are good surgical in solving clinical problems, a new understanding of risks and able to comply with chronic immunosup- basic immunologic processes has emerged. The pur- pressive . Clinical transplantation has pose of this review is not only to discuss new agents that may be part of the coming revolution in clinical advanced significantly, with most transplant centers transplantation, but also to discuss what these reporting 1-yr renal allograft survival rates of better agents have taught us about basic immunologic proc- than 80%. Nevertheless, rejection and a progressive esses. We will also examine new insights into several loss of allogratts over time continue to occur. The established agents. In the near future, the challenge immunosuppressive agents currently used may lead to transplant nephrologists and surgeons will be to to the development of life-threatening infections, use new, powerful agents intelligently. This will re- malignancies, and advanced atherosclerosis as a quire understanding their mechanisms of action. consequence of some of their side effects. This re- view examines the mechanisms involved in allograff MECHANISMS OF RENAL ALLOGRAFT rejection as currently understood. The recent knowl- REJECTION edge into the mechanism of action of cyclosporine, Before discussing the new immunosuppressive FK506, and rapamycin on activation is pre- drugs and monoclonal , we briefly outline sented. Information recently available on some of the mechanisms of allograft rejection. Processes in- the established therapies such as steroids, antime- hibited by specific drugs or monoclonal antibodies tabolites and monoclonal antibodies as well as the will be discussed in greater detail later in this review. newer agents is also discussed. The interaction be- Two different classes of T lymphocytes are neces- tween clinical transplantation and basic research in sary for the most vigorous allograft rejection (2). One is the CTL or cytotoxic T lymphocyte. These T cells immunology continues to result in exciting advances have a cell-surface marker called CD8. An Interac- in both fields. tion of a naive CTL with its target cell is by Itself not Key Words: Transplantation, , rejection, sufficient to result In lysis of the target cell. The aio graft. kidney naive CTL must receive accessory signals from a helper T cell. The latter cells have a cell-surface Received February 4, 1993. Accepted July 2. 1993. 2Coffespondence to Dr. C. Lu, University Transplant Program at Parkiand Me- marker called CD4. These accessory signals include mortal Hospital, Division of Nephrology, University of Texas Southwestern Medi- interleukin (IL)-2, gamma, and IL-6. After cal School, 5323 Harry Hines Boulevard, Dallas, TX 75235-8856. receiving these accessory signals, the naive CTL be- 1046-6673/0406- 1239$03.00/0 comes fully competent to kill the allograft kidney cell. Journal of the American Society of Nephrology Copyright © 1993 by the American Society of Nephrology The accessory signals are secreted by helper T cells.

Journal of the American Society of Nephrology 1239 Prevention and Treatment of Renal Allograft Rejection

Note that two signals are necessary to stimulate helper T cells (for a review, see references 9 and 10). craft cell There is an antigen-specific interaction between the T cell receptor and Its specific antigen. There must also be stimulation of the T cell by accessory signals

such as B7 or IL- 1 . The activated T cell then secretes additional lymphokines, including IL-2, IL-4, granu- locyte-macrophage colony stimulating factor, and in- terferon gamma. These lymphokines activate mac- rophages and B cells and change the biology of the allograft so that it is more susceptible to rejection. As an example of the latter, interferon gamma increases the amount of MHC antigens on the allograft and thus makes it better able to stimulate host T cells. In addition, the lymphokines stimulate bone marrow to stimulate bone marrow produce more inflammatory cells. Thus, the activated to produce inflammatory cells activate rnacrophages T cell performs many activities that coordinate the activate B cells complex process of allograft rejection. In addition, increase MHC ags on allograft the entire process is amplified when the activated T cell proliferates. DRUGS INTERFERING WITH BIOSYNTHESIS & THUS DNA SYNTHESIS AND PROLIFERATION . RS61443. IMMUNOSUPPRESSIVE DRUGS MIZORIBINE Drugs Binding lmmunophilins: Cyclosporines, Figure 1 . Outline of allograft rejection-activation of CD4 FK5O6, Rapamycin, and Their Analogs helper T cells. Reprinted with permission from reference 151. Ia, class II MHC antigen (ag). See Text. IFN, interferon The story of this family of drugs and how they gamma; IL2R, 11-2 receptor; GM-CSF, granulocyte-macro- work is an elegant example of the dialogue between phage colony-stimulating factor; IcR, I cell receptor. clinical transplantation and basic immunology. This dialogue is resulting not only In new, possibly safer

In Figure 1 , we review the activation of the helper and more effective, immunosuppressive drugs, but T cell in greater detail. As shown at the top of Figure also in a more profound understanding of T cell

1 , host helper T cells may be activated by interacting activation. directly with Class II major histocompatibility anti- These drugs may be grouped together because they gens (Class II MHC or Ia) on allograft cells. This Is the all bind cytosolic proteins called Immunophilins so-called “direct” pathway of T cell activation and (1 1 , 1 2). This binding Is critical for the immunosup- may occur either when host T cells encounter allo- pressive effect. The active intracellular inhibitor is graft cells in the allograft kidney (“peripheral sensi- not the drug alone, but a drug-immunophilin corn- tization”) or when donor dendritic cells (“passenger plex. It is therefore appropriate to briefly review the leukocytes”) emigrate to the host spleen and encoun- immunophilins in the next paragraph. ter host T cells at that site (“central sensitization”) Cyclosporines bind a group of immunophilins (3,4). Alternatively, antigens from the allograft may called . FK506 and rapamycin bind a be internalized by host macrophages or dendritic different group called FK506-binding proteins (ab- cells and digested into short peptides. These peptides breviated FKBP). In this review, we will focus on associate with host Ia molecules and are displayed A (12) and the 1 2-kd FKBP because they on the cell surfaces of these host cells. These then are the best understood of each immunophilin fam- stimulate the helper T cell. This is the so-called “in- ily. Proteins with amino acid sequences similar to direct” pathway of T cell activation (for example, see that of cyclophilin are present throughout nature references 5 and 6). The relative importance of these from Eschertchta colt to humans. This suggests that two alternative pathways in the activation of helper cyclophilin has such an important function that it T cells is not well established at this time. Note that, has been conserved through eons of evolution. Cyclo- in addition to presenting antigens to helper T cells, philins are present in all tissues, further suggesting macrophages also secrete important accessory mole- an important biologic function. The immunophilins cules, including IL-2, IL-6, and tumor necrosis factor- are peptidyl, prolyl, cis-trans . This alpha (TNF-ce). Among the large number of effects of means that they catalyze cis-trans conformations of these cytokines is their ability to activate the allograft peptides, perhaps allowing them to fold Into their endothelium (7,8). This results in the recruitment of active conformations and be transported to the ap- host lymphocytes, macrophages, and polymorpho- propriate places in the cell. For example, this occurs nuclear leukocytes into the allograft. In the development of the Drosophila eye. In this

1240 Volume 4 Number 6. 1993 Lu et al

model system, mutations in the cyclophilin called (17,18). Calcineurin is a calcium-acti- “ninaA” prevent the transport of rhodopsin Rh 1 from vated phosphatase. Its inhibition may prevent T cell the endoplasmic reticulum to the appropriate place activation in the following way. Normally, after the In the photoreceptor cell, and visual impairment re- T cell receptor is stimulated, intracellular calcium suits (13). rises. This activates calcineurln, which removes The discovery that cyclosporine, FK506, and ra- phosphate(s) from the nuclear factor of activated T pamycin inhibit the activity of immuno- cells (NFAT). This dephosphorylatlon allows NFAT to philins led to the idea that these drugs inhibited T enter the nucleus, where it combines with another cell activation by inhibiting isomerase activity. How- protein to form an active nuclear factor. The active ever, this idea is not true for the following reasons NFAT binds to and activates the gene for IL-2. ThIs (14,15). One, the immunophilins are broadly distrib- ultimately results In IL-2 secretion ( 1 4, 1 9). By Inhib- uted, and if these drugs acted by inhibiting isomerase iting the phosphatase activity of calcineurin, cyclo- activity, they should inhibit the activation of all cells. sporine-cyclophilin and FK506-FKBP complexes pre- Yet, the immunophilin-binding drugs usually only vent NFAT from moving from the cytoplasm to the inhibit T cells. Two, the concentrations of cyclospor- nucleus. Activation of the IL-2 gene does not occur me or FK506 required to inhibit T cells are far below (1 7, 1 8). Supporting this hypothesis is the correlation the concentrations needed to inhibit isomerase actlv- between the inhibition of T cell activation by analogs ity. Three, there is no correlation between the ability of FK506 or cyclosporine and the ability of the ana- of analogs of cyclosporine and FK506 to inhibit iso- log-immunophilin complex to inhibit calcineurin merase activity and the analog’s ability to inhibit T phosphatase activity (20). This hypothesis also ex- cell activation. Four, this idea fails to explain the plains the specificity of cyclosporine and FK506 for absence of correlation between the biochemIcal T cell activation because the nuclear binding factor structure and the immunosuppressive ability of these NFAT is found only in T cells. drugs. In other words, cyclosporine, a cyclic polypep- Also supporting this hypothesis is an observation tide, and FK506, a antibiotic, have com- of potential clinical importance. FK506 and rapa- pletely different biochemical structures, yet they in- mycin inhibit each other’s immunosuppressive ac- hibit the same step in T cell activation. The inhibited tivities (11,12,14). This is illustrated in Figure 3. step Is activation through the T cell receptor. In con- When FK506 binds to FKBP, the isomerase Is inhib- trast, rapamycin and FK506 are structurally related macrolide antibiotics and inhibit the same isomerase. However, despite their biochemical similarities, ra- pamycin and FK506 inhibit completely different steps of T cell activation (Figure 1). FK506 inhibits T cell activation via the T cell receptor, but not via the IL-2 receptor. On the other hand, rapamycin inhibits 7 Component X inhibited. activation via the IL-2 receptor but not via the T cell FKSO6 1-cell response to stimulation of IcR receptor (16). inhibited. No binning A hypothesis that explains all of the currently isomerase inrsbited to component Y available data states that the active intracellular im- 506BD munosuppressant is not the immunophilin-binding > cornponentsX or drug itself, but a drug-immunophilin complex i-n r’---’-i isomerase inhibited (11,12,14) (Figure 2). Thus, complexes of FK506- FKSO6 binding ______protein IFKBP) FKBP or cyclosporine-cyclophilin bind to and Inhibit Component Y inhibited. rapamycin T-cell response to lL-2 ANTIGEN inhibited. Binding to component X prevented. TcR

isomerase inhibited CALONEURIN rtL NT’ iTi NFAT.A8-.-I.ACflVATE 1-2 Figure 3. The complex of FK5O#{212}and FKBP inhibits I cell activation via the I cell receptor (IcR), whereas the com- Figure 2. The complex of cyclosporine (cya) and cyclo- plex of rapamycin and FKBP inhibits I cell activation via the philin inhibits I cell activation by inhibiting calcineurin. IcR, 11-2 receptor. This model illustrates how FK5O6 and rapa- T cell receptor; CA, calcium; Pi, phosphorus; NFAT-AB, ac- mycin might antagonize each other’s immunosuppressive tive heterodimer of nuclear factor of activated I cells. See activity. Component V. calcineurin. Reprinted with permis- Text. sion from reference 151. See Text.

Journal of the American Society of Nephrology 1241 Prevention and Treatment of Renal Allograft Rejection

ited. However, the important fact, as discussed Steroids above, is that the FK506-FKBP complex binds to and inhibits calcineurin (“component X” in Figure 3). On Steroids remain important immunosuppressive the other hand, when rapamycin binds to FKBP, the drugs. Although they are old agents, there is a new rapamycln-FKBP complex binds to a different pro- understanding of how steroids work. Steroids have tein, “component Y.” This inhibits the T cell response significant inhibitory actions at a number of differ- to stimulation via the IL-2 receptor, but not via the T ent stages of allograft rejection. cell receptor. By binding up all of the FKBP, rapa- As discussed in the previous section, cyclosporine mycin may prevent the formation of FK506-FKBP and FK506 prevent the activation of the IL-2 gene by complexes and thus prevent the inhibition of calci- preventing the formation of NFAT, the nuclear factor neurin (“component X” in Figure 3). This explains that activates this gene. Steroids also prevent the the ability of rapamycin to antagonize the immuno- activation of the IL-2 gene, but by an entirely differ-

suppressive effect of FK506 and vice versa (2 1 ). The ent mechanism (32,33). Steroids act, not by prevent- effects of 506BD, an analog of FK506, are particu- ing the formation of NFAT and other nuclear factors, larly instructive In this regard. 506BD binds to the but by preventing appropriate amounts of these flu- isomerase and InhIbits It. However, the 506BD-FKBP clear factors from activating the IL-2 gene. complex does not bind to either calcineurin (“compo- Steroids also inhibit T cell activation indirectly by nent X”) “component Y.” Thus, 506BD inhibits the inhibiting macrophage functions. Steroids inhibit the isomerase but has no immunosuppressive activity. expression of Ia molecules on macrophage cell sur- Indeed, by binding up to FKBP, 506BD prevents the faces (34-36). Thus, macrophages ingest antigens formation of FK506-FKBP complexes and thus an- shed by allograft cells, process these antigens, and tagonizes the immunosuppressive effects of FK506 then present the antigenic peptides to T cells in the (16,2 1). context of Ia molecules. This is the “indirect” pathway

Because cyclosporine binds to different immuno- of T cell activation (Figure 1 ) (5,6). T cell activation philins than FK506 and rapamycin, cyclosporine requires that the T cell receptor interact with the should not be antagonized by FK506 or rapamycin. antigen in the context of Ia. If steroids inhibit the Furthermore, because cyclosporine and rapamycin expression of Ia molecules, they will also inhibit the inhibit different stages in T cell activation (Figure 1), ability of macrophages to present antigen to and these two drugs are predicted to synergize with one activate T cells.

another. This prediction is valid in animal models Steroids inhibit the production of TNF-a IL- 1 , and (22). eicosanoids by macrophages (37,38). This has impor- “Component Y” in Figure 3, the target of the rapa- tant implications (8,39). A major event during allo- mycin-FKBP complex, has not been Identified. How- graft rejectIon is the Infiltration of allograft tissue by ever, rapamycin does selectively inhibit the activa- host inflammatory cells. In other words, the inflam- tion of p70 S6 kinase by IL-2 (23). This kinase may matory cells that are in the host’s bloodstream must be important in the response of T cells to IL-2. interact with the endothelium, such that the T cells, We have focused on the effects of cyclosporine, neutrophils, and monocytes translocate across the FK506 and rapamycin on T cell activation because endothelium into the allograft tissue, where rejection

these are the best-understood immunosuppressive then occurs. TNF-a, IL-i , and eicosanoids (prosta- activIties of these drugs. These drugs also have mul- glandins, leukotrienes, thromboxanes, and lipoxins) tiple other effects on the immune system, but a dis- activate aliograft endothelial cells such that they ex- cussion of them is beyond the scope of this review. press adhesion molecules, such as intercellular adhe- The reader is referred to several excellent recent sion molecule 1 (ICAM- 1), and selectins (for a review, reviews (15,24,25). see references 8, 40, and 41). These molecules cause This review is focused on the immunology of im- inflammatory cells to adhere to the endothelium and munosuppressive agents, and the clinical use of cy- then to move across it. In addition, eicosanoids are closporine, FK506, and rapamycin is not discussed. chemotactic agents that attract leukocytes to sites of There are several excellent recent reviews on the inflammation. Indeed, the antiinflammatory effects clinical use of cyclosporine (26-28). Cyclosporin G of nonsteroidal antiinflammatory agents, such as (29), an analog of cyclosporin A, may have similar aspirin and indomethacin, are the result of their immunosuppressive activities but less nephrotoxic- ability to inhibit the production of the eicosanoid ity. FK506 has been used successfully in transplan- prostaglandin E2. tation (30) but has not yet been compared with cyclo- The mechanisms by which steroids inhibit the pro- sporine in randomized, blinded clinical trials. Rapa- duction of the above proinflammatory molecules is mycin has not yet been tested in humans, but there now understood at a molecular level. Steroids inhibit is extensive preclinical experience (for a review, see both the transcription and the translation of the gene reference 31). encoding phospholipase A2 (42). Phospholipase A2 is

1242 Volume 4 Number 6 1993 Lu et al

important because it is the first in metabolic immunodeficiency occurring In patients whose pur- pathways leading to the production of all eicosanoids. inc metabolism is disrupted by an inherited defi- The idea that steroids act on lipocortin to inhibit ciency of adenosine deaminase or purine nucleoside phospholipase A2 (43) is not consistent with some phosphorylase (5 1 ,52). recent evidence (44,45). In addition, steroids also Azathioprine. The Nobel Prize-winning research Inhibit the gene for prostaglandin synthetase at the of Elion, Hitchings, and Black demonstrated that 6- translational level (46,47). The major inhibitory ef- mercaptopurlne was an effective antileukemic agent fects of steroids on the production of TNF-a and IL-i by virtue of its ability to interfere with the purine occur after these genes have been transcribed into metabolism of rapidly dividing leukemic cells. Robert mRNA. Steroids inhibit the translation of the mRNA Schwartz realized that lymphoblasts formed during into TNF-a (37) and IL-i fi (48,49) protein and de- an immune response resembled leukemic lympho- crease the stability of mRNA for IL-i f3 (38). cytes and demonstrated that 6-mercaptopurine was also an effective . Roy Calne successfully applied this insight to kidney transpian- Drugs Interfering With Purine and Pyrimidine tatlon (for reviews, see references 1 and 57). Metabolism Subsequent studies showed that azathioprine, the This class of drugs includes azathioprine, which is i -methyl-4-nitro-5-imidazolyl derivative of 6-mer- a well-established immunosuppressive agent, and captopurine, was even more effective than the parent the newer experimental drugs RS6 1443, mizoribine, compound (1). Azathioprine is slowly converted into and brequinar. These drugs all interfere with the 6-mercaptopurine and methylnitrolmidazole by glu- metabolism of either purine or pyrimidine nucleo- tathione and other sulfhydryl-containing molecules sides. Their effects may not be limited simply to the In red blood cells. This results in more sustained inhibition of DNA synthesis and the proliferation of levels of 6-mercaptopurine and methylnitroimidazole immune cells. As shown in Figure 4, nucleotides are in serum, both of which are important in the immu- important in a number of additional cellular proc- nosuppressive effect of azathioprlne. The effect of esses (50). UDP and GDP are Important in the glyco- the 6-mercaptopurine moiety of azathioprine will be sylation of proteins. Such glycoproteins Include the discussed first. and other adhesion molecules Important in Although the metabolic pathways are intricate, allograft rejection (see “Antibodies to Adhesion Mol- they must be reviewed because they have Important ecules” section). ATP is the universal currency of clinical implications. In an initial critical step. 6- energy for cellular processes. ATP and GTP, via its mercaptopurine is converted to 6-thioinosinic acid effects on G proteins, are important intermediates in (TIMP) by the enzyme hypoxanthine-guanine phos- intracellular signalling. The importance of purines phoribosyltransferase (HGPRT) (Figure 5). TIMP is in lymphocyte function is illustrated by the severe then converted to thionucleotides, which inhibit the

FORMATiON OF DNA A.SD I THUS LYMPHOCYTE PROLIFERATION

/ I FORMATIONOFGTP ADE NUCLEOTIDLS I’ #{188} ARE COMPONENES OF TIlE COVZYMES NAD+, FAD, & REGULATES LYMPHOCYTE & POWERS MOVLML OF CoA) ThIPORTANT i MACROPHAGE ACTIVATION MACROMOLECULES METABOLISM OF PROTE4S. VIA “G-PROTES T4 CELLS CARBOHYDRATES, & FATS

Figure 4. Importance of purine and pyrimidine metabolism on various cellular functions. CoA, coenzyme A; FAD, flavin adenine nucleotide. See Text.

Journal of the American Society of Nephrology 1243 Prevention and Treatment of Renal Allograft Rejection

RThOWS’PHOSPHATE of 6-mercaptopurine ( 1 ). The mechanisms underlying the greater sensitivity of lymphocytes and neutro- R1$OSE PHOSPHATE PYROPBOSPHOIC4ASE phils to azathioprine, as opposed to other rapidly (PR?? SYN’I’IlETASE) replicating cells such as gut epithella, are not known. It is important to emphasize the potential clinical importance of the salvage pathway (see HGPRT in Figures 5 and 6) in the immunosuppression by 6- mercaptopurine. After prolonged therapy with aza- thioprine, some T lymphocytes mutate and become deficient in HGPRT. These T lymphocytes do not convert 6-mercaptopurine to TIMP and thus are not inhibited. The appearance of such mutated HGPRT- deficient lymphocytes may result in acute rejection (53). In a similar fashion, patients with Lesch-Nyhan syndrome, who lack HGPRT on a genetic basis, also SALVAGE PATHWAYr resist the immunosuppressive effects of 6-mercap- HYPOXANTRP4E-GUANP4E PHOSPHORIBOSYL

TRANSFERASE (HGPRT) topurine (53,54). I 6-Mercaptopurine may be inactivated in two ways ADENP4E GUANPIE before it is converted by HGPRT to TIMP (Figure 6). (6.MERCAFTOI’UR1NE (6MP)j 6-Mercaptopurine may be inactivated by the enzyme thiopurine methyltransferase (TPMT). This is clini- Cor&D cally important because there is a wide range of TPMT activity among patients. Those with high Figure 5. Inhibition of purine metabolism by azathioprine. TPMT activity will have reduced availability of 6- RS61443, and mizoribine. See reference 50. De novo syn- mercaptopurine for conversion by HGPRT to TIMP. thesis of purines. The first committed step of the de novo Those very rare patients with low TPMT activity will pathway is the conversion of ribose-5-phosphate to 5-phos- be at risk for myelotoxicity (55,56). 6-Mercaptopurine phoribosyl- 1-pyrophosphate (PRPP) by the enzyme ribose may also be inactivated when converted to thiouric phosphate pyrophosphokinase (also known as PRPP synthe- acid by xanthine oxidase. Although this pathway has tase). PRPP is converted to inosinate (IMP) and then to adenylate (AMP) or guanylate (GMP). These products of de little genetic variation (55), it is important for the novo synthesis inhibit PRPP synthetase and thus prevent transplant nephrologIst because it is inhibited by excessive production of AMP and GMP (152). Some authors allopurinol. Thus, the administration of allopurinol (153) have suggested that adenine and guanine nucleo- to patients receiving azathioprine is dangerous be- tides differ in their effect on the PRPP synthetase of human lymphocytes. Large amounts of the former inhibit PRPP syn- thetase, whereas large amounts of the latter stimulate PRPP AZATHIOPRDIE synthetase. Salvage pathway. The key enzyme of the sal- vage pathway is HGPRT. HGPRT converts adenine, 6-mer- IMIDAZOLE IMITHI-METHYL captopurine (6MP). and guanine into their respective nu- -MERCAFTOPURPlE MERCA?TOPURPJE cleotides. 6MP does not inhibit lymphocyte proliferation I unless it is converted into its nucleotide thioinosinate by ELECTRON.AFFPE ACID HGPRT. Sites of drug inhibition. (the IMIDAZOLE DERIVATIVES I OASE active moiety of mycoph.nolate mofetll (RS61443)) and mizoribine phosphate (the active metabolite of mizorlbine) inhibit IMP dehydrogenase, the enzyme converting inosi- CELLULAR TOXICiTY I HypoxANrRP4E-GuANuJE nate to guanylate. 6-MP is converted into thioinosinate by I PHOSPHORIBISYL HGPRT and then into other thionucleotides. These inhibit L PRPP synthetase and the conversion of inosinate into both THIO-NUCLEOTIDES adenylate and guanylate (1). The most marked inhibitory rrioit OF DENOVO effect is on the production of adenylate (59). PURPlE BIOSYNTHESIS (ESPECIALLY ADENOSD’t) Figure 6. Metabolic pathways of azathioprine. Azathioprine is converted to methylnitroimidazole and 6-mercatopurine, formation of 5-phosphoribosyl- 1 -pyrophosphate in each of which has immunosuppressive activities. The inac- the first committed step of de novo purine biosyn- tivation of 6-mercaptopurine by thiopurine methyltransfer- thesis and which also prevent the formation of aden- ase has some genetic variability. The inactivation of 6- ylate nucleotides by inhibiting inosinate dehydrogen- mercaptopurine by xanthine oxidase by allopurinol may ase. Very little of the thionucleotides are incorporated lead to increased toxicity. The inhibition of I cell activation into RNA or DNA, and such incorporation is unlikely by 6-mercaptopurine requires the purine salvage pathway. to be a major mechanism for the immunosuppression See Text and Figure 5.

1244 Volume 4 Number 6 ‘1993 Lu et al

cause there will be a marked increase in immuno- osine nucleotides than do other cell types. This view suppression and myelotoxicIty (57). Is supported by the normal lymphocyte function in Transplant patients taking azathioprine can have patients with Lesch-Nyhan syndrome, who have an successful pregnancies, although there is an mci- Inherited deficiency of HGPRT and therefore no pur- dence of congenital malformations and allograft fail- me salvage pathway (63). However, lymphocytes do ure. Although 6-mercaptopurine does cross the pla- use the purine salvage pathway, and the activity of centa, fetal tissues contain little HGPRT and the drug this pathway does increase with activation (67,68). cannot be converted into its active agent, thioinosine Indeed, the effect of 6-mercaptopurine on lympho- (57). cyte activation depends upon its conversion to TIMP Although one mode of azathioprine’s action de- by the purine salvage pathway, as discussed above pends upon the 6-mercaptopurine moiety, the meth- (Figures 5 and 6). There are at least two isoforms of ylnitroimidazole moiety Is also important (Figure 6). IMP dehydrogenase (69). It is possible that these have Because 6-mercaptopurine ultimately inhibits the differential sensitivity to mycophenolic acid and that biosynthesis of purine, particularly adenosmne (Fig- the sensitive isoform Is preferentially expressed In ure 5), the addition of adenine or hypoxanthmne re- activated lymphocytes. stores the depleted adenosmne pools and reverses the The long-term safety of mycophenolic acid in hu- inhibitory effect of 6-mercaptopurine in vitro. In con- mans has previously been demonstrated by its use in trast to 6-mercaptopurine, adenmne and hypoxan- treating psoriasis (70). At this point, a small clinical thine did not completely reverse the inhibitory effects trial with RS6 1 443 In human recipients of cadaveric of azathioprine. This indicates an additional inhibi- renal allografts has shown promising results when tory mechanism that is presently not well understood RS6 1 443 is used in concert with cyciosporine and

(59-62). steroids to prevent rejection (7 i ). Of special Interest Drugs Inhibiting Inosine Monophosphate Dehy- are anecdotal reports that RS6 1 443 may be used drogenase and the Production of Guanosine Nude- successfully to rescue allografts after other therapies otides-RS61443 (Mycophenolate Mofetil or My- have failed to reverse rejection. In animal studies, cophenolic Acid Morpholinoethylester) and Mizo- RS6 1 443 wIll successfully reverse rejection. even ribine (Bredinin). RS6 1 443 and mizoribine are two when treatmcnt Is delayed by several days (63). new drugs that inhibit inosine monophosphate (IMP) Mizoribine is another new immunosuppressive dehydrogenase. This enzyme is Important in the de agent that is similar to RS6i443. It must be phos- novo synthesis of guanosmne and deoxyguanosmne nu- phorylated before it becomes an inhibitor of IMP cleotides (Figure 5). dehydrogenase (Figure 5). Like RS61443, its Inhibi- RS61443 is the morpholmnoethyl ester of myco- tion of lymphocyte activation in vitro Is reversed by phenolic acid (63); the latter is the active inhibitor of guanosmne nucleotides (59). Mizorlbine phosphate IMP dehydrogenase. RS6 1 443, rather than myco- does not inhibit early events in T lymphocyte acti- phenolic acid itself, is used clinically because it has vation (activation of c-myc, c-myb, or IL-2 genes) (72). greater bioavailabillty after oral administration. High Three different small Japanese series of patients concentrations of RS6 1443 are present in the small Indicate that mizoribine in combination with steroids bowel, and this agent may therefore be especially and cyclospormne may be used successfully to prevent useful in future small bowel transplantation (64). rejection (73-75). RS6 1443 is converted by the liver into mycophenolic Brequinar Sodium. Brequmnar (BQR, DUP 785, NSC acid glucuronide and is secreted into the bile. The 368390) noncompetitlvely inhibIts dlhydro-orate de- glucuronide is then converted by intestinal glucuron- hydrogenase, a key enzyme in the de novo synthesis idase back into active mycophenolic acid, and a high of pyrimidmnes. This results In the depletion of thy- concentration is present in the bowel. This may also midine, uridine, and cytosine nucleotides, which are explain the gastroIntestinal toxicity of RS6i443, Important in DNA and RNA synthesis as well as in which is especially prominent in dogs. several other processes (Figure 4). Brequmnar has By inhibiting the de novo synthesis of guanosmne undergone extensive evaluation as a chemotherapeu- and deoxyguanosmne nucleotide. RS6 1443 has pow- tic agent to treat human malignancies. The drug is a erful inhibitory effects on rodent and dog lymphocyte powerful immunosuppressant In animal models (76). activation both in vitro and in vivo (63,65,66). This Agents Interfering With Delivery of Costimula- is expected, given the importance of nucleotides in tory Signals to T Cells: CTLA4Ig, Steroids, Anti- many cellular processes (Figure 4). The inhibiting ICAM-i, and Antilymphocyte function-associated effects may be reversed by the addition of guanosmne antigen-i. As reviewed in “Mechanisms of allograft nucleotides in in vitro systems (59). The effects of rejection-a brief outline” and Figure 1, T helper cells RS6 1443 appear to be relatively selective for lym- require two signals to become activated (77). Signal phocytes. Some suggest that this Is because lympho- One is delivered by the T cell receptor interacting cytes depend more on the de novo synthesis of guan- with alloantigen. This signal by Itself will tolerize the

Journal of the American Society of Nephrology 1245 Prevention and Treatment of Renal Allograft Rejection

T cell (78). However, activation does occur if a costi- mulatory signal, in addition to Signal One, is also INSIDE OUTSIDE received by a T cell. Several costimulatory signals CELL CELL have been Identified. One such signal is B7, a mole- cule found on the cell surfaces of antigen-presenting accessory cells (iO). This signal Is specifically inhib- CD3 c chain ited by a genetically engineered molecule called CTLA4Ig (79). In animal models, injections of CTLA4Ig cause antigen-specific tolerance to pan- CD3c chain creatic islet transplants (80) and prevent the rejection of cardiac allografts (8 i ). More preclmnical work will be needed before CTLA4Ig can be used in human I trials. ‘ychain Another costimulatory signal is IL-i (9). As dis- IL UI cussed earlier, steroids inhibit the production of IL-i and could thus prevent antiallograft T cell activation. TcR (3 chain ICAM- i , a cell surface molecule, may also act as a FI’t ii costimulatory signal. We will discuss the effects of monoclonal antibodies against this molecule below. ti J variable 14TCR a chain region

Other Drugs In addition to the agents discussed above, i 5-deox- L1aur yspergualmn effectively prevents and treats rejection ij in animals (82) and In human trials (83). SK&F 105685 (azaspIrene). prostaglandin E analogs, lipox- ygenase inhibitors, somatostatin, and tE1Echan are also immunosuppressive agents (for a review see reference 84). These drugs will not be discussed here Figure 7. The I cell receptor (IcR) complex. Shaded boxes because their effects on the immune system are not represent invariant regions between various I cell clones. yet well understood. Open boxes represent variable (idiotypic) regions. See Text. MONOCLONAL ANTIBODIES PRESENT AND FUTURE ognize different antigens. In addition to their variable A wide variety of monoclonal antibodies are in or idiotypic regions, the a and (3 chains of the T cell various stages of clinical use, trials, or preclmnical receptor also have “framework” regions. These are testing. It Is not possible to discuss each individually the scaffolding that support the variable regions and within this review. Instead, we have concentrated on are the same in most T cells. In an ideal world, the a few that illustrate new strategies or that are ac- transplant nephrologist would administer mono- tually In clinical use either In humans or in subhu- clonal antibodies that recognize the idlotypic regions man primates. For a full discussion of all of the of the T cell receptor a and /3 chains and thus destroy current agents, see reviews in references 85 through only T cells recognizing the allograft. Other T cells, 90. with other idiotypes that recognize pathogens such as tuberculosis, would be spared. Unfortunately, Monoclonal Antibodies Against the T Cell such monoclonal anti-idiotypic antibodies are not Receptor Complex available for clinical use. Instead, the available The T cell receptor complex consists of several monoclonal antibodies recognize invariant struc- proteins residing on the cell surface of T lymphocytes tures common to all T cells. These monoclonal anti- (for reviews, see references 9 1 and 92) (Figure 7). bodies thus inhibit not only those T cells recognizing This complex has two major functions. One is to the allograft, but also those recognizing infectious recognize specific alloantigens. This function is ful- agents and malignant cells, hence, the increased sus- filled by the “variable” regions of the a and fi chains ceptibility of transplant recipients to infections and of the T cell receptor. Together, the “variable” regions some malignancies. of the a and fi chains form the structure (idiotype) The other function of the T cell-receptor complex that recognizes alloantigens on the transplanted kid- is to transmit the signal that an antigen has been ney. These regions are “variable” in that they are recognized from the cell surface to the inside of the different in populations of T cells (clones) that rec- T cell so that intracellular biochemical events, such

1246 Volume 4’ Number 6’ 1993 Lu et al

as lymphocyte activation, can take place. This signal- of the molecule (“domains”) forming the ends of the transducing function resides in the other chains of “Y” bind to antigen. These are the variable regions of the T cell receptor complex or the CD3 complex. The the molecule. The stem of the “Y” forms a CD3 complex consists of peptides called e, #{244},‘y, and . structural support, activates complement. and allows These peptides perform the same function and do the antibody molecule to bind to neutrophils. lym- not vary from T cell to T cell. phocytes, and monocytes (for a review, see reference OKT3: the Use of Genetic Engineering To Over- 2). This is the constant region, or Fc region, of the come the Problems of OKT3 and Other Murine antibody molecule. Much of the human antibody Monoclonal Antibodies. OKT3 is the only mono- against OKT3 is directed against the constant region clonal antibody presently approved by the Food and of mouse monoclonal antibodies. By the use of ge- Drug Administration for use in transplantation. netic engineering, the immunogenicity of murine OKT3 is a mouse that binds to monoclonal antibodies such as OKT3 is decreased by the e chain of the T cell receptor complex. The clinical the construction of a “chimeric” molecule (Figure 8). efficacy of OKT3 has been reviewed previously Such “chimeric” antibodies have a human constant (87,93,94) and will not be discussed here. Instead, region and a mouse variable region. we will focus on new approaches to solve the two “Chimeric” monoclonal antibodies can be further major problems associated with this important agent. improved. Only the “hypervariable” regions of the Problem I. The development of antibodies against “variable” regions are critical for binding to antigen. OKT3 may be prevented by the construction of “Humanized” monoclonal antibodies constructed by human-mouse “chimeric” monoclonal antibodies genetic engineering consist of human amino acid and “humanized” monoclonal antibodies. As a mu- sequences, except at the “hypervariable” regions, rine protein, OKT3 Is foreign to humans and is there- where the original murine amino acid sequences are fore immunogenic. Patients make antibodies against present (Figure 8). Such “humanized” monoclonal an- OKT3. These human anti-murine OKT3 antibodies tibodies are not immunogenic for humans. In other generally peak 1 to 2 wk after the first course of words, patients produce few antimonoclonal anti- therapy and may reduce the efficacy of a second body antibodies and the “humanized” monoclonal an- course of OKT3 and the subsequent administration tibodies therefore have a very long half-life in the of any other monoclonal antibody derived from mice human circulation (88,89). HumanIzed OKT3 has (94,95). been constructed, but clinical trials have not yet been In the future, this problem may be prevented by reported (96). producing genetically engineered “chimeric” human- Problem II. The potentially dangerous “capillary mouse OKT3 or “humanized” OKT3 (88,89). Figure 8 leak” or “first-dose” syndrome may be prevented shows the Y-shaped structure of immunoglobulmn by new monoclonal antibodies that have Pc regions (Ig)G antibody molecules such as OKT3. The regions that do not bind Fc receptors on monocytes. Despite its effectiveness, OKT3 is often associated with fever, myalgia. vomiting, or diarrhea after the first doses. Occasionally, there are serious life-threatening com- plications such as pulmonary edema, hypotension, variable [ region and aseptic meningitis (93,97). This “first-dose” re- action results when OKT3 activates T cells and Fc 00 hypervariable regions Ec portion from mouse receptor-bearing monocytes by cross-linking the two II cell types (96). Activation results in the release of MOUSE ANTIBODY all other regions TNF-a, interferon gamma, and IL-2 (97-100). These human cytokmnes cause the symptoms of the first-dose re- mouse I action, as evidenced by the ability of these lympho- (‘II (‘\ variable iJ iJ region “HUMANIZED” ANTIBODY kines to produce a first-dose-like reaction when in- II jected into experimental animals (101). TNF-a may I human be particularly important because antl-TNF-a anti- Fc portion bodies prevent the first-dose syndrome in a murine

model (102). It is remarkable that IL-i fi and IL-i a are CHIMERIC not released during the first-dose response. MOUSE-HUMAN ANTIBODY It is important to note that cross-linking, and thus Figure 8. Structure of mouse antibody, chimeric mouse- activation, of T cells and monocytes depends criti- human antibody, and “humanized’ antibody. White areas cally upon the ability of the Fc region of OKT3 to are from the murine antibody genes. Dark areas are from bind to Fc receptors on monocytes (86,96). In a num- human antibody genes. See Text and reference 88 for ber of experimental systems, the first-dose reaction details. is alleviated if the Fc region of OKT3 is altered such

Journal of the American Society of Nephrology 1247 Prevention and Treatment of Renal Allograft Rejection

that it no longer binds to the . Examples Monoclonal Antibodies Specific for Activated include the destruction of the Fc region during the T Cells preparation of OKT3 (103), with an IgA Isotypic var- Iant of OKT3 where the Fc portion does not bind Fc Although the monoclonal antibodies against var- receptor ( 1 04), and the genetic engineering of the Fc bus components of the T cell receptor complex are region of OKT3 so that it does not bind Fcreceptors effective immunosuppressive agents, they are also (96). nonspecific in Inhibiting immune responses against At this time, the newer forms of OKT3 are not not only the allograft but also infectious agents. How- available. However, the first-dose reaction of OKT3 ever, monoclonal antibodies against antigens (“acti- can be alleviated by the administration of high-dose vation” antigens) found only on the surface of acti- steroids 1 h before OKT3. The timing of the steroid vated T cells may theoretically inhibit only antlallo- dose may be important. Steroids given simultane- graft responses (for reviews, see references 1 14- ously with OKT3 or 4 h after OKT3 were not effective 1 1 6). Presumably, at the time of transplant, only (99,105,106). those T cells against the kidney will be activated. Other Monoclonal Antibodies Against the T Cell These T cells will have cell-surface antigens unique Receptor Complex. In addition to OKT3, there are a to activated T cells. These would be killed by mono- number of other monoclonal antibodIes against the clonal antibodies against these activation antigens. T cell receptor complex that are undergoing clinical Desirable T cells against antigens, such as tubercu- trials to determine If their theoretical advantages losis, might be spared because these T cells are not prove true in clinical practice (for reviews, see refer- activated by the allograft. ences 85 to 87). Several of these antibodies have a The best-understood T cell activation antigen is the different Fc region than OKT3 and, because of this p55 IL-2 receptor (see references 1 1 5 and 1 1 6 for difference. cause a less severe first-dose reaction reviews). We will briefly discuss its biology so that than that discussed above. T 1 0B9. 1 A-3 1 is a mono- monoclonal antibodies directed against it can be clonal antibody against a framework region of the a! understood. There are three forms of the IL-2 recep- f3 chains of the T cell receptor complex (107-1 10). tor. First, resting T cells have a low-affinity receptor (See “Monoclonal Antibodies Against the T Cell on their cell surfaces. This is a single protein called Receptor Complex” for a discussion of “framework” p75 because it has a molecular mass of 75 kd. After versus “variable” regions of the T cell receptor.) the T cell is activated, an additional IL-2 receptor T 1 0B9. 1 A-3 1 and OKT3 have different Fc regions. appears on the cell surface. This is called p55 be- The Fc region of T1OB9. 1 A-3 1 is 1gM Fc, whereas cause of its molecular weight. The p55 molecule may OKT3 has an IgG2a Fc regIon. This may offer three exist as an individual cell surface molecule, and this advantages. First, T1OB9.1A-3i does not activate T Is the second form of the IL-2 receptor. The p55 cells in vitro, and this is correlated with a reduced molecule may also bind to the p’75 molecule and form “first dose” reaction in vivo. Second, most human a heterodimer with an extremely high affinity for IL- antIbodies against Ti 0B9. 1 A-3 1 do not cross-react 2. This is the third form of the IL-2 receptor. The with OKT3. Thus, it may be possible to give high-affinity IL-2 receptor allows the activated T cell Ti 0B9. i A-3 i to patients who have previously re- to be stimulated by IL-2 and further differentiate into ceived OKT3 and vice versa. Finally, the 1gM Fc effective cytotoxic T cells, for example. The pSS/p’75 region of Ti 0B9. 1 A-3 1 activates human comple- IL-2 receptor is also called CD25. The p55 protein is ment, and this results In the lysis of T cells. This also called “Tac.” may make Ti 0B9. 1 A-3 1 more effective than OKT3, From the point of view of the transplant nephrol- which has an IgG2a Fc region that does not activate ogist, an Important point of the above paragraph is human complement. that resting T cells do not have the p55 IL-2 receptor BMAO31 (85,111) is also a murine monoclonal an- on their cell surfaces. Only activated T cells have tibody against a framework region of the a/fl chains p55. Anti-p55 IL-2 receptor antibodies should thus of the T cell receptor complex. Unlike OKT3, BMA be ideal agents to use Immediately after transplant 031 has an IgG2b Fc region and may not elicit as in an “induction” or “sequential” protocol, instead of severe a first-dose reaction (112). the currently used antilymphocyte globulin or OKT3. Monoclonal antibodies that have the same Fc re- In theory, the only activated T cells present immedi- gion as OKT3 also elicit a strong first-dose response ately after a kidney transplant should be those di- and may therefore offer no advantages over OKT3. rected against the transplant. Those T cells would be For example, WT32 Is similar to OKT3 both in being destroyed by monoclonal antibodies against the p55 a murlne antl-CD3 monoclonal antibody and in hav- IL-2 receptor. Resting T cells, including those against Ing an IgG2a Fc region. As expected, WT32 is an infectious agents, would not express the p55 IL-2 effective immunosuppressive agent and also causes receptor and should not be destroyed by these mono- a severe first-dose reaction (113). clonal antibodies. According to this theory, there

1248 Volume 4’ Number 6’ 1993 Lu et al

should be fewer infectious complications in patients treated with anti-p55 IL-2 receptor antibodies. __000A In rodent experimental systems, monoclonal anti- p55 antIbodies have been effective immunosuppres- sive agents (for reviews, see references 114 and 117). There are a number of monoclonal antibodies against the human p55 IL-2 receptor that are undergoing clinical trials. These include 33B3. 1 (118,119), AED B which Is as effective as rabbit antithymocyte globulin in preventing rejection in a large trial of renal trans- EXAMPLES OF IMMUNOTOXIN plant recipients. Smaller numbers of patients have received anti-Tac (120), Campath-6 (or YTH-906) PARTS II AND III (121,122), and (BB1O)BT 563(123). PART I (RECOGNITION STRUCTURE) (INTRACELLULAR GUIDANCE SYSTEM & ACTUAL TOXIN) lmmunotoxins and Chimeric IL-2 PE4O Domain I II;o:n:::s ii & UI A difficulty with murine monoclonal antibodies against the pS5 IL-2 receptor, used In the above clinical trials, is the inefficiency of most murine IgG monoclonal anti-IL-2 recetj antibodies in fixing human complement and thus lysing T cells. This problem may be overcome by the attachment of a toxin to the monoclonal antibody. The monoclonal antibody thus delivers a toxic mole- interleukin-2 PE4O Domains II 8, Ii cule to the target T cell, which is killed by intoxication rather than by complement-mediated iysis. B Natural toxins produced by plants and bacteria are Figure 9. (A) Structure and function of natural toxins. Natural among the most deadly known substances (for re- toxins have three parts. ‘A-Part T binds to a specific views, see references 89 and 124 through 127). If as receptor on cell surfaces. The entire toxin is then endocy- few as one toxin molecule reaches the cytoplasm of tosed by the cell and resides temporarily within an endo- a cell, the protein synthetic machinery will not func- some, ‘B.’ Part ‘II’ then guides the endotoxin to the specific tion and the cell will die. Such toxins Include bacte- intracellular compartment, C, where Part ‘lIr exerts the toxic effect, ‘D.” Alternatively, the toxin may stay within the rial toxins, such as Pseudomonas exotoxin and diph- endosome and eventually be destroyed (‘Efl). The latter therla toxins, and plant toxins, such as ricin. These may be the fate of improperly constructed synthetic im- toxins all have three functional parts. One part of munotoxins. See Text and reference 126 for details. (B) the toxin molecule (Part I) recognizes and binds to Examples of immunotoxin. Domain III is the actual toxic part the surfaces of all cells. A second part (Part II) causes of a molecule of wild-type Pseudomonas exotoxin (PE4O). the toxin to be transported to the appropriate part of However, Domain Ill must reach the appropriate intracellu- the cell where the third part (Part III) of the toxin lar compartment to be active. This occurs because Domain molecule actually kills the cell (Figure 9). By various I binds to cell surfaces and facilitates endocytosis of the chemical and genetic engineering techniques, “Im- entire PE4O toxin complex. Furthermore, Domain II guides munotoxins” have been made where Part I of the Domain III to the appropriate intracellular compartment. Immunotoxins are constructed so that a monoclonal anti- toxin molecule has been replaced by a monoclonal body, such as that for the 11-2 receptor, is substituted for antibody. The toxin will now bind to and kill only Domain I. Domain Ill will now be directed only to I cells with cells recognized by the monoclonal antibody. Thus, 11-2 receptors. Similarly, 11-2 may also be substituted for Parts II and III of the Pseudomonas exotoxln and Domain I. See Text and reference 126. diptheria toxin have been attached to monoclonal antibodies against the p55 IL-2 receptor (126). This immunotoxin is undergoing clinical trials in the antIbody had an unanticipated cross-reactivity treatment of T cell leukemias, where the leukemic against neurons. The other concern is that some cells have a high expression of the p55 IL-2 receptor. functional reminants of the recognition structure of In general, Immunotoxins have been reserved for the toxin (Part I) might cause toxicity to normal cells, cancer patients who are in desperate clinical situa- as has been noted in one toxin-monoclonal antibody tions because of two concerns (89). One is toxicity conjugate against the p55 IL-2 receptor. If the effI- due to an unexpected cross-reactivity of the mono- cacy and safety of these immunotoxins can be dem- clonal antibody. For example, a monoclonal anti- onstrated in cancer patients, then these agents may body-toxin conjugate directed against breast cancer eventually be used in transplantation. caused severe neurotoxicity because the monoclonal A related approach has been to replace the recog-

Journal of the American Society of Nephrology 1249 Prevention and Treatment of Renal Allograft Rejection

nition part of the toxin molecule with IL-2, instead of an important event In T cell activation. Some mono- a monoclonal antibody against the p55 IL-2 receptor. clonal anti-CD4 antibodies prevent this event. The IL-2 part of the chimeric IL-2 toxin molecule will (3) Some monoclonal antI-CD4 antibodies induce bind to activated T cells and be internalized into the long-lasting antigen-specific tolerance in animal cytoplasm, where the toxin will kill the T cell. Such models when administered at the time of transplan- a chimeric immunotoxin (for reviews, see references tation (1 28). In other experiments, long-term allograft 1 1 4 and 1 1 7) has been used successfully in animal tolerance occurred when anti-CD4 was given with a models to treat rejection and autoimmune diseases. pretransplant transfusion of blood or membrane Another approach has been to attach a radioactive fragments containing donor MHC molecules. The atom to a monoclonal antibody and thus kill the mechanisms underlying this tolerance induction are target cell. Although this strategy has been used in not understood. One possibility is that separate liga- cancer patients, It has not yet been used in solid tion of CD4 and the T cell receptor results in the organ transplantation (88-90). death of mature T cells (1 32). Alternatively, there are at least two different subpopulations of CD4-positive T cells (Th 1 and Th2), one of which may inhibit Antibodies to CD4 antlallograft responses by secreting inhibitory lym- CD4 has characteristics that make it an attractive phokines such as IL- 1 0. It is possible that some anti- target for monoclonal antibodies in the prevention CD4 antibodies differentially affect this “suppressor” and treatment of rejection. Although CD4 is found on CD4-posItlve T cell subpopulatlon (for reviews, see many cells, including monocytes, macrophages. Lan- references 1 33 and 134). gerhans’ cells, eoslnophils, endothelial cells of he- The various anti-CD4 monoclonal antibodies rec- patic sinusoids, and sperm, the most important cell ognize different parts of the CD4 molecule and, not bearing CD4, from the transplant nephrologist’s unexpectedly, have different effects on T cell func- point of view, Is the helper T lymphocyte (for reviews, tion. Not all antibodies (1 30, 1 35) cause all of the see references 1 28 to 1 30). The following properties effects listed above. Some anti-CD4 monoclonal an- make CD4 an attractive target for monoclonal anti- tibodies remove CD4 T cells from the circulation; body therapy. others do not but still have potent immunosuppres- ( 1 ) CD4 on the surface of T cells binds to Ia (Class sive effects. II MHC) molecules on the surface of antigen-present- Infusions of OKT4A monoclonal anti-CD4 Into Ing cells and thus stabilizes T cell antigen-presenting Cynomolgus monkeys resulted in renal allograft sur- cell Interactions. In many cases, the antigen-pre- vival for 40 to 50 days (85, 1 36). Monoclonal anti- senting cell may be an allograft kidney cell. To un- CD4 has been used in small numbers of patients with derstand this role of CD4, one must first review the rheumatoid arthritis, vasculItis, multiple sclerosis, structure of the Class II MHC molecule (reviewed In and mycosis fungoides (for reviews, see references reference 131). The Class II MHC molecule consists 86 and 1 35). An early Phase I trial with BL4 antI- of two polypeptides. The portion of the molecule CD4 In human kidney transplantation in association (“membrane proximal”) closest to the cell surface has with prednisone and azathioprine has been reported an amino acid sequence that is conserved between (1 29). Chimeric anti-CD4 with human constant re- individuals. The portion of the molecule furthest gions and murine anti-CD4 (M-T4 12) has been used from the cell surface (“membrane distal”) contains in 22 human heart transplant patients in conjunc- regions where the amino acid sequences vary be- tion with prednisone, azathioprine, and cyclosporine. tween Individuals. This “membrane distal” portion (See Figure 8 for an explanation of chimeric antibod- also has a cleft that contains peptides from the inside ies.) This therapy resulted in decreased allograft re- of the antigen-presenting cell. The T cell receptor jections, fewer side effects, and fewer Infections than binds to the “membrane distal” portion of the Class in the control group (137). Other anti-CD4 mono- II MHC molecule. CD4 binds to the “membrane prox- clonal antibodies are either in or about to be tested imal” portion and stabilizes the interaction between In clinical trials (85). T cell and antigen-presenting cell. Some monoclonal Another useful feature of some anti-CD4 mono- anti-CD4 antibodies are immunosuppressive because clonal antibodies Is their ability to inhibit xenospe- they prevent, or disrupt, this Interaction. cific antibody responses. In other words, a major (2) Interaction between CD4 and the “membrane problem in using mouse antibodies against human proximal” portion of the Class II MHC molecule may lymphocyte antigens is the development of human cause CD4 to deliver an activating signal to the T cell. anti-mouse (xenospecific) antibodies. Such xenospe- The cytoplasmic portion of CD4 is associated with a cific antibodies may inactivate the murine mono- tyrosine protein klnase, p56lck. Interaction of the ex- clonal antibody. Some anti-CD4 antibodies prevent tracellular portion of CD4 with Class II MHC may the formation of such xenospecific antibodies. In one change the activity of ps6lck, which in turn may be animal model, the injection of anti-CD4 prevented

1250 Volume 4’ Number 6’ 1993 Lu ef al

the development of xenospeclfic antibodies against a and thus decrease damage during the reperfuslon of later injection of monoclonal antibodies against the the allograft (86). p55 IL-2 receptor (1 35). This increased the half-life In light of all of these considerations, it is not and effectiveness of the anti-p55 IL-2 antibody. This surprising that monoclonal antibodies against ICAM- strategy has yet to be tested In clinical trials. 1 and LFA- 1 should have Important immunosuppres- sive activities. For example, cardiac allografts sur- Antibodies Against Adhesion Molecules vive indefinitely In mice after treatment with mono- ICAM- I and Lymphocyte Function-Associated clonal antibodies against LFA-1 and ICAM-1 (145). Antigen-I Monoclonal antibodies against ICAM- 1 have been used both to prevent rejection and to treat ongoing ICAM- 1 (also called CD54) and lymphocyte func- rejection of renal allografts in Cynomolgus monkeys tion-associated antigen- 1 (LFA- 1 , also called CD 11 (1 39, 1 46). Although ICAM- 1 is on the surfaces of a/CD18fl) are important In allograft rejection (138). host and allograft endothelium and other cells, no First, they act as adhesion molecules. They stabilize vasculitis or other damage due to the anti-ICAM- 1 interactions between CD4-positlve T cells and alloan- antibodies was detected (139). tigen-presenting cells allowing T cell activation. An LFA- 1 molecule on one cell binds to an ICAM- 1 mol- Other Monoclonal Antibodies ecule on the surface of the other cell (Figure 10). In the same manner, LFA- 1 and ICAM- 1 also stabilize Most rodent monoclonal antibodies do not fit hu- interactions between CTL and their targets, allowing man complement well and do not destroy the human the targets to be killed. Furthermore, LFA- 1 and lymphocyte target. An important exception Is the ICAM- 1 are important adhesion molecules that allow Campath- 1 series of rat monoclonal antibodies, which recognizes the abundant antigen CD52 on the translocation of inflammatory cells into the allo- graft. In other words, these molecules cause leuko- lymphocyte surfaces and destroys these cells via the cytes to adhere to the capillary endothelium and then classic complement pathway. The IgG2b form, Cam- move across the endothelium into the allograft path-1G, has been used successfully to treat human (8,40,41). In addition to acting as adhesion molecules allograft rejection (1 47), and a humanized form has that stabilize cell-cell interactions, LFA- 1 and ICAM- been produced (148). 1 may also deliver activating signals from the extra- A virtually infinite number of monoclonal antibod- cellular environment into T cells. The levels of ICAM- ies could potentially be used to treat or prevent rejec- 1 on human allograft cell surfaces increase during tion. These include antibodies to the various lympho- the course of rejection in response to lymphoklnes kines, such as interferon gamma or TNF-a ( 1 49), as (139-143). This provides a positive feedback loop in well as other molecules on the surfaces of lympho- the lymphocyte-allograft interactions, which may ul- cytes. such as CD8, CD2, CD7, or CD5. We have timately lead to the destruction of allograft tubule limited our discussion to monoclonal antibodies ac- cells (144). Antibodies against ICAM- 1 may also pre- tually in use or undergoing successful human or vent neutrophils from adhering to the endothelium subhuman primate trials. The reader Is referred to other reviews for information on other monoclonal antibodies that may be used in human transpianta- A. Killer T lymphocyte Interaction tion (85-90,150).

CONCLUSION At this time, the only Food and Drug Administra- tion-approved agents to prevent and treat renal al- lograft rejection are steroids, azathloprine, cyclospor- me, OKT3, and polyclonal equine antithymocyte Im- B. munoglobulin. Although 1 -yr allograft survival is 80% or better In most centers, a significant number of patients still lose their transplants, particularly after 1 yr. The current agents have a number of severe side effects. Transplant nephrology stands at the threshold of a new era In which new agents and a new under- standing of basic immunologic processes may allow improved medical management. The intelligent use of the new armamentarlum will require an under- Figure 10. The roles of LFA-1 and ICAM- 1. IcR, I cell receptor. standing of how the new agents affect the immune See Text. Reprinted with permission from reference 138. system. This review has attempted to outline some

Journal of the American Society of Nephrology 1251 Prevention and Treatment of Renal Allograff Rejection

of these new agents and what they have taught us cell signal transduction. J Biol Chem 1992; about the immune response. 267:131 15-13118. 1 3. Colley NJ, Baker EK, Stamnes MA, Zuker CS: The cyclophilmn homolog ninaA is required In ACKNOWLEDGMENTS the secretory pathway. Cell 1991:67:255-263. 14. DeFranco AL: Signal transduction: Immuno- During the writing of the manuscript. C.Y. Lu was supported by the suppressants at work. Nature (Lond) 1991; Baxter Extramural Grant Program and by Grants ROl -HD242792, 352:754-755. RO1-DK43634. and K04-HD00862 from the NIH. MA. Vazquez was 1 5. Sigal NH, Dumont FJ: Cyclospormn A, FK-506, supported by an NIH Minority Faculty Award and a National Kidney and rapamycin: Pharmacologic probes of lym- Foundation Clinical Investigator Award. S.C. Sicher was supported phocyte signal transduction. Annu Rev Immu- by an NIH Institutional National Research Service Award to the nol 1 992; 10:519-560. Division of Nephrolojr. University of Texas Southwestern Medical 16. Bierer BE, Mattila PS, Standaert RF, et al.: School (2T32-DK07257-1 1A2). We acknowledge the support of our Two distinct signal transmission pathways In transplant surgeons. Drs. I. Dawidson, A. Sagalowsky. and P. Peters; T lymphocytes are inhibited by complexes our fellow transplant nephrologists. Drs. B. Toto. J. Thompson, K. formed between an Immunophilin and either Brinker, L. Melton, P. Vergni. A. Hull, and B. Palmer: our resident FK506 or rapamycin. Proc Nati Acad Sci USA and fellow staff; and our transplant coordinators and inpatient 1990:87:9231-9235. transplant nursing staff. 1 7. Clipstone NA, Crabtree GR: Identification of calcineurin as a key signalling enzyme In T- lymphocyte activation. Nature (Lond) 1992; REFERENCES 357:695-697. 1 8. O’Keefe SJ, Tamura J, Kincaid RI, Tocci MJ,

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1256 Volume 4 . Number 6. 1993