~r,). OSSEVOORT ET AL.--b~-------------------------------

~~~

Use of in Vitro Correlates for Selection ofCandidate Immunosuppressive Antibodies Priorto a Primate Transplant ModelMiriam A. Ossevoort', Maria C. Noort', Almuth Radzikowski', Yvon van der Hout', Katrien Lorre', PeterDe Waele' and Margreet Jonker''Dept. ofImmunotherapy, Biomedical Primate Research Centre, NL-Rijswijk, 2Fresenius AG, D-Grafelfing, JInnogenetics NV, B-Gent

SummaryThe selection of possible candidate immunosuppressiveantibodies to prevent graft rejection is performed in vitro.Additionally, due to the species specificity of these monoclonalantibodies (MABs), pre-clinical studies in non-human primatesare necessary. If a positive correlation between the in vitro andin vivofindings would exist, these tests can act as a pre-screening before new reagents are tested in vivo. The correlati-on of the in vitro and in vivo efficacy of an anti- T-lymphocyteglobulin (ATG) and an anti-CD80 MAB is evaluated in arhesus monkey skin transplant model. The results show thatlymphocytotoxic titers (NIH-test) do not predict the outcome ofin vivo effectiveness of ATG in rhesus monkeys. Additionally,no evidence of tolerance to a skin allograft could be shown tocorrelate with inhibition of a secondary mixed lymphocyteculture (MLC) by anti-CD80 and cyclosporin A (CsA). Thus,these in vitro assay used can not predict the in vivo efficacy ofnew immunosuppressive antibodies.

Keywords: transplantation, antibodies, in vitro, primates

1 Introduction

Immunological rejection of an allograftthrough T cell dependent mechanisms isan evitable result after organ transplanta-tion between genetically non-identical in-dividuals. Therefore, administration ofdrugs suppressing the recipient's T cellfunction is required for successful trans-plantation of allogeneic organs. Nowadayscalcineurin inhibitors and glucocortico-steriods are used clinically as a routine.Even though acute rejection can effectivelybe prevented with these drugs and rejec-tion episodes can often be reversed byincrease of the dose or administration ofT-cell antibodies (ATG,OKT3), many

ALTEX 15, Suppl./98

Zusammenfassung: Korrelation von in vitro Tests mit einemPrimaten-Transplantationsmodell zur Auswahl geeigneterimmunsuppressiver AntikorperDie Auswahl geeigneter immunsuppressiver Antikorper zurVerhinderung der Transplantat-Abstoj3ung wird in vitrodurchgefuhrt. Zusatzlicn sind, in Abhangigkeit von derArtspezifitdt dieser monoklonalen Antikorper priiklinischeStudien an nichthumanen Primaten erforderlich. Sollte es einepositive Korrelation zwischen in vitro und in vivo Ergebissengeben, konnten diese Tests als Prdscreening eingesetzr werden,bevor neue Antikorper in vivo getestet werden. Die Korrelationzwischen in vitro und in vivo Wirksamkeit eines Anti- T-Lymphozyten-Globulins (ATG) und eines anti-CD80 monoklo-nalen Aruikorpcrs wird in einem Rhcsusaffcn Hauttransplanta-tionsmodell geprii{t. Die Ergebnisse zeigen, daj3 sich mit denlymphotoxischen Titern die Ergebnisse der in vivo Wirksamkeitdes ATGs bei Rhesusaffen nicht voraussagen lassen. Dariiber-hinaus konnten keine Hinweise auf eine Toleranz gegeniiberdem Hauttransplantat gefunden werden, die mit einer Hem-mung der sekunddren Mixed Lymphocyte Culture (MLC) durchAnti-CD80 und Zyklosporin A korrelieren. Damit kann mitdiesem in vitro Test die in vivo Wirksamkeit neuer immunsup-pressiver Antikorper nicht vorhergesagt werden.

transplant patients suffer late graft lossusually caused by chronic rejection. Alt-hough many cells participate in the pro-cess of transplant rejection, only T cellsappear to be absolute required (Krenskyet al., 1990). In a recent review, the roleof T cell costimulatory activation pa-thways in transplant rejection has beendescribed (Sayegh et al., 1998). The T cellrequires beside triggering via the antigen-receptor, a costimulatory signal via CD 154or CD28, to become fully activated(Chamber et al., 1997; Springer et al.,1987). When the T cell encounters an an-tigen-specific stimulus in the absence ofcostimulation, the T cells becomes un-re-sponsive or tolerant to that particular an-

tigen (Mueller et al., 1989). Several stra-tegies using different monoclonal antibo-dies directed against costimulatory mole-cules (anti-CD80, anti-CD86, anti-CD40LMABs and CTLA4-Ig), have proven toinduce tolerance/non-responsiveness toallografts in rodents and non-human pri-mates (Larsen et al., 1996; Lenschow etal., 1995; Kirk et al., 1997).

In organ transplantation new approa-ches are investigated that are aimed at theinduction of complete non-responsiven-ess towards the allograft or at least at thereduction of standard immunosuppressi-on. The selection of possible candidate an-tibodies is performed in vitro. Additional-ly, due to the species specificity of these

23

_O_SS_E_V_OO_R_T_E_T_AL_. ~!~----c'~,

MABs, pre-clinical studies in non-humanprimates are necessary. To be able to pre-dict the in vivo efficacy of the MABs, invitro assays are developed. If a positivecorrelation between the in vitro and in vivofindings would exist, these tests can act asa pre-screening before new reagents aretested in vivo. The correlation of the in vi-tro and in vivo effectivity of ATG and ananti-CD80 MAE is evaluated in a rhesusmonkey skin transplant model.

T-cell specific antibodies have been eva-luated extensively, and a well known ex-ample is anti- T-Iymphocyte globulin(ATG). To investigate the potency of rab-bit ATG in vitro, lymphocytotoxic titerswere determined in rhesus monkeys andwere correlated to the in vivo effects ofATG on skin allograft survival time.

A recent valid approach for immune sup-pression after solid organ transplantationis the blockade of the costimulatory pa-thways, such as CD80-CD28 pathway.This costimulatory signal is relatively re-sistant to cyclosporin A (CsA). Therefore,it is suggested to use anti-CD80 MAB as aCsA-sparing agent. Additionally, in vitroa combination of anti-CD80 MAB withCsA can induce allo-antigen specific non-responsiveness in human mixed lympho-cyte cultures (MLC), whereas reactivity to"third party" antigens remains intact (Co-moli et al., 1995; Van Gool et al., 1994).These promising data initiated a study toexplore the use of anti-CD80 in combina-tion with a suboptimal dose of CsA as aprophylactic treatment to prevent skintransplant rejection in a rhesus monkeymodel. The in vivo effects of this treat-ment were correlated to the effect of thiscombination of drugs to mixed lympho-cyte culture using rhesus monkey peri-pheral blood mononuclear cells (PBMC).

2 Animals, material and methods

2.1 AnimalsRhesus monkeys (Macaca mulatta) wereborn and raised at the Biomedical PrimateResearch Centre or were purchased froman outdoor station. The animals were bet-ween 4 and 10 years of age and typed forMamu-A, B, DR antigens and ABO anti-gens. Recipients had no history of alloim-munisation (pregnancy, blood transfusion,allografts) and had not received murineantibodies. Male and female animals wereused. The protocol of the study and the care

24

was in accordance with guidelines of theAnimal Care and Use Committee instal-led by Dutch law.

on days -2, 0, 2, 5 and 7. Two skin graftsfrom two donors each are transplanted onday 0, Skin graft survival is scored byvisual inspection. The day on which com-plete crust formation of the skin grafts isobserved, is taken as graft survival time.The mean survival time of the 4 grafts isaveraged. During the experiment, leuko-cyte and lymphocyte counts are determi-ned.

2.2 ATG skin transplant experimentPrior to the transplantation experiment,each batch of ATG is tested in vitro in aIymphocytotoxicity assay using unsepa-rated PBMC (NIH-test) and B- or T-cellenriched suspensions (B or T-test) of therhesus monkey selected for the skin graftexperiment (Mittal et al., 1968; Roger etaI., 1976), Each batch of ATG is tested inone rhesus monkey for batch release pur-poses. ATG (60 mg/kg) is administered

2.3 anti-CD80 MAB and CsA skintransplant experimentTo test the in vitro efficacy of anti-CD80MAB in combination with CsA, a prima-

A: ATG evaluation in monkeyscorrelation graft survival and T-Iymphocytotoxic titer

90008000 0 0 0

7000•.. 6000~ 5000(ijo 4000E0.'13 3000e

2000 IIDOOO 0 DO

1000 o 0 O!:IIIIIl 0 [D] 0 0 o 0

1D8mlD 800 0 000 5 10 15 20 25 30 35 40

skin graft survival in days

8: correlation graft survival and duration lymphopenia

50

Vl 40;.,('(l

"0 <> <> <>.s 30('(l S'2 <> <>Q)0.0 <>s: 20 <> <> e <>0.

E~ <> <>coa:&o ~ gl <> e ec: <>000 10~ <>e <>e (I)C)::l <>"0 <>

00 5 10 15 20 25 30 35 40

, skin graft survival in days

Figure 1: Evaluation of ATG batches in rhesus monkeys; each dot represents the meanskin graft survival time of one monkey and the Iymphocytotoxicity titer of the ATGon T-cell enriched suspensions (A) or the duration of the lymphopenia (8)

ALTEX 15, Suppl./98

____ ~~------------------------------------------------O-S-S-EV-O-O-R-T-ET-A--L.~~~

ry one-way mixed lymphocyte culture(MLC) was performed (Ossevoort et aI.,in press). The rhesus monkey PBMC werestimulated with an irradiated rhesus mon-key Herpes-B virus-transformed B cellline for 5 days in the presence of anti-CD80 MAB (10 ug/ml) and/or CsA (400ng/ml). The proliferative response wasmeasured as 3H-thymidine incorporation.For the secondary MLC, the cells werecultured for 5 days in the presence of anti-CD80 MAB and CsA. Then, the cultureswere washed to remove the reagents andleft unstimulated for 2 days after whichthe cultures were restimulated with samestimulator cells or rIL- 2. No reagents wereadded to the second culture. The secon-dary cultures were harvested after 3 days,the last 18 hours in the presence of 3H_thymidine. The immunosuppressive pot-ency of the combination of anti-CD80MAB (0.5 mglkg) and CsA (5 mglkg) wastested in a rhesus monkey skin transplantmodel. The anti-CD80 MAB was giveni.v. daily for 10 days starting at day -I.CsA was given i.m. daily from day -2 un-til rejection was scored (Ossevoort et aI.,in press).

3 Results

3.1 ATG skin transplant experimentUntreated rhesus monkeys reject the graf-ted skin between day 9 and 12, with a meansurvival time of day 10 post-transplanta-tion. Figure 1 shows the results of the im-munosuppressive efficacy of different bat-ches of ATG displayed as skin graft survi-val times. The lymphocytotoxic titers weredetermined using T-cell enriched suspen-

sions and the duration of lymphopenia(lymphocytes less than 50% of pre-treat-ment value)

No correlation between lymphocytoto-xic titers on T-cell enriched suspensionsand the survival was found (correlationcoefficient = 0.11). Similar results werefound for the lymphocytotoxic titers usingB-cell enriched suspensions or unsepara-ted lymphocytes (data not shown). Theduration of the lymphopenia showed aweak correlation (correlation coefficient= 0.33) with the skin graft survival.

3.2 anti-CD80 MAB and CsA skintransplant experimentIn vitro results demonstrated that the in-duction of allo-antigen specific rhesusmonkey T cells can be prevented usingCsA in combination with anti-CD80MAB. To this end primary MLC assayswere performed in the presence or absenceof anti-CD80 MAB and / or CsA. After 5days tritiated thymidine incorporation wasmeasured (Table 1). Addition of anti-CD80 MAB or CsA alone resulted in amean inhibition of the proliferative re-sponse of 52 or 77 % respectively. Additi-on of both anti-CD80 MAB and CsA re-sulted in a complete abrogation of the al-loantigen-specific response. These prima-ry cultures were then washed, left unsti-mulated for 2 days and then restimulatedwith the same stimulator cells. No anti-CD80 MAB or CsA were added to the se-condary cultures (table 1). Now the cul-tures that had previously been exposed toCsA in combination with anti-CD80 MABdid not proliferate, whereas the culturespreviously exposed to medium, CsA or

Table 1: The immunosuppressive efficacy of anti-COSO mAb (10 ~g/ml) in combinationwith CsA (400 ng/ml) in a primary and secondary MLC using a B cell line stimulatorcells

experiment 1 primary MLC secondary MLC

medium O' 0

anti-COSO 30 38CsA 86 46

anti-COSO + CsA 98 89experiment 2 primary MLC secondary MLC

medium 0 0

anti-COSO 54 32CsA 61 0

anti-COSO + CsA 89 51. .

* % inhibition

ALTEX15,Supp1.l98

anti-CD80 MAB could be restimulated.The T cells in the primary cultures werestill able to respond since restimulationwith the same antigen in the presence ofrIL-2 did result in proliferation (data notshown). This suggested that the T-cell hadbeen silenced during the first phase of theexperiment. The in vivo immunosuppres-sive effects of the combined treatment witha suboptimal doses of CsA (trough levels100-150 ng/ml) and anti-CD80 MAB wasexamined in a skin allograft model (Osse-voort et aI., 1998). The skin graft survivaltime of untreated or CsA-treated rhesusmonkeys was 10 days. Treatment withCsA in combination with anti-CD80MAB resulted in a significantly increasedskin graft survival time to 14 days. Thus ashort term immunosuppression, resultingin prolonged skin graft survival could beobtained using CsA and anti-CD80 MABas a prophylactic treatment in a rhesusmonkey skin transplant model.

4 Conclusions

Since many of the anti-human MABs alsoreact with lymphocytes of macaques, therhesus monkey skin transplant model is avalid model to test the immunosuppressiveefficacy of monoclonal antibodies specificfor human T cell subsets (Jonker et al., 1983;Jonker et al., 1986; Nooij et al., 1987).

The application of a valid in vitro assaythat predicts the immunosuppressive po-tential of a new reagent in organ transplan-tation, implies a significant reduction ofthe number of animals used for in vivoexperiments. In this study, the in vitro andin vivo outcome of the immunosuppressi-ve activity of ATG and an anti-CD80 MABwere compared. The results show that lym-phocytotoxic titers do not predict the out-come of in vivo effectiveness of ATG inrhesus monkeys. Additionally, in this pro-tocol, no evidence of tolerance to a skinallograft could be shown to correlate withinhibition of a secondary MLC. Thus, un-til now no in vitro assay has been esta-blished to predict the in vivo efficacy ofnew immunosuppressive antibodies toprevent graft rejection.

References

Chambers, C. A. and Allison, J.P. (1997).Co-stimulation in T-cell responses. Curr:Opin. Immunol. 9, 396-404 .

25

OSSEVOORT ET AL. wr:"\-------------------------------~~~~--",~,

Comoli, P., Montagna, D., Moretta, A.,Zecca, M., Locatelli, F. and Maccario,R. (1995). Alloantigen-induced humanlymphocytes rendered non-responsiveby a combination of anti-CD80 mono-clonal antibodies and Cyclosporin-Asuppress mixed lymphocyte reaction invitro. 1. Immunol. 155,5506-5511.

Jonker, M., Goldstein, G. and Balner, H.(1983). Effects of in vivo administrati-on of monoclonal antibodies specific forhuman T cell subpopulations on the im-mune system in a rhesus monkey mo-del. Transplantation 35,521-526.

Jonker, M., Nooij, F. J. M., Suylichem van,P., Neuhaus, P. and Goldstein, G. (1986).The influence of OKT8F treatment onallograft survival in rhesus monkeys.Transplantation. 41,431-435.

Kirk, A. D., Harlan, D. M., Amstrong, N.N., Davis, T.A., Dong, Y., Gray, G. S.,Hong, X., Thomas D., Fechner, J. H. Jr.and Knechtle, S. J. (1997). CTLA4-Igand anti-CD40 ligand prevent renal al-lograft rejection in primates. Proc. Natl.Acad. Sci. USA 94, 8789-8794.

Krensky, A. M., Weiss, A., Crabtree, G.,Davis, M. M. and Parham, P. (1990). Tlymphocyte-antigen interaction in trans-plant rejection. N. Eng. 1. Med. 322,510-517.

Larsen C. P., Elwood E. T., Alexander D.Z., Ritchie, S. A., Hendrix, R., Tucker-Burden, c.,Cho, H. R., Aruffo, A., Hol-lenbaugh, D., Linsley, P. S., Winn, K. J.and Pearson, T. C. (1996). Long-termacceptance of skin and allografts after

blocking CD40 and CD28 pathways.Nature 381, 434-438.

Lenschow, D. J., Zeng, Y, Hatchcock, K.S., Zuckerman, L. A., Freeman, G.,Thistlethwaite, J. R., Gray, G. S., Ho-des, R. J. and Bluestone, J. A .. (1996).Inhibition of transplant rejection follo-wing treatment with anti-B7.1 and anti-B7.2 antibodies. Transplantation 60,1171-1178.

Nooij, F. J.M. andJonker,M. (1987). Theeffect on skin allograft survival of amonoclonal antibody specific for a po-lymorphic CD3-like cell surface mole-cule in rhesus monkeys. Eur. 1. Immun-01.17,1089-1093.

Mittal K. K., Mickey M. R., Singal D. P. andTerasaki, P. I. (1968). Serotyping for homo-transplantation. XVIII. Refinement ofmicrodroplet lymphocyte cytotoxicity test.Transplantation 6,913-917.

Mueller, D. L., Jenkins, M. K. andSchwartz, R. H. (1989). Clonal expan-sion versus clonal inactivation: a costi-mulatory signal pathway determines theoutcome of T cell antigen receptor oc-cupancy. Annu. Rev. Immunol. 7, 445-480.

Ossevoort, M. A., de Boer, M., Lorre, K.,VandeVoorde, A. and Jonker, M. (1998).Blocking of co-stimulatory pathwaysusing monoclonal antibodies as a newstrategy to prevent transplant rejectionin a non-human primate model. Transpl.Proc. 30, 1061-1062.

Ossevoort, M. A., Lorre, K., Boon, L., vanden Hout, Y., de Boer, M., De Waele,

26

P., Jonker, 1. and VandeVoorde, A.(1998). Prolonged skin graft survival byadministration of anti-CD80 monoclo-nal antibody with Cyclosporin A. 1. Im-munother. in press.

Roger J. J. and van Leeuwen A. (1976).The major histocompatibility complexof rhesus monkeys. VI. Serology and ge-netics of Ia-like antigens. Tissue anti-gens 8, 67-86.

Sayegh, M. H. and Turka, L. A. (1998).The role ofT-cell costimulatory activa-tion pathways in transplant rejection. N.Eng. 1. Med. 338, 1813-1821.

Springer, T. A., Dustin, M. L., Kisshimo-to, S. and Marlin, D. (1987). The lym-phocyte function associated LFA-1,CD2, and LFA-3 molecules: cell adhe-sion receptors in the immune system.Annu. Rev. Immunol. 5, 233-252.

Van Gool, S. w., Ceuppens, J. L., Walter, H.and de Boer, M. (1994). Synergy betweencyclosporin A and a monoclonal antibodyto B7 in blocking alloantigen-induced T-cellactivation. Blood 83, 175-180.

AcknowledgementThis work was in part supported by a grantfrom "Platform Altematieven voor Dier-proeven", PAD 96-19.

Correspondence addressDr. Margreet JonkerDepartment Immunotherapy, BiomedicalPrimate Research CentreLange Kleiweg 151NL-2288GJ Rijswijk

ALTEX 15, Suppl./98