`
`R.L. Kirkman. M.E. Shapiro, C.B. Carpenter, E.L. Milford. E.L. Ramos, N.L. Tilney, T.A. Waldmann,
`
`C.E. Zimmerman, and T.B. Strom
`
`HE SEARCH for more efl‘ective and less broadly toxic
`irnmunosuppressive agents remains a major goal of
`transplantation research. One approach to achieving more
`specific immunosuppression is to target only those lympho-
`cytes responding to an allograft by directing therapy at
`activation antigens. Of these antigens,
`the interleukin-2
`receptor (IL-2R) has proven of particular interest, both
`because of its important biological role in the activation
`T-cells‘ and because of extensive animal model experience
`with anti-IL-2R monoclonal antibody (Mab) therapy.”
`Anti-Tac is a murine Mab with specificity for the 55KD
`beta subunit of the human IL-2R.7 Anti-Tac blocks binding
`of IL-2 to its receptor and prevents association of the alpha
`and beta chains of the receptor to form the high affinity
`IL-2R.” Recent work from our laboratory has shown that
`anti-Tac as a single agent will significantly delay rejection of
`renal allografts in cynomolgus monkeys.‘ Encouraged by
`these results, we have initiated a randomized trial of prophy-
`lactic therapy with anti-Tac in clinical renal transplantation.
`This study presents our early experience with three protocols
`for the use of this agent.
`
`In the second protocol, no cyclosporine was used in the experimen-
`tal group in the first week. Patients were randomized to receive
`either anti-Tac 20 mg/day for 10 days, azathioprine 2 mg /kg/ day,
`and prednisone 30 mg/day, with cyclosporine 3 mg/kg/day added
`on day 8., or conventional triple therapy with cyclosporine 8 mg/
`kg/day, azathioprine 2 mg/kg/day, and prednisone 30 mg/day.
`This protocol was terminated prematurely, as noted below.
`In the third protocol, low dose cyclosporine is being used in the
`experimental group. Patients are randomized to receive either
`anti-Tac 20 mg/day for ten days, cyclosporine 4 mg/kg/ day,
`azathioprine 2 mg / kg/day, and prednisone 30 mg / day, or conven-
`tional triple therapy as in the second protocol. In the experimental
`group, the cyclosporine dose is increased to 8 mg/kg/day at the
`conclusion of anti-Tac treatment. This protocol is ongoing, with nine
`patients entered in the experimental group and ten in the control
`group to date.
`During treatment, serum and peripheral blood mononuclear cells
`
`were obtained on days 0, 2, 4, 6., 8, 10, and 14 to monitor anti-Tac
`serum levels, the development of anti-mouse immunoglobulin anti-
`bodies, T-cell subsets and expression of IL-2R on circulating lym-
`phocytes, and the effect of therapy on the ability of circulating
`lymphocytes to participate in mixed lymphocyte reactions and
`cell-mediated lympholysis.
`
`MATERIALS AND METHODS
`
`Preparation and Administration of Anti-Tac
`
`RESULTS
`
`Protocol 1
`
`Anti-Tac, a murine IgG2a Mab has been extensively characterized
`elsewhere.” The antibody was purified from ascites of Balb/c mice
`inoculated with the hybridoma, suspended in saline at a concentra-
`tion of 2 mg / ml, sterilized by filtration, and stored at -20°C. Prior
`to the first administration of the Mab, all patients were injected
`intradermally with 0.1 ml of a 1:l000 dilution of anti-Tac in saline to
`exclude hypersensitivity. Each dose of 20 mg was infused intrave-
`nously over two hours in 50 ml normal saline containing 1% human
`serum albumin.
`
`Patient Protocols
`
`Three protocols for the use of anti-Tac were examined. In each
`protocol, only patients receiving a first cadaver allograft were
`eligible, and patients were randomized to experimental or control
`groups by a sealed envelope technique. There were no significant
`differences between groups with respect to age, sex, or degree of
`HLA AB or DR matching. All protocols were approved by the
`clinical studies committtees of both hospitals.
`In the first protocol, patients were randomized to receive anti-Tac
`plus conventional
`immunosuppression (n - 12) or conventional
`immunosuppression alone (n - 9). Anti-Tac was given at a dose of
`20 mg qd for 10 days beginning on posttransplant day 1. Conven-
`tronal immunosuppression consisted of either cyclosporine 12 mg/
`kg/day and prednisone 30 mg/day or cyclosporine 8 mg/kg/day,
`azathioprine 2 mg/kg/day, and prednisone 30 mg/day. In both
`groups cyclosporine doses were adjusted by blood level and clinical
`evidence of nephrotoxicity. First rejection episodes were treated with
`a methylprednisolone pulse 1 gm IV qd for 3 days.
`
`The first protocol was designed to ascertain the safety of
`anti-TAC administration and to obtain preliminary evidence
`of efficacy. Patients were randomized to receive anti-TAC
`plus conventional immunosuppression (n - 12) or conven-
`tional immunosuppression alone (n - 9). In the group receiv-
`ing anti-TAC, all skin tests were negative and no complica-
`tions of antibody administration were identified.
`.
`In this protocol, administration of anti-TAC reduced the
`frequency of early rejection episodes and delayed the onset of
`the first rejection episode. In the first ten days posttrans-
`plant, during which anti—TAC was given to the treatment
`group, only one of 12 patients receiving anti—TAC experi-
`enced a rejection episode, compared with five of nine patients
`in the control group (p -c: 0.05). The single treated patient
`with rejection had primary nonfunction of the graft, and
`
`From the Brigham and Women's and Beth Israel Hospitals,
`
`Harvard Medical School, Boston, Massachusetts and the Metab-
`
`olism Branch, National Cancer Unit, National Institutes of Health,
`
`Bethesda, Maryland.
`
`Address reprint requests to Dr Robert L. Kirkman, Department
`
`of Surgery. Brigham and Women’s Hospital. 75 Francis Street,
`
`Boston, Massachusetts 021 15.
`
`Supported by grants from the National Institutes of Health.
`
`© 1969 by Appleton Lange, lnc.
`
`0041-1345/89/$03.00! +0
`
`1766
`
`Transplantation Proceedings, Vol 21, No 1 (February), 1969: pp 1766-1766
`
`PETITI()NER'S EXHIBITS
`
`Exhibit 1035 Page 1 of3
`
`
`
`EARLY EXPERIENCE WITH ANTI-TAC
`
`1767
`
`rejection was diagnosed by biopsy on postoperative day 7.
`patient was subsequently demonstrated to have cyclo-
`sporine nephrotoxicity, and it
`is uncertain if a clinical
`rejection episode would have been noted had there been
`immediate renal function.
`
`Rejection episodes eventually occurred in seven of twelve
`patients receiving anti—TAC and in eight of nine control
`patients (p -= ns). However,
`the mean time to the first
`rejection episode was 24.7 days in the anti-TAC group,
`compared with 9.4 days in the control group (p «:1 0.01,
`Mann-Whitney rank sum testing). Four patients with rejec-
`tion in the anti-TAC group responded to a steroid pulse,
`while three required OKT3; one of the latter patients eventu-
`ally lost his graft to uncontrolled rejection. A single control
`patient also lost his graft, with accelerated acute rejection
`leading to removal of a ruptured allograft on postoperative
`day 4.
`All patients in this initial protocol have now been followed
`12-21 months, with no subsequent rejection episodes or graft
`losses. Giiaft survival is 92% in the treated group and 89% in
`the control group. Mean creatinine at last follow-up was 2.1
`mg/dl, for the anti-TAC group and 1.7 mg/dl for the control
`group (p = ns). No deaths have occurred in either group.
`
`Protocol 2
`
`As the initial protocol suggested that anti-TAC would pre-
`vent rejection during its administration, the second protocol
`was designed to determine if the use of cyclosporine could be
`avoided in the early posttransplant period. Patients were
`randomized between quadruple therapy, consisting of anti-
`TAC for ten days, azathioprine and prednisone, with cyclo-
`sporine added on day 8, or triple therapy. By happenstance,
`five of the first six patients entered in this protocol were
`randomized to the anti-TAC group. None of these patients
`completed the anti-TAC protocol, one because of an appar-
`ent reaction to anti-TAC and four because of rejection
`during therapy.
`The reaction to the antibody was the development of fever
`and pulmonary edema on the fifth day of treatment. The
`patient was not volume overloaded and there was no evidence
`of infection or rejection. Anti-TAC was discontinued and
`cyclosporine begun, with resolution of the symptoms and
`continued good graft function.
`i
`The four patients with rejection were managed by cessa-
`tion of anti-TAC and initiation of cyclosporine. One of the
`rejection episodes was easily reversed with a single steroid
`pulse, while the others required more than one steroid pulse
`or OKT3. One of these patients subsequently died of dissemi-
`nated CMV. Because this protocol did not appear to obviate
`the need for cyclosporine in the early posttransplant period, it
`was terminated.
`
`Protocol 3
`
`The current protocol was established to determine if anti-
`TAC will allow the use of a lower dose of cyclosporine in the
`early posttransplant period. Patients are being randomized to
`
`receive anti-TAC for ten days plus low dose cyclosporine or
`full dose cyclosporine. The cyclosporine dose in the experi-
`mental group is increased following conclusion of anti-TAC
`treatment; all patients in both groups receive azathioprine
`and prednisone. To date, nine patients have been entered in
`the treated group, ten in the control. There has been one
`rejection episode within ten days of transplantation in the
`treated patients, compared with eight
`in the control
`(p < 0.05). There have been no immunological graft losses in
`either group, but follow-up is less than four months in all
`cases. One patient who received anti-TAC, and who was not
`treated for rejection, died at four months from CMV and
`pneumocystis pneumonia. A single patient receiving anti-
`TAC developed pruritis, which was managed symptomati-
`cally and did not require cessation of therapy.
`
`Anti-TAC and OKT3
`
`As noted above, six patients treated with anti-TAC subse-
`quently required therapy with OKT3. All six patients had
`rapid reversal of their rejection episodes, although one subse-
`quently had a recurrent rejection and represents the sole
`graft
`loss from rejection in all
`three treatment groups.
`Details of the anti-mouse immunoglobulin response and
`clearance of circulating CD3 positive cells in these patients
`are described in a separate manuscript in this volume.’
`
`Monitoring Studies
`
`None of the monitoring studies performed revealed signifi-
`cant differences between the treated and control patients. Of
`particular note, treatment with anti-TAC did not prevent
`expression of the IL-2 receptor on the surface of circulating
`T-cells following transplantation.”
`
`DISCUSSION
`
`The data presented here demonstrate that anti—TAC, a Mab
`directed against the human IL-2R, will reduce the frequency
`of early rejection episodes following transplantation and
`delay the onset of those which do occur, when used in
`combination with cyclosporine. The early results with proto-
`col 3 suggest that the dose of cyclosporine can be signifi-
`cantly reduced compared with standard immunosuppression
`regimens. This finding is in accord with animal models, in
`which significant synergy between anti-IL-2R Mab’s and
`cyclosporine has been shown} This reduced dose of cyclospo-
`rine simplifies the management of renal transplant recipients
`by decreasing the incidence of nephrotoxicity. When com-
`bined with the lower incidence of rejection, the early course
`of anti-TAC-treated patients is remarkably uncomplicated.
`These results differ from those of Soulillou et al using
`another anti-IL-2R Mab, 33B3.1." In that experience,
`excellent results were obtained without cyclosporine; but
`with a higher prednisone dose. Further experimentation with
`anti-IL-2R therapy will be required to determine optimal
`antibody characteristics and protocols.
`A critical finding of the current study was the successful
`use of OKT3 to treat rejection in patients previously receiv-
`
`l,’ETITIONER'S EXHIBITS
`
`
`
`
`
`1768
`
`KIRKMAN, SHAPIRO, CARPENTER ET AL
`
`ing anti-TAC. This observation permits the design of proto-
`cols employing sequential use of monoclonal antibodies of
`dilferent idiotypes directed against the same or different
`targets. In particular, it allows the use of one or more Mab’s
`for rejection prophylaxis without precluding use of other
`antibodies for rejection therapy. Moreover, when this finding
`is combined with the increasing variety of Mab’s defining
`targets and subsets of immunologic interest, the opportunity
`for more detailed manipulation of the immune response is
`evident.
`
`REFERENCES
`
`1. Waldmann TA: Science 232:727, 1986
`
`2. Kirkman RL, Barrett LV, Gaulton GN, et al: J Exp Med
`162:358.. 1935
`
`3. Kirkman RL, Barrett LV, Koltun WA, et al: Transplant Proc
`19:618., 1987
`
`4. Kupiec-Weglinski JW, Diamantstein T, Tilney NL et al: Proc
`Natl Acad Sci USA 83:2624, 1986
`
`5. Kupiec-Weglinski JW, Hahn HJ, Kirkman RL, et al: Trans-
`plant Proc 20:207. 1988
`6. Reed MH, Shapiro ME, Strom TB, et al: Transplantation (in
`press)
`
`7. Uchimaya T, Broder S, Waldman TA: J Immunol 12621393,
`1931
`
`8. Wang HM, Smith KA: J Exp Med 166:1055, 1987
`
`9. Ramos EL, Wood IG, Rollins MR, et al: Transplant Proc (in
`press)
`
`10. Ramos EL, Wood IG, Rollins MR, et al: Transplantation
`(data unpublished)
`
`11. Soullilou JP, Lemauff B, Olive D, et al: Lancet 1:l339, 1987
`
`‘H.-nu-.
`
`PETITIONER'S EXHIBITS
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`Exhibit 1035 Page 3 of