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`J Transplantation
`
`voLuME 41 • NUMBER s May 1986
`
`BIOEPIS EX. 1027
`Page 1
`
`

`

`May 1986
`Volume 41
`Number 5
`
`Contents
`OVERVIEW
`
`Tr nspl ntatior
`
`Official Journal of the Transplantation Societ
`
`Suppressor T cells in allogeneic models.
`I.V. Hutchinson. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
`
`EXPERIMENTAL TRANSPLANTATION
`
`lifelong reversal of the metabolic abnormalities of advanced diabetes in rats by whole(cid:173)
`Pancreas transplantation.
`M ~.J. Orloff, G.E. Greenleaf, P. Urban, and B. Girard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556
`on~clonal antibody analysis of canine hematopoietic cells: Role of la-like and Thy-1 antigens
`In bone marrow engraftment.
`·
`M.M. Prendergast, K.F. Bradstock, A.F. Broomhead, W.G. Hughes, A. Kabral, M.C. Berndt,
`and K. Tiver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
`
`CLINICAL TRANSPLANTATION
`
`. . . . . . . . . . 572
`
`Monoclonal antibody therapy: Anti-idiotypic and non-anti-idiotypic antibodies to OKT3 arising
`despite intense immunosuppression.
`ALG G.J. Jaffers, T.C. Fuller, A.B. Cosimi, P.S. Russell, H.J. Winn, and R.B. Colvin
`treatment of steroid-resistant rejection in patients receiving cyclosporine.
`VA.J. Matas, V.A. Tellis, T. Quinn D. Glichlick, R. Soberman, R. Weiss, G. Karwa, and F.J.
`eith .
`'
`Severe apl~~t;~ ~~~~·i~ ·a·s~~~i~~~~ ·~i~~ ~~~~~i~ ·~~~~~~t~~~~~~ ~~~~;d·i~~i~:· ;~~~~~;~~i~
`
`SH
`
`and hematologic reconstitution after allogeneic bone marrow transplantation.
`t.J. Deeg, LG. lum, J. Sanders, G.J. Levy, K.M. Sullivan, P. Beatty, E.D. Thomas, and R.
`~
`Antigen-s~~~i~i~ ·a·n~i~~~~ ·r~~~~~~~~ ~~ ·~~~~~~~~t~~ ·t~ ·t~~~~~s· ~~x~;~ ~~t~~ ·h~~~~ ~a·r~~~
`transplantation.
`o· S. Shiobara, L.G. Lum, R.P. Witherspoon, and R. Storb. . . . . . . . . . . . . . . . . . . . . . . . . . . 587
`lagnosis of tubular injury in renal transplant patients by a urinary assay for a proximal
`tubular antigen, the adenosine-deaminase-binding protein.
`~E. Tolkoff-Rubin, A.B. Cosimi, F.L. Delmonico, P.S. Russell, R.E. Thompson, D.J. Piper,
`.P. Hansen, N.H. Bander c L Finstad C. Cordon-Cardo, L.H. Klotz, L.J. Old, and R.H.
`R
`ubin
`'
`· ·
`'
`· · · · · · · ....................................................... 593
`A
`crit~cal look at survival of diabetics with end-stage renal disease: Transplantation versus
`dialysis therapy.
`~.:· Khauli, D.R. Steinmuller, A.C. Novick, C. Buszta, M. Goormastic, S. Nakamoto, D.G.
`1
`t, M. Magnusson, E. Paganini, and M.J. Schreiber. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
`P
`lasma
`exch~nge for platelet alloimmunization.
`W
`1
`· · Bens1nger, C.D. Buckner, R.A. Clift, S.J. Slichter, and E.D. Thomas. . . . . . . . . . . . . . . 602
`R
`esults of r ·
`.
`lvmg-related kidney transplantation in Puerto R1co.
`ih E.A. Santiago-Delpin, z. Gonzalez, E. Rive-Mora, J.H. Amadeo, and R. Ramirez-Gonzalez.
`e ~ffects of cyclosporine on Toxoplasma gondii in vivo and in vitro.
`.E. McCabe, B.J. Luft, and J.S. Remington. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
`A
`utonomic system dysfunction and polyneuropathy in nondiabetic uremia: A one year follow-
`~P study after renal transplantation.
`· Solders, A. Persson, and H. Wilczek. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
`
`579
`
`5~
`
`606
`
`BIOEPIS EX. 1027
`Page 2
`
`

`

`IMMUNOBIOLOGY
`Allospecificity of activated T cells grown from endomyocardial biopsies from heart transplant
`patients.
`A. Zeevi, J. Fung, T.R. Zerbe, C. Kaufman, B.S. Rabin, B.P. Griffith, R.L. Hardesty, and R.J.
`Duquesnoy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620
`Induction of transplantation tolerance in rats by spleen allografts: I. Evidence that rats tolerant
`of spleen allografts contain two phenotypically distinct T suppressor cells.
`W.R. Duncan, S.M. Stepkowski, and H. Bitter-Suermann. . . . . . . . . . . . . . . . . . . . . . . . . . 626
`Skin allograft rejection by both L3/T4+ and Lyt-2+ T cell subsets.
`S. Cobbold and H. Waldmann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
`Improved renal allograft function and survival following nonspecific blood transfusions: I.
`Induction of soluble suppressor factors inhibiting the mitogenic response.
`R. Roy, J. Lachance, R. Noel, J.H. Grose, and R. Beaudoin.
`. . . . . . . . . . . . . . . . . . . . . . . 640
`Treatment and prevention of acute graft-versus-host disease with thalidomide in a rat model.
`G.B. Vogelsang, A.D. Hess, G. Gordon, and G.W. Santos.. . . . . . . . . . . . . . . . . . . . . . . . . 644
`BRIEF COMMUNICATIONS
`Transfusion of donor class I MHC antigen prior to kidney transplantation in dogs.
`P.F. Gores, A. Sitges-Serra, U.J. Hesse, J.S. Najarian, and D.E.R. Sutherland.
`. . . . . . . . . . 648
`The influence of the transplantation technique on the duration of endocrine function of
`pancreas allografts in the rat model.
`W. Timmermann, T. Schang, and A. Thiede. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650
`The effect of exchange blood transfusions on the cytotoxic alloantibody response in AS
`strain rats.
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . 652
`N. Mistry, T. Horsburgh, P.S. Veitch, and P.R.F. Bell.
`Optimization by timing of oral cyclosporine to prevent acute kidney allograft rejection in dogs.
`M. Cavallini, F. Halberg, D.E.R. Sutherland, G. Cornelissen, J. Heils, and J.S. Najarian.
`. . . . 654
`Determination of cyclosporine concentration in bile.
`L. Scanlon, R. Baloh, B. Gridelli, K.M. Rao, B. Shaw, T. Starzl, and A. Sanghvi.
`Encapsulation of cyclosporine by phosphatidylinositol-cholesterol liposomes.
`L. Stuhne-Sekalec, J. Chudzik, and N.Z. Stanacev . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659
`Effect of cyclosporine and low-temperature culture on prevention of rejection of islet xeno(cid:173)
`grafts (rat-to-mouse).
`R. Terasaka, P.E. Lacy, R.P. Bucy, and J.M. Davie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661
`Positive direct antiglobulin tests associated with intravenous gamma globulin use in bone
`marrow transplant recipients.
`C.F. Whitsett, J.A. Pierce, and L.E. Daffin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663
`Hematuria and hypercalciuria following renal transplantation.
`J.E. Springate, R.D. Fildes, S.M. Mauer, and L.G. Feld. . . . . . . . . . . . . . . . . . . . . . . . . . . . 664
`Successful treatment of persistent post-transplant hyperparathyroidism with diphosphonate.
`K.M.L. Leunissen, F-J.F.E. Vismans, R.J.M. van Leendert, and J.P. van Hooff. . . . . . . . . . . 666
`
`. . . . . . . . . 657
`
`BIOEPIS EX. 1027
`Page 3
`
`

`

`Transplantation
`
`Official Journal of the Transplantation Society
`
`,], R. BATCHELOR
`London, England
`PETER ,J. MORRIS
`Oxford, En~-:land
`
`EUROPEAN EDITORIAL OFFICE:
`Department of Immunology
`Royal Postgraduate Medical School
`Hammersmith Hospital
`Ducane Road
`London W12, England
`
`Editors
`E. ,J, EICIIWALD
`Salt Lake City, Utah
`DAVID STEINMULLER
`Ann Arbor, lvfichtl;an
`
`ANTHONY P. MONACO
`Boston, Massachusetts
`MARY L. WOOD
`Boston, Massachusetts
`
`NORTH AMERICAN EDITORIAL OFFICE:
`New England Deaconess Hospital
`185 Pilgrim Road
`Boston, Massachusetts 02215
`
`A. G. BIRTCH, Sprinf.:field, Illinois
`M. M. BORTIN, Milwauhee, Wisconsin
`A. B. COSIMI, Boston, Massachusetts
`C. S. DAVID, Rochester, Minnesotu
`M.A. HARDY, New Yorh. New Yorh
`I. V. HUTCIIINSON, Oxford, England
`I{, H. KERMAN, Houston, Texas
`W. S. LAPP, !vfontreal, Canada
`
`Editorial Board
`Class of 1987
`D. W. MASON, Oxford, En~-:land
`R. W. MELVOLD, Chicauo, Illinois
`.J. MILLim, Miami, Florida
`R. PICIILMAYR, Hannover, Germany
`,J. C. ROSENBERG, Detroit, Michiuan
`D. H. SACHS, Bethesda, Maryland
`,J. H. SOUTHARD, Madison, Wisconsin
`T. STARZL, Pittsburuh. Pennsylvania
`
`R. STORB, Seattle, Washington
`T. B. STROM, Boston, Massachusetts
`R. N. 'I'AUB, New Yorh, New Yorh
`E. THORSBY, Oslo, Norway
`A. 'I'ING, Oxford, England
`H. V/AGNEH, Ulm, Germany
`T. G. WEGMANN, Edmonton, Canada
`D. WHITE, Cambridge, England
`
`/
`
`E. ALBERT, Munich, Germany
`F. 0. BELZER, Madison, Wisconsin
`R E. BILLINGHAM, Dallas, Texas
`C. BUNCH, Oxford, England
`S. ,J. BUHAKOFF, /Joston, Massachusetts
`R. EPSTEIN, Ohlalwma City, {}/dalwma
`R B. E'IVI'ENmm, Los Arzueles, California
`,J. FABRE, East (lrinstcad, England
`R. P. GALE, Los Anueles, California
`N. E. GoEKEN, Iowa City, Iowa
`
`Class of 1 988
`B. M. HALL, Camperdown, N.S. W., Australia
`B. S. HANDWERGER, Rochester, Minnesota
`,J. M. Hows, London, Enuland
`H. ,JEEKEL, Rotterdam, The Netherlands
`T. E. MANDEL, Melbourne, Australia
`C. MILLER, Worcester, Massachusetts
`T. MOIIANAKUMAR, Richmond, Virginia
`E. MOLLER, Stochholm, Sweden
`D. E. PEGG, Cambridue, Enuland
`B .. J. ROSER, Cambridue, Enuland
`
`C. F. BARKER, /'hi/adelphia, l'ermsylvania
`S. I. CliO, l3ostorz, Massachusetts
`D. V. CRAMER, l'itt.~buruh. l'erznsylvaniu
`R. A. DA YNES, Salt Lahe City, Utah
`H .• J. DEEG, Seattle, Washinuton
`R. M. FEHGUSON, Columbus, Ohio
`.J. A. FRELINGER, Chapelllill,
`North Carolina
`M. H. GAIWVOY, San Francisco, California
`T. ,J. GILL, l'ittsburuh, l'ennsylvania
`E. C. GOHDON-SMITII, London, En~-:land
`
`Class of 1989
`E. GOULMY, Leiden, The Netherlands
`C. G. GROTH, Stochlwlm, Sweden
`R D. GU'lvi'MANN, Montreal, Canada
`B. D. KAHAN, Houston, Texas
`H. A. KREIS, Paris, France
`,J. W. KUPIEC-WEGLINSKI, Boston,
`M as,~achusetts
`I. McKENZIE, Melbourne, AuBtralia
`P. McMASTEH, Birmin~-:lwm, England
`G. OPELZ, Heidelberg, Germany
`L. C. PAUL, Lciden, The Netherlands
`
`R. H. RUBIN, Boston, Massachusetts
`F. SANFILIPPO, Durham, North Carolina
`G. W. SANTOS, Baltimore, Maryland
`J. W. STREILEIN, Miami, Florida
`P. I. 'I'ERASAKI, Los Angeles, California
`N. L. TILNEY, Boston, Massachusetts
`J.D. TYLER, St. Louis, Missouri
`G. M. WILLIAMS, Baltimore, Maryland
`R. F. M. WOOD, London, England
`
`I. PENN, Cincinnati, Ohio
`J. R. SALAMAN, Cardiff, Wales
`R. L. SIMMONS, Minneapolis, Minnesota
`E. SIMPSON, Harrow, England
`H. W. SOLLINGER, Madison, Wisconsin
`R. M. STEINMAN, New Yorh, New Yorh
`D. E. R. SUTHERLAND, Minneapolis,
`IV! innesota
`J. M. THOMAS, Greenville, North Carolina
`P. J. TUTSCHKA, Columbus, Ohio
`D. A. VALLERA, Minneapolis, Minnesota
`
`Transplantation OSSN 0041-1:l:J7) is published monthly by Wil(cid:173)
`liams & Wilkins, 42B E. Preston Street, Baltimore, MD 21202. Second
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`liams & Wilkins.
`
`BIOEPIS EX. 1027
`Page 4
`
`

`

`Transplantation
`INSTRUCTIONS FOR AUTHORS
`
`Articles dealing with completed research, and brief or prelimi(cid:173)
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`Overview articles should cover a topic of special interest in the
`field of transplantation. The overview should not exceed 4000
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`1. Winearls C.J, Fabre .JW, Millard PH, Morris PJ. Use of
`cyclophosphamide and enhancing serum to suppress renal
`allograft rejection in the rat. Transplantation 1979; 28: 271.
`2. Osler AG. Complement: mechanisms and functions. Engle(cid:173)
`wood Cliffs: Prentice Hall, 1976.
`3. National Center for Health Sciences. Acute conditions: in(cid:173)
`cidence and associated disability, United States July 1968-
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`tistics, 1972. (DHEW publication no. (HSN) 72-1036).
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`
`BIOEPIS EX. 1027
`Page 5
`
`

`

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`AUTHOR INDEX
`
`Amadeo, ,J. H., 606
`
`Baloh, R., G57
`Bander, N. H., 59:3
`Beatty, P., 583
`Beaudoin, R., 640
`Bell, P. R. F., 652
`Bensinger, W. I., G02
`Berndt, M. C., 5G5
`Bitter-Suermann, H., 626
`Bradstock, K. F., 5G5
`Broomhead, A. F., 565
`Buckner, C. D., G02
`Bucy, R. P., 661
`Buszta, C., 598
`
`Cavallini, M., ()54
`Chudzik, .J., 659
`Clift, R. A., 602
`Cobbold, S., 634
`Colvin, R. H., 572
`Cordon-Cardo, C., 59:3
`Cornelissen, G., 654
`Cosimi, A. H., 572, 593
`
`Daffin, L. E., 663
`Davie, J. M., 661
`Deeg, H .• J., 583
`Delmonico, F. L., 59:l
`Duncan, W. R., 626
`Duquesnoy, R. .J., 620
`
`Feld, L. G., 664
`Fildes, R. D., 664
`Finstad, C. L., 593
`Fuller, T. C., 1572
`Fung, J., 620
`
`Girard, B., 556
`
`Glichlick, D., 579
`Gonzalez, Z., GOG
`Goormastic, M., 598
`Gordon, G., G44
`Gores, P. F., G48
`Greenleaf, G. E., 556
`Gridelli, B., G57
`Griffith, B. P., 620
`Grose, ,J. H., 640
`
`Halberg, F., 654
`Hansen, W. P., 593
`Hardesty, R. L., 620
`Heils, ,J., 654
`Hess, A. D., G44
`Hesse, U. ,J., 648
`Horsburgh, T., 652
`Hughes, W. G., 565
`Hutchinson, I. V., 547
`
`Jaffers, G .• J., 572
`
`Kabral, A., 565
`Karwa, G., 579
`Kaufman, C., 620
`Khauli, R. B., 598
`Klotz, L. H., 593
`
`Lachance, J., 640
`Lacy, P. E., 661
`Leunissen, K. M. L., 666
`Levy, G .• J., 583
`Luft, B. J., 611
`Lum, L. G., 583, 587
`
`Magnusson, M., 598
`Matas, A. ,J., 579
`Mauer, S. M., 6G4
`McCabe, R. E., 611
`
`Mistry, N., 652
`
`Najarian, J. S., G48, G54
`Nakamoto, S., 598
`Noel, R, 640
`Novick, A. C., 598
`
`Old, L. J., 593
`Orloff, M. J., 556
`
`Paganini, E., 598
`Persson, A., 616
`Pierce, J. A., 663
`Piper, D. J., 593
`Prendergast, M. M., 565
`
`Quinn, T., 579
`
`Rabin, B. S., 620
`Ramirez-Gonziilez, R., 606
`Rao, K. M., 657
`Remington, J. S., 611
`Rive-Mora, E., 606
`Hoy, R, 640
`Rubin, R. H., 593
`Russell, P. S., 572, 593
`
`Sanders, J., 583
`Sanghvi, A., G57
`Santiago-Delpin, E. A.,
`606
`Santos, G. W., 644
`Scanlon, L., 657
`Schang, T., 650
`Schreiber, M. J., 598
`Shaw, B., 657
`Shiobara, S., 587
`Sitges-Serra, A., 648
`Slichter, S. J., 602
`
`Soberman, R., 579
`Solders, G., GIG
`Springate, J. E., GG4
`Stanacev, N. Z., G59
`Starzl, T., G57
`Steinmuller, D. R., 598
`Stepkowski, S. M., G26
`Storb, R., 583, 587
`Stuhne-Sekalec, L., 659
`Sullivan, K. M., 583
`Sutherland, D. E. R., 648,
`654
`
`Tellis, V. A., 579
`Terasaka, R., 661
`Thiede, A., 650
`Thomas, E. D., 583, 602
`Thompson, R. E., 593
`Timmermann, W., 650
`Tiver, K., 565
`Tolkoff-Rubin, N. E., 593
`
`Urban, P., 556
`
`Van Hooff, J. P., 666
`Van Leendert, R. J. M.,
`666
`Veitch, P. S., 652
`Veith, F. J., 579
`Vidt, D. G., 598
`Vismans, F-J. F. E., 666
`Vogelsang, G. B., 644
`
`Waldmann, H., 634
`Weiss, R., 579
`Whitsett, C. F., 663
`Wilczek, H., 616
`Winn, H. J., 572
`Witherspoon, R. P., 587
`
`Zeevi, A., 620
`Zerbe, T. R, 620
`
`BIOEPIS EX. 1027
`Page 6
`
`

`

`0041-1:!:37 /86/4105-0572$02.00/0
`TJtANSPLANTATION
`Copyright 19 19813 by The Williams & Wilkins Co.
`
`Vol. ·11, No.5
`Printed in U.S.A.
`
`MONOCLONAL ANTIBODY THERAPY
`
`ANTI-IDIOTYPIC AND NON-ANTI-IDIOTYPIC ANTIBODIES TO 0KT~3 AHISING DESPITE INTENSE
`lMMUNOSUPPHESSION 1
`
`GHEGOHY .]. JAFFEHS, THOMAS C. FULLER, A. BENEDICT COSIMI, PAUL S. RUSSELL,
`HENHY .J. WINN, AND HOBEHT B. COLVIN~
`
`Transplantation-Immunulogy and lmrmmopathology Units, Departments of Surf!ery and I'atho/of!y, Massachusetts Genera! Hospital
`and Ilaruard Medical Sc/u;o/, /Joston, Massachusetts 0211·1
`
`The frequency, timing, and specificity of the humoral
`antibody response to a murine monoclonal antibody
`(OKT3, IgG2a) were measured in 21 consecutive renal
`allograft recipients. These patients received i.v. OKT3,
`1-5 mg/day for 10-20 days as treatment for acute graft
`rejection. Maintenance immunosuppression consisted of
`azathioprine and corticosteroids. Using three different
`assays, an antibody response was detected in 75% of the
`20 patients with adequate samples. The ELISA assay of
`the overall lgM and lgG reactivity to OKT3 revealed
`that lgM anti-OKT3 appeared in 65% and lgG anti(cid:173)
`OKT3 in 50% of the patients, reaching a peak 20-33
`days after the last dose of OKT3. The IgM prccceded the
`lgG in most cases (P<0.02) and in 8 cases was detected
`during therapy. One patient had high levels of lgM anti(cid:173)
`OKT3 before therapy, yet responded normally to OKT3.
`Interference with the therapeutic effectiveness was ev(cid:173)
`ident in one patient who developed IgG antibodies dur(cid:173)
`ing therapy. His serum blocked the binding of F-OKT3
`to normal lymphocytes in the presence of normal
`BALB/c serum. The blocking assay, done by flow cytom(cid:173)
`etry, measured anti-idiotypic (ld) reactivity since the
`sera did not affect the binding of OKTS (another IgG2 .. )
`or anti-Leu4 (another anti-T3), and the blocking activ(cid:173)
`ity remained after affinity absorption with normal
`mouse lgG. Using this assay, 60% of the pateints made
`an anti-ld response. One made only anti-ld, and several
`had anti-Id at times when other reactivities were unde(cid:173)
`tectable. Antibodies to non-idiotypic, presumably iso(cid:173)
`typic, determinants represented on OKTS occurred in
`only 44%, while other reactivity (OKT4; lgG2bK) was
`less common (12%) and weaker.
`While no adverse allergic reactions occurred in this
`group of patients, the anit-Id antibodies, which arc a
`prominent feature of the immune response to this and
`probably other monoclonal antibodies, can block their
`therapeutic effectiveness and can arise despite intense
`immunosuppression. This response may require the use
`of different idiotypes for prolonged or repeated courses
`of therapy and may be the major obstacle to the use of
`human monoclonal antibodies.
`
`The parenteral administration of foreign serum proteins has
`been known since the early days of serotherapy to stimulate an
`immune response. The typical allergic manifestations include
`fever, skin rash, anaphylaxis, and serum sickness. This phe-
`
`nomenon has acquired new relevance with the application of
`murine hyhridoma-derived antibodies to clinical medicine. De(cid:173)
`spite some initial optimism, the injection of these murine
`proteins into man, as expected, regularly provokes an immune
`response by the host ( 1-11 ).
`We have had extensive experience with the usc of one mono(cid:173)
`clonal antibody, OKT:l (IgGc,,;.;, BALB/c), used as an adjuvant
`to azathioprine and prednisone for treatment of acute renal
`allograft rejection (I, 2, II). This antibody, reactive with an
`invariant component ('I':l)* of the T cell antigen receptor com(cid:173)
`plex ( 12), has proved to be dramatically effective in reversing
`acute cellular rejection when administered at doses of 1-5 mg/
`day over 10-20 days. This report describes in detail the speci(cid:173)
`ficity of the antibody response to OKT3 in 21 renal allograft
`recipients, and the implications for therapy. A preliminary
`report of the first 11 patients has been presented (6, 7).
`
`MATEHIALS AND METHODS
`Patients. The 21 consecutive OKT:l-treated patients studied
`were initially treated with prednisone and azathioprine after
`receiving a cadaver donor renal allograft ( 1, 2). They were given
`OKT:l (Ortho Pharmaceuticals, Raritan, N.J) when they had
`their first episode of renal allograft rejection. The antibody was
`given once daily as a 1-5-mg i.v. bolus. No patient manifested
`a skin reaction to intradermal OKT:l (0.1 pg) prior to treatment.
`In an attempt to modify the immune response to OKT3, G
`patients also received cyclophosphamide (patients Nos. 10 and
`17-21).
`Serum samples. l3lood samples were collected in acid-citrate(cid:173)
`dextrose (ACD) at least twice weekly beginning at the time of
`transplantation and continuing for up to ten months after
`OKT:l therapy. An average of 19 samples per patient were
`analyzed in 20 patients (range 12-24). One patient (No. 3) had
`only :3 posttreatment samples (all negative) and therefore was
`not included in further analysis. The samples were centrifuged
`at 1500 rpm and the plasma was stored at -20°C. Peripheral
`blood lymphocytes from these samples were used for immuno-
`logic monitoring as described previously ( 1 ).
`Enzyme-linhed immurwsorbent assay (ELISA). This assay,
`performed by a slight modification of published methods (13),
`was used to detect both anti-Id and non-anti-Id anti-OKT~l
`antibodies. Polyvinyl vinyl plastic microtiter plates (lmulon I,
`
`1 This work was supported by US PHS Grant HL-18646 and by funds
`provided by the Ortho Pharmaceutical Corporation.
`2 Reprint requests should be addressed to H.B. Colvin, M.D., Im(cid:173)
`munopathol06'Y Unit, Department of Pathology, Massachusetts Gen(cid:173)
`eral Hospital, Boston, MA 02114.
`
`• Abbreviations used: ACD, acid citrate dextrose; CAl•\, (BALB/
`cxAj.J)I•\; ELISA, enzyme-linked immunosurbent assay; F-, t1uores(cid:173)
`cein-conjugated (e.g., F-OKT:l); Id, idiotype; MFC, median t1uorescence
`channel; PBS, phosphate-buffered saline; PHA, phytohemagglutinin;
`T:l, the 20-25 kilodalton surface antigen of mature T cells.
`572
`
`BIOEPIS EX. 1027
`Page 7
`
`

`

`May, 1986
`
`JAFFERS ET AL.
`
`573
`
`Dynatech, Alexandria, VA) were coated with 1 J.Lg/ml of purified
`parenteral grade OKT3, 0.05 M NaHCO:; buffer (pH 9.6). The
`plates were incubated overnight at 4 oc then washed with phos(cid:173)
`phate-buffered saline containing .05% (v/v) Tween-20 (0.01 Na
`phosphate, 0.14 M NaCI, (pH 7.4) (PBS-Tween). Each serum
`sample was diluted 1:240 in PBS-Tween (5 J.Ll/1.2 ml buffer).
`200 Ill of this mixture was added to the wells. The plates were
`incubated for 2 hr at room temperature, washed with PBS(cid:173)
`Tween, and 200 J.Ll of a 1:500 dilution of alkaline-phosphatase(cid:173)
`conjugated goat antihuman IgG (Kirkegaard-Perry, Gaithers(cid:173)
`berg, MD)-or goat antihuman IgM (both heavy chain specific)
`was added at a similar concentration. Anti-IgG with light chain
`reactivity was used in early studies as a screening test. These
`solutions were incubated for 2 hr at room temperature and
`washed again using PBS-Tween. Finally, 200 J.Ll of 1 mg/ml p(cid:173)
`nitrophenyl phosphate in 10% diethanolarnine was added. The
`resultant color change of substrate was read on an ELISA plate
`spectrophotometer (Bio-Tek Instruments) at 405 nM. Sera
`from 15 healthy volunteers and assays without OKT3, serum,
`or anti-Ig were used as controls. All assays were done in
`triplicate. Each daily run was standardized by running aliquots
`of a known postive control. This serum had a net OD.o:; (test(cid:173)
`blank) of about 650 and 70 for the IgG and IgM assays,
`respectively. Standardized values for IgG were obtained by
`multiplying the net OD4o:> of the test sample by 650/(0D.ws of
`standard) on that day (or 70/0D.10,, of standard for IgM). A
`sample was considered positive if the corrected OD4os was 2 SD
`above the mean of the controls (95th percentile) in the case of
`the pretreatment samples, or if the OD.10,., was more than double
`the pretreatment OD4'" in the case of subsequent samples
`(provided the OD.1or. was >100).
`P HA blast preparation. The preparation of phytohemagglu(cid:173)
`tinin (PHA)-stimulated T cell blasts was accomplished by
`methods previously described (14). Brief1y, normal peripheral
`mononuclear cells were isolated on Ficoll-Hypaque gradients
`and stimulated with 1 J.lg of PHA per 10'1 lymphocytes in 1 ml
`of RPMI 1640 medium containing 5% normal human serum,
`antibiotics, and 5 J.LM mercaptoethanol. Cells were maintained
`hy dilution to 10" /ml in T cell growth factor medium containing
`interleukin-2 about every third day.
`Enhancin{i assay for rwn-Anti-Id antibodies. Using flow cy(cid:173)
`tometry, non-anti-Id human antibodies that included isotype(cid:173)
`specific reactivity to murine IgGz, were detected by the binding
`to OKT8-coated (IgG 2,K,CAI•\) PHA blasts. Antibodies not
`specific for Id or isotype were similarly measured with OKT4-
`c<Jated OgG2bK,CAF 1) targets. OKT3-coated targets were used
`as a positive control. PHA blasts (which contained both T3+T4+
`and T:J+Ts+ cells) were suspended at a concentration of 10G/ml
`in Iu>MI 1640. Nonf1uoresceinated OKT8, OKT4, or OKT3
`ifJ.5 J.Lg) or PBS was added to each tube containing 50 J.Ll of
`PHA blasts. The suspensions were incubated at 4 oc for 30 min
`and washed twice with PBS. 50 J.LI of test or normal serum was
`added and the mixture incubated and washed as before. Flu(cid:173)
`oresceinated goat antihuman IgG (heavy and light chain reac(cid:173)
`tive) was then added and the tubes were incubated and washed
`as before. The median intensity of the positive staining was
`evaluated using the Spectrum III f1ow cytometer (Ortho Diag(cid:173)
`nostics System, Westwood, MA) (1.5). The percentage of in(cid:173)
`crease in the median f1uorescence channel (MFC, linear units)
`above uncoated blasts (no monoclonal antibody) for each sam(cid:173)
`ple was calculated. This corrects for any contribution by anti(cid:173)
`T-cell antibodies that may be present in the patient's serum,
`
`e.g., anti-HLA. Positive values were defined as 2 SD units
`above the mean of 21 normals.
`Affinity columns. OKT3 and normal mouse IgG (Miles) were
`coupled to CNBr-activated Sepharose 4B (Pharmacia) by
`standard techniques, with approximately 10 mg of protein/ml
`swollen gel. Columns were 12X0.9 em (mouse IgG) and 4.5X0.9
`em (OKT3). Fractions were eluted in PBS and then 3M KSCN,
`dialyzed, and reconcentrated to the starting volume by positive
`pressure filtration (Amicon XM-50).
`Anti-idiotype (!d) assay. The basis of this assay is that certain
`anti-Id block the binding of the Id-bearing antibody with an(cid:173)
`tigen. The test was done by flow cytometry using f1uorescein(cid:173)
`ated OKT3 (F-OKT3) as the Id and T3+ T cell blasts (or
`normal peripheral blood lymphoc