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Transplantation voLuME 41 • NUMBER s May 1986

PFIZER 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 whole­Pancreas 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

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 . . . . . . . . . . 572 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 . ' 579

Severe apl~~t;~ ~~~~·i~ ·a·s~~~i~~~~ ·~i~~ ~~~~~i~ ·~~~~~~t~~~~~~ ~~~~;d·i~~i~:· ;~~~~~;~~i~ and hematologic reconstitution after allogeneic bone marrow transplantation.

SHt.J. Deeg, LG. lum, J. Sanders, G.J. Levy, K.M. Sullivan, P. Beatty, E.D. Thomas, and R. ~ 5~

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,

R .P. Hansen, N.H. Bander c L Finstad C. Cordon-Cardo, L.H. Klotz, L.J. Old, and R.H.

ubin ' · · ' A · · · · · · · ....................................................... 593

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.

P 1

t, M. Magnusson, E. Paganini, and M.J. Schreiber. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598 lasma

W 1 exch~nge for platelet alloimmunization.

R · · Bens1nger, C.D. Buckner, R.A. Clift, S.J. Slichter, and E.D. Thomas. . . . . . . . . . . . . . . 602 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. 606

e ~ffects of cyclosporine on Toxoplasma gondii in vivo and in vitro. A .E. McCabe, B.J. Luft, and J.S. Remington. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611

utonomic system dysfunction and polyneuropathy in nondiabetic uremia: A one year follow-~P study after renal transplantation.

· Solders, A. Persson, and H. Wilczek. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616

PFIZER 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. N. Mistry, T. Horsburgh, P.S. Veitch, and P.R.F. Bell. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652

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. . . . . . . . . . 657

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 xeno­grafts (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

PFIZER EX. 1027 Page 3

,], R. BATCHELOR London, England

PETER ,J. MORRIS Oxford, En~-:land

Transplantation Official Journal of the Transplantation Society

Editors E. ,J, EICIIWALD

Salt Lake City, Utah

DAVID STEINMULLER Ann Arbor, lvfichtl;an

ANTHONY P. MONACO Boston, Massachusetts

MARY L. WOOD Boston, Massachusetts

EUROPEAN EDITORIAL OFFICE: NORTH AMERICAN EDITORIAL OFFICE:

Department of Immunology Royal Postgraduate Medical School Hammersmith Hospital

New England Deaconess Hospital 185 Pilgrim Road Boston, Massachusetts 02215

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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

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

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

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

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

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. 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

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 Wil­liams & Wilkins, 42B E. Preston Street, Baltimore, MD 21202. Second class postage paid at Baltimore, MD, and at additional mailing offices. Postmaster, send address changes (form :l!i7~l) to Williams & Wilkins, 42B E. Preston Street, Baltimore, MD 21202. Subscription rates

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PFIZER EX. 1027 Page 4

Transplantation INSTRUCTIONS FOR AUTHORS

Articles dealing with completed research, and brief or prelimi­nary reports designated as Brief Communication will be consid­ered for publication. The latter should present new ideas or techniques or findings of special timeliness and significance. Brief Communications should not be divided into sections; it is recommended that the last paragraph be a summary and the entire double-spaced typewritten manuscript including tables and figures should not exceed 10 pages. Letters to the Editor, whether in commentary on articles appearing in TRANSPLAN­TATION or about other pertinent matters will also be considered. Overview articles should cover a topic of special interest in the field of transplantation. The overview should not exceed 4000 words and should not be divided in sections. The references should be done according to the instructions below for Refer­ences. If and when manuscripts are accepted for publication, it will be necessary for The Williams & Wilkins Company to receive in writing the assignment of copyright from all authors of all manuscripts. Forms for this purpose will be supplied. Submit an original, 2 copies, and 3 sets of illustrations. Avoid extensive handwritten alterations. Failure to observe the for­mat requested may delay processing of the manuscript. Typescript must be double-spaced throughout. Use bond paper for the original copy. Maintain a minimum left margin of approximately 4 em and a right margin of 2.5 em. Number the pages in sequence. Title page should carry the title, author's name under the title, and institution and address under the author's name. Include postal zone or "zip code" in the address. To avoid confusion in bibliographic indexing, it is preferable for authors to state given names in full. Title should be concise (preferably not more than 15 words). Summary of not more than 250 words is to be typed on a separate sheet. Footnotes for the text should be typed double-spaced on a separate page, but all footnotes may be typed on the same page. Footnotes should be designated by superscript Arabic numbers and numbered in sequence. Footnotes should include source of support (cited as footnote to the title), current address, corre­spondence address, and abbreviations. Abbreviations used in the text and their meanings should be listed alphabetically and typed double-spaced on a separate sheet of paper or on the page with other footnotes. References should be typed double-spaced on a separate sheet. References should be numbered in the order in which they are cited in the text. Six or fewer authors should be listed. If there are seven or more, only the first three and "et al." should be listed. The titles of articles are to be included, and the journal names are to be abbreviated according to the Index Medicus style. Text citations of the references are placed in parentheses. Only papers that have been accepted for publication should be listed in the references. Manuscripts in preparation, unpub­lished observations, and personal communications should be referred to as such in the text. Completed manuscripts that have been submitted for publication may be cited as footnotes to the text. If the manuscript is subsequently in press, appro-

priate changes can be made in the galleys. The following are examples of the reference style: 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. Engle­wood Cliffs: Prentice Hall, 1976.

3. National Center for Health Sciences. Acute conditions: in­cidence and associated disability, United States July 1968-,June 1969. Hockville, Md.: National Center for Health Sta­tistics, 1972. (DHEW publication no. (HSN) 72-1036).

Tables should be typed double-spaced throughout (including column heads, footnotes, and data), each table on a separate sheet. Number the tables in sequence with Arabic numerals. For each table supply a concise descriptive heading. Designate explanatory footnotes by superscript lowercase Latin letters. Type footnotes immediately below the table to which they refer. All columns must carry concise headings descriptive of the data in the column. Photographs of tables are not acceptable. All tables are cited in the text in numerical order. Figures (i.e., photomicrographs, charts, and graphs) are to be supplied as unmounted glossy photographic prints. Lettering on graphs should be large enough so that when the figure is reduced to the width of a single column (8.8 em), the lettering is still legible (1.5 mm high for an 8.8-cm-wide figure). Photo­micrographs and other photographs should be no larger than 8.8 em in width, if possible. Number the figures in sequence in Arabic numbers. Figures should be labeled on the back with the author's name, figure number, and the word "top." All figures are cited in the text in numerical order. Figure legends, numbered to correspond with the figures, typed double-spaced, and assembled on a separate sheet are to be supplied for all figures. These legends should explain all symbols used on the figures and should indicate staining and magnification for photomicrographs. Page charges. Authors will be charged $40.00 for each printed page per article. Under exceptional circumstances, page charges will be waived by the editors.

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AUTHOR INDEX

Amadeo, ,J. H., 606 Glichlick, D., 579 Mistry, N., 652 Soberman, R., 579 Gonzalez, Z., GOG Solders, G., GIG

Baloh, R., G57 Goormastic, M., 598 Najarian, J. S., G48, G54 Springate, J. E., GG4

Bander, N. H., 59:3 Gordon, G., G44 Nakamoto, S., 598 Stanacev, N. Z., G59

Gores, P. F., G48 Starzl, T., G57 Beatty, P., 583 Noel, R, 640 Steinmuller, D. R., 598 Beaudoin, R., 640 Greenleaf, G. E., 556 Novick, A. C., 598 Stepkowski, S. M., G26 Bell, P. R. F., 652 Gridelli, B., G57 Storb, R., 583, 587 Bensinger, W. I., G02 Griffith, B. P., 620 Stuhne-Sekalec, L., 659 Berndt, M. C., 5G5 Grose, ,J. H., 640 Old, L. J., 593 Sullivan, K. M., 583 Bitter-Suermann, H., 626

Orloff, M. J., 556 Sutherland, D. E. R., 648, Bradstock, K. F., 5G5 Halberg, F., 654 654

Broomhead, A. F., 565 Hansen, W. P., 593 Paganini, E., 598 Buckner, C. D., G02 Hardesty, R. L., 620 Persson, A., 616 Tellis, V. A., 579 Bucy, R. P., 661 Heils, ,J., 654 Pierce, J. A., 663 Terasaka, R., 661 Buszta, C., 598 Hess, A. D., G44 Piper, D. J., 593 Thiede, A., 650

Hesse, U. ,J., 648 Prendergast, M. M., 565 Thomas, E. D., 583, 602

Cavallini, M., ()54 Horsburgh, T., 652 Thompson, R. E., 593

Chudzik, .J., 659 Hughes, W. G., 565 Quinn, T., 579 Timmermann, W., 650

Clift, R. A., 602 Hutchinson, I. V., 547 Tiver, K., 565 Tolkoff-Rubin, N. E., 593

Cobbold, S., 634 Rabin, B. S., 620 Colvin, R. H., 572 Jaffers, G .• J., 572 Ramirez-Gonziilez, R., 606 Urban, P., 556 Cordon-Cardo, C., 59:3 Rao, K. M., 657 Cornelissen, G., 654

Kabral, A., 565 Remington, J. S., 611 Cosimi, A. H., 572, 593 Rive-Mora, E., 606

Van Hooff, J. P., 666 Karwa, G., 579 Van Leendert, R. J. M., Kaufman, C., 620 Hoy, R, 640 666

Daffin, L. E., 663 Khauli, R. B., 598 Rubin, R. H., 593 Veitch, P. S., 652 Davie, J. M., 661 Klotz, L. H., 593

Russell, P. S., 572, 593 Veith, F. J., 579 Deeg, H .• J., 583 Vidt, D. G., 598 Delmonico, F. L., 59:l

Lachance, J., 640 Sanders, J., 583 Vismans, F-J. F. E., 666

Duncan, W. R., 626 Sanghvi, A., G57 Vogelsang, G. B., 644

Duquesnoy, R. .J., 620 Lacy, P. E., 661 Santiago-Delpin, E. A., Leunissen, K. M. L., 666 Levy, G .• J., 583

606 Waldmann, H., 634

Feld, L. G., 664 Luft, B. J., 611 Santos, G. W., 644 Weiss, R., 579

Fildes, R. D., 664 Lum, L. G., 583, 587 Scanlon, L., 657 Whitsett, C. F., 663

Finstad, C. L., 593 Schang, T., 650 Wilczek, H., 616

Fuller, T. C., 1572 Schreiber, M. J., 598 Winn, H. J., 572

Fung, J., 620 Magnusson, M., 598 Shaw, B., 657 Witherspoon, R. P., 587

Matas, A. ,J., 579 Shiobara, S., 587

Girard, B., 556 Mauer, S. M., 6G4 Sitges-Serra, A., 648 Zeevi, A., 620 McCabe, R. E., 611 Slichter, S. J., 602 Zerbe, T. R, 620

PFIZER 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 anti­OKT3 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 anti­OKT3 before therapy, yet responded normally to OKT3. Interference with the therapeutic effectiveness was ev­ident in one patient who developed IgG antibodies dur­ing 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 cytom­etry, 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 activ­ity 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 unde­tectable. Antibodies to non-idiotypic, presumably iso­typic, 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-

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., Im­munopathol06'Y Unit, Department of Pathology, Massachusetts Gen­eral Hospital, Boston, MA 02114.

nomenon has acquired new relevance with the application of murine hyhridoma-derived antibodies to clinical medicine. De­spite 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 mono­clonal 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 com­plex ( 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 speci­ficity 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-citrate­dextrose (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,

• Abbreviations used: ACD, acid citrate dextrose; CAl•\, (BALB/ cxAj.J)I•\; ELISA, enzyme-linked immunosurbent assay; F-, t1uores­cein-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

PFIZER 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 phos­phate-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 PBS­Tween, and 200 J.Ll of a 1:500 dilution of alkaline-phosphatase­conjugated goat antihuman IgG (Kirkegaard-Perry, Gaithers­berg, 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 p­nitrophenyl 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:; (test­blank) 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 phytohemagglu­tinin (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 cy­tometry, non-anti-Id human antibodies that included isotype­specific 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,CAF1) 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. Flu­oresceinated goat antihuman IgG (heavy and light chain reac­tive) was then added and the tubes were incubated and washed

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 an­tigen. The test was done by flow cytometry using f1uorescein­ated OKT3 (F-OKT3) as the Id and T3+ T cell blasts (or normal peripheral blood lymphocytes) as the antigen in the presence of the test serum. Normal BALB/c mouse serum was added to block non-anti-Id antibodies. The controls included normal human serum and fluoresceinated OKT8 (F-OKT8). The amount of F-OKT3 required to saturate PHA blasts or peripheral lymphocytes was determined by titration with a constant number of cells, plotting the MFC-vs.-concentration of F-OKT3 used (Fig. 1). Saturation was achieved at 8 J.Lg/ml ofF -OKT3; beyond this point no further shift in the MFC was achieved. A point of about 30% saturation was selected (2.5 J.Lg/ ml) for use in the assays to maximize sensitivity. A similar point was determined for F-OKT8.

The assay was performed by adding 25 J.Ll of test or normal serum to 20 J.Ll of BALB/c serum and incubating for 30 min at room temperature. Subsaturating amounts of F-OKT3 or F­OKT8 were added and these were incubated for 30 min on ice. Normal huffy coat peripheral blood lymphocytes (25 J.LO, pre­viously prepared from ACD blood, were added to each tube with further incubation for 30 min at 4 oc. Residual erythro­cytes were lysed with NH4Cl and the cells were washed twice in PBS. Samples were analyzed using the Spectrum III f1ow cytometer gated on the lymphocyte or lymphoblast cluster. The MFC of the positive cells above the positive threshold channel was taken as the index of staining intensity. Pretreatment samples had inhibition of 7±7%. Therefore a decrease in the MFC of greater than 20% of the normal serum control, provided there was not a concommitant inhibition ofF-OKT8, was taken as evidence for the presence of anti-Id antibody. Samples

10 20 40 80 160 320 640 1280 2560

DILUTION

as before. The median intensity of the positive staining was evaluated using the Spectrum III f1ow cytometer (Ortho Diag­nostics System, Westwood, MA) (1.5). The percentage of in­crease in the median f1uorescence channel (MFC, linear units) above uncoated blasts (no monoclonal antibody) for each sam­ple was calculated. This corrects for any contribution by anti­T-cell antibodies that may be present in the patient's serum,

FIGUHE I. Plot of t1uorescence intensity (MFC, linear units) vs. dilution of F-OKT3 on PHA lymphoblasts. A point of about 30% saturation was selected for the anti-Id inhibition assay.

PFIZER EX. 1027 Page 8

574 TRANSPLANTATION Vol. 41, No.5

containing no BALB/c serum were analyzed in parallel. F -anti­Leu 4 (Becton Dickinson Monoclonal Center, Mountain View, CA) was used for comparison.

Statistical analysis. Values given are mean ± SD unless otherwise noted. Student's t and Fisher's exact tests were used for evaluating statistical significance. An antibody response was considered positive if two or more sample values were above the defined threshold, either a doubling of the pretreat­ment value or above the 95th percentile of normal controls.

RESULTS

ELISA: total Anti-OKT3 response. Using the ELISA with OKT3 as the antigen, the lgG and lgM antibodies of all specificities to OKT3 were determined. Except for patient No. 18, the ELISA reactivity of the patient's pretreatment serum was similar to that of the 15 controls, both for lgG and for lgM (Table 1) and the anti-lg with light chain reactivity gave essentially the same result as anti-y chain.

Patient No. 18 had the highest pretreatment reactivity, which was exclusively lgM. This binding could be completely elimi­nated by preincubation of the test serum with BALB/c serum, and partially blocked with preincubation in normal goat serum. A rheumatoid factor assay with human lgG was within normal limits (<60 I.U.). The level of the lgM antimouse lgG antibody did not change by more than 5% during treatment, and the patient responded normally to the OKT3 therapy (Table 2).

An anti-OKT3 antibody response, defined as doubling of the pretreatment OD4os, was detectable at various times posttreat­ment in 14 (70%) of those individuals (Tables 2 and 3). Four had only lgM and one had only lgG. The lgG was detected significantly later than the lgM by an average of 10 days, although the time of the peak level was the same (20-23 days) (Tables 2-4). Antibodies were detected during OKT3 adminis­tration in 8 patients. All 8 had lgM antibodies and 2 also had lgG. Only one patient (No. 4), who had lgG as well as IgM anti-OKT3, showed resistance to the pharmacologic effects of OKT3. The other 7 cleared their T cells normally despite the presence of lgM anti-OKT3. Only 10% (lgG) and 33% (lgM) of the responders had persistent antibodies when checked 6 weeks or longer after the last dose. Two had detectable levels 14 weeks posttherapy (Nos. 7, 15).

Enhancing assay. This assay was used to detect isotype and light chain reactivity by assaying T8+ and T4+ PHA blasts coated with OKT8, OKT4, or OKT3 antibodies. Three patients (Nos. 10, 12, and 14) had pretreatment reactivity above the 95th percentile (Tables 1 and 2). Of the 16 tested, 7 (44%) showed a two-fold or greater increase in reactivity to OKT8 (lgG2nK)-coated cells. Only two (12%) bound (both weakly) to OKT4 (lgG2bK)-coated cells, suggesting that the reactivity de­tected was primarily to isotype specificities on the heavy chain. The timing of the anti-isotype was closer to that of the lgM than the lgG (Table 4).

OKT8 and OKT:3 enhancing reactivity were disparate in 2 patients. The serum from patient No. 1 reacted with OKTS coated, but not OKT:3 coated cells. She had blocking anti-Id antibodies and it is possible that these displaced the OKT3 from the target cell, giving a false negative. A similar phenom­enon may explain the negative OKT3-enhancing results in patients Nos. 2 and 17 who also had blocking anti-Id. A second patient (No. 14) had the opposite pattern, reacting with OKT3-coated, but not OKT8-coated cells. The restricted reactivity in this case may have been due to anti- ld of the non blocking type.

Bloching anti-Id assay. None of the pretreatment sera specif­ically blocked the binding of F-OKT3 or F-OKT8 to normal lymphocytes (Table 1). However, after the course of OKT:J therapy, 12 patients (()0%) developed antibodies that selectively blocked the antigen-binding of F-OKT3. The sera blocked F­OKT:l in the presence of normal BALB/c serum and did not

block F-OKT8 or F-anti-Leu 4, arguing that the specificity was restricted to idiotypic-and not allotypic or isotypic-deter­minants on OKT:l (Fig. 2).

TABLE 2. Onset of anti-OKT:l antibodies and effect on OKT3 response"

ELISA­OI('I':l

Patient

19 2 15 :lh 4 () 5 11 6 7 8 5 9 G

10 11 12 1:1 14 15 w 17 18 19 20 21

:n -1

10

Ir:M

4 -4

-2 7 8

-:3 -8

28

2 -10

-f>

p--10

-()

Anti· Id

8 2f>

()

2 5

2 10

2 8

14 8

4f>

Hesponse to Enhancing OKT:l (cells/

llllll3)d

~~~-

OKT:l OI<TS OKT·I T:l THT8

8 3 15:3 15 276 7 402

477 52·!

2 7 0 ·ll 0 15:3

118

2 2 3 191

4 0 1:10 p- p- p- 5 5G

45 5 80 p- p- 0 :H

2 2 72 1 392 p:l p- p- 1G 379

23 242 4 129

17 436 17 274 :lO 2:31 24 598

105

• The day when antibody was first detected (0 = last day of OKT:l therapy; negative numbers-days before therapy ended).

6 Only :l posttreatment samples. 'Abbreviations: p = pretreatment sample positive, >95th percentilt>

of normal controls; - = negative; blank = not done. d Last blood values on OKT:l.

TABLE 1. Pretreatment reactivity

ELISA (0D405 )" Enhancing assay Blocking assay

(% increase) (% inhibition)

lg lgG IgM OKT3 OK'l'8 OK'l'4 OKT3 OKT8

Patients 369±219 339±147 60±92 59±76 144±297 38±80 7±7 8±11 Controls 322±125 198±151 29±30 27±:!4 16±21

• Excludes patient No. 18 whose values were 1384 (lg), 118 (lgG), and 1718 (lgM) on the :3 ELISA assays. lg was a screening test using goat antihuman immunoglobulin reactive to 'Y heavy chain and both light chains, while lgG and lgM were detected by heavy-chain-specific antisera.

PFIZER EX. 1027 Page 9

May, 1986 ,JAFFEHS ET AL. 575

TABLE 3. Maximum antibody levela

ELISA-OKT3: OD,or. Blocking: Enhancing (% increase

MFC) Patient

lgG lgM Anti-ld (% In- OKT:l OKT8 OKT.t hibition)

G:l4(19) 596(59)" 75( 8) - 1122(19)

2 1761(15) 142(75) 89(25)

:J 4 1:314(14) 4GO( 5) 8:l(l4) :l58( 1) 5G9( 2)

5 172:3(15) 22G(l0) 91(12) 489( 7) 178( 7)

G 292( 8) 92(14) 111G(l4) 118:l(19)

7 8 1971(12) 5583(12) !JI( 8) 6-10(22) 200(22) 9 782(10) 881(10) 33(10) 400(13) 263(13)

10 11 599(39) 608(.15) - 94(52)

12 1:l - 8G9(13) 89( 2) 1200(76) 1500(76) G7(7G)

14 900(55) 5:38(29) 55(14) 10G7( 3)

15 G55(-1) 385(-1) Jfj

17 88(22)

18 8G5(30) 92(1G)

19 597( 0)

20 19!)(45) 58(45)

21

"Day of maximum (after OKT:J therapy ended) in parentheses. ''Assays in italics were positive in the last sample tested (more than

fj weeks from the last OKT:J dose).

TABLE 4. Summary of time course of antibody response

Number Onset•

IgM 1:3 0±3'' lgG 10 IO±:l Idiotype 12 11±4 Isotype 7 4±1

"Day after last OKT:J dose, mean ± SK • Preceeds JgG, P<.02.

Peuko

2:3±7 20±5 1G±3 2:3±9

Further experiments were performed to document whether the effect was indeed due to anti-Id antibodies. The presence <;f residual free OKT3 in the serum was ruled out by the lack of detectable mouse lg on normal lymphocytes after incubation in the blocking serum. In all patients OKT3 antibody was undetectable in the serum by 2 days after therapy had ended (data not shown). For this reason only sera taken at least 48 hr after the end of therapy were analyzed (except for patient No. 4 who was demonstrated to clear the OKT3 within 7 hr). Serum that blocked OKT3 did not block F-anti-Leu 4, an IgG1 antibody to a different epitope on the same molecule (Fig. 2). This argues strongly against free or complexed T3 antigen itself as a significant source of antigenic competition in the blocking assay.

Sera from two of the patients were analyzed by affinity ehromatography using columns with immobilized OKT3 or normal mouse IgG. The sera from patients Nos. 4 (Fig. 2 and :l) and 8 (Figs. 4 and 5) were absorbed with mouse IgG on Sepharose. The eff1uent and the dialyzed 3M KSCN eluate were tested for blocking activity. The eluate from normal mouse IgG (patient No. 8, day 5) showed equivalent blocking ofF­OKT:3 and F-OKT8 of 88% and 83%, respectively, in the

absence of BALB/c serum. The analogous eluate from patient No. 4 had no blocking activity. In contrast, the effluent blocked F-OKT3 completely but had little activity against F-OKT8, even in the absence of added BALB/c serum (Fig. 4). The eft1uent (patient No. 4) and the starting serum were then absorbed on an OKT3 column. This removed the blocking activity, which was undetectable even after reconcentration. Taken together, these data indicate that the blocking activity was due to antibodies that reacted selectively with OKT3 and not to other mouse immunoglobulins, satisfying the criteria for anti-Id specificity.

The time course of the anti-Id antibody production was variable, but generally paralleled that of the IgG (Table 4). The anti-Id antibodies first became detectable at an average of 11 days (range 0-45) after therapy had ended. The most rapid antibody response was manifested at 12 days after the start of therapy in patient No. 4 (Fig. 3). At this time he became refractory to the T lymphopenic effect of OKT3. The only clinical signs of an allergic reaction were an eosinophilia of 12% and a fever upon administration of the OKT3. No serum sickness lesions (glomerulonephritis, vasculitis, mouse lg de­posits by immunofluorescence) were seen in renal biopsy taken during the OKT3 treatment. The patient suffered no adverse systemic effects from this response, although the rejection process became reactivated and the allograft was eventually rejected. This was the only patient whose therapy was stopped because of anti-OKT3 antibodies. Three other patients had anti-lei antibody in the first sample analyzed, 2 days after therapy ended. Anti-Id antibody persisted in 33% of patients after 6 weeks; the longest documented was 6 months (No. 8).

Of the 12 patients that made a blocking anti-Id antibody, 3 (25%) had no IgG and 2 (17%) had no lgM reactivity detectable by ELISA. Similarly, 30% of those with blocking anti-Id had no isotype/light chain reactivity detectable by the enhancing assay. Patient No. 17 made exclusively an anti-Id response, detectable only in the blocking assay. Furthermore, the anti-Id assay detected antibody on one or more occasions in 7 other patients when the other assays were negative. This can be seen, for example, in the time course of the antibody response in patient No.8 (Fig 4), in which the anti-Id response was persis­tently positive when the ELISA and enhancing assays were negative. Two patients (Nos. 11 and 15) made anti-OKT3 antibody detected on ELISA that did riot block antigen binding or have reactivity to OKT8. These antibodies may include nonblocking anti-Id.

To estimate the portion of the antibody response that was anti-Id, serum from patient No. 8 at the peak of the antibody response (day 12, Fig. 4) was analyzed by ELISA using normal BALB/c serum as a competitive inhibitor for non-anti-Id anti­bodies. Normal mouse serum (100 Jl!) was able to block 69% of the IgG binding to OKT3, while 100% could be blocked by 10 Jlg OKT3, so that anti-Id constituted about 31% of the reactiv­ity.

Correlations with clinical course. With the exception of the one patient (No. 4) who developed anti-Id antibodies that neutralized the in vivo effectiveness of OKT3, no adverse effects were associated with the antimouse lg or anti-OKT3 idiotype response. The response to therapy and outcome of the graft were not correlated with the presence or absence of an antibody response. However, of the 6 patients who received cyclophosphamide (Nos. 10 and 17-21) only 17% made an lgG anti-OKT3 response detectable in ELISA, whereas 9 of 14 of

PFIZER EX. 1027 Page 10

576 THANSPLANTATION Vol. 41, No.5

NORMAL C4/13

OKT3 OKT3

18 ,-r 1- ,--, · 11-,. •· T-,-n

FIC:UHE 2. Fluorescence histograms of blocking anti-ld assny. Normal lymphocytes stained with fluoresceinated monoclonal antibodies in the presence of normal BALB/c serum and either normal human serum (left column) or serum from patient No. 4 (right column). The patient\ serum blocks F-OKT:l, hut not F-01\TS (both are lgG 2.) or F-anti-Leu 4 (IgGI, reactive to the T:l antigen). Cell number is on the y-axis and fluorescence intensity is on the x-axis. The num· her in the lower right of each histogram is the median fluorescence channel of the positive cell:;.

OKT8

Leu4

~~ .. 120

Patient 4

ELIS.4. a OK T3 (OD405J

lgM _ 20

lgO 274

BLOCKING aid 17

" inhlblllon

1000

1.0 200

Skin tnt 8 Bx: Rejection

'

2.0 10 40

Bx: Rejection 8MioG

'

10

PREDNISONE !¥] ]~0 mg/d /"///ij/,.,.)n,)

AZATHIOPRINE ·' ]~omg/d

25 235 358

156 257 573

83

OKT3

5 tO 15 20 25

DAYS POST TRANSPLANT

460

787

87

30

Leu4

120

FIGUHE 3. Clinical course of patient No. 4, who developed blocking anti-Id during therapy and became resistent to the T lymphopenic effects of OKT3. lgG and lgM anti-OKT3 was detected during therapy. Blocking anti-Id appearance was accompanied by resistance to the T lymphopenic response to OKT3 (day 22).

the non-cyclophosphamide group made such antibodies (?<0.076). Cyclophosphamide had little effect on the blocking anti-Id response, which still occurred in 50% of patients­although somewhat later than the others, peaking at 16-45 days. No correlation with the recipient's HLA phenotype and the antibody response was found.

J.O ~~Q ~·-~

II~ 160 * 240

...._ "' 0

~ ~ !jj

:2

~

1000

!5000 (\ P•llenl <#8

4000

3000

2000

1000

·:: l 00

40

20 ·::.:;fZ:/:'::>-:· .'·:'·:.::;:·:.:;::,;'!/},:'/) . .. ·.:u .\·;·;.:.:.::;{;;;::;).' :~\:.\ :~;HJ.

BLOCKING aid

oL..~~~~~~~....,..~­·20 ·10 0 10 20 30 40 50 60 70 80

/OKT3/ DAY AFTER LAST OKT3

FIGUHE 4. Time course of antibody response in patient No. 8, as detected by each assay in this study. The horizontal bar is the thresh­hold of positivity (2 SD above the mean of normals). Note the early rise of IgM and the presence of both blocking anti-Id and anti-isotype (OKT8 enhancing assay). No antibodies to OKT4 were detectable.

DISCUSSION

In this series 60% of the patients treated with OKT3 devel­oped anti-Id antibodies and 44% anti-isotypic antibodies, de­spite intense immunosuppressive treatment (Table 5). The

PFIZER EX. 1027 Page 11

lvfay, 1986 ,JAFFERS ET AL. 577

T3 T8

11

:! u I'

JJ'T -, . , -y-y-0 ~r-~• -, ., -r·r r·r·r • -, -r· , .. , - '1 l lO 10 1~0 UO 201 ~HI

F!GUHE 5. Blocking assay of the mouse IgG absorbed serum from patient No. 8, day 5. Fluorescence histograms ofT cell blasts stained with F-OKT:l (T3) or F-OKT8 (T8) in the presence of the effluent (E) that passed through the mouse IgG column or the control (C) which was the ultrafiltrate of the effluent (passed through XM-50). (U) unstained cells. No BALB/c serum was added. The effluent completely blocks F-OKT:l, in contrast to its slight inhibitory effect on F-OKT8.

TABLE 5. Summary of response pattern

N Anti-OKT3 Blockin~-: Antiisotype" by ELISA anti-Id

7 + + + 4 + + 3 +

+ 5

" Four patients were not tested for antiisotype.

response was transient and, with one exception, did not affect therapeutic outcome even though lgM antibodies frequently appeared before the completion of therapy.

A number of experiments indicated that these patients did make antibody with anti-Id specificity. The patient's serum blocked the antigen binding of OKT3, but not OKT8, a mono­clonal of the same isotype, and the blocking occured even in the presence ofBALB/c serum and after absorption with mouse immunoglobulin. These data indicate that the activity is spe­cific for OKT3 and not due to reactivity with other mouse immunoglobulin. Three other possibilities for this blocking activity were considered: free OKT3 antibody, free T3 antigen, and autologous anti-T3 antibody (anti-anti-ld). Residual free OKT:3, able to bind T:3 antigen, was ruled out by the inability to detect mouse lg binding to normal1' cells by flow cytometry and the ability of OKT3 affinity columns to remove the activity. The presence of free T3 antigen was rendered unlikely by the inability of the test sera to block the binding of anti-Leu 4, a monoclonal antibody to a different epitope on the same antigen complex. Similarly, no inhibition was obtained with 12F6, another lgG2• monoclonal antibody to T3 (unpublished data). One would have to postulate blocking by a fragment or altered T3 molecule. Finally, an anti-anti-Id response with human anti-T3 activity was considered, as has been described in pa­tients treated with an antitumor monoclonal antibody (9). This was unlikely because of the inability of the test serum to bind normal T lymphocytes (the control for the enhancing assay) and the ability of an OKT3 affinity column to remove the blocking activity.

The nonidiotypic reactivity detected by the binding of OKT8 was probably isotypic, because little reactivity was found to OKT4. We cannot exclude the possibility that a portion of the OKT8 reactivity may be to allotypic determinants not ex­pressed on OKT4. OKT3 was derived from a BALB/c mouse, and both OKT8 and OKT4 were obtained from (BALB/cXA/ J) F\ mice (16). The strains of origin of the heavy chains in OKT4 and OKT8 are unknown. Several lgG2• allotypic deter­minants of BALB/c are expressed by A/J but none are appar­ently expressed on lgG2b (17). Anti-isotype specificities have been detected in other patients treated with OKT3 (10).

The presence of some degree of reactivity to mouse immu­noglobulin in the pretreatment sera and in normal controls was expected. Antibodies to equine ATG have been detected by passive hemagglutination in up to 50% of patients awaiting renal transplantation with no previous exposure to ATG (18). These antihorse immunoglobulin antibodies were found to cross react with goat and rabbit immunoglobulin. The specificity of these antibodies has not been established. They may be crossreactive antibodies to immunoglobulin antigens absorbed orally (beef, pork, etc.) or rheumatoid factors. We found no correlation between preexisting antibodies and the severity of side effects from monoclonal therapy. The one patient with a high titer prior to treatment responded fully to OKT3 therapy for rejection. A further analysis of these antibody crossreactiv­ities will be necessary to determine the importance of these interactions.

Although early studies were optimistic that monoclonal anti­bodies might have low immunogenicity (1, 3, 19), following our initial report (6), several other studies have documented the presence of both non-anti-Id and anti-ld responses (4, 5, 7, 9, 10, 14, 15). The production of such antibodies may be influ­enced by the particular monoclonal antibody used and the immune reactivity of the individuals treated. More than half the patients treated for lymphoreticular malignancies with a nonmitogenic antibody (anti-Leu 1) produce antimouse anti­body, similar to that of our transplant recipients. Although these antibodies do not cause immune complex disease, the response effectively neutralized the monoclonal antibody, as in our patients (15).

OKT3 does not seem particularly more or less immunogenic than other anti-T-cell or antitumor antibodies, despite its known mitogenic activity. The one distinctive feature of the immune response to OKT3 may be the predominance of the anti-Id response. Only 5% of the antibodies to the murine immunoglobulin have been to idiotypic determinants in lym­phoma patients treated with anti-Leu 1 (20), whereas in one of our patients only anti-ld antibodies were detected, and in another they accounted for 31% of the antibody response. Overall, 75% of our patients who had any antibody response made anti-Id that was able to block antigen binding. Why OKT3 should be particularly effective at promoting an anti-Id response is unknown, but one could speculate that this might be related to the target molecule, a part of the T cell antigen receptor complex. However, the strong anti-Id response is not unique to OKT3, as anti-Id constituted about one-third of the antibody response of goats immunized with myeloma proteins (21 ). Furthermore, we have observed blocking anti-Id in mon­keys treated with anti-Leu 2a (Cosimi et al., manuscript in preparation).

That the recipients of murine monoclonal antibodies should develop an immune response to these foreign proteins is, of

PFIZER EX. 1027 Page 12

578 TRANSPLANTATION Vol. ·11, No.5

course, expected. Such responses have been described in recip­ients of antithymocyte (or lymphocyte) globulin (ATG) made in horses or rabbits (22, 23). However, with more recent prep­arations of ATG that have been deaggregated and administered i.v., the antibody response is typically inconsequential and does not preclude repeated use of ATG (24, 25).

Two likely possibilities exist for the differences between the intensity of the response to monoclonal and polyclonal anti­bodies. The broadly reactive ATG may be more effective at destroying the T cells and/or B cells needed to make an antibody response. If this is the case, a monoclonal antibody­or drug, such as cyclophosphamide-might be added that would interfere with B cell function. A second possibility is that the substantial amount of nonreactive immunoglobulin in the pol­yclonal preparations acts to promote tolerance in the recipients. It is likely that more intense deaggregation of the monoclonal antibody will be ineffective, since essentially all of the soluble monoclonal antibody becomes "aggregated" when it binds to the target cell surface. Indeed, some years ago, Lance and Dresser demonstrated that rabbit ATG was more immuno<tenic in mice than normal, nonimmune, rabbit IgG (21 ). Bas;d on this observation, a possible solution is to give a loading dose of nonreactive monoclonal antibody of the same isotype.

At this time, anti-immunoglobulin and anti-Id antibodies represent barriers to the prolonged or repeated application of murine monoclonal antibodies. The anti-Id barrier will likely also exist for human monoclonal antibodies: the magic "bullets" are stopped by equally magic "shields." Although monoclonal antibodies have exceptional promise for specific modulation of immune responses, the antibody response will need to be elim­ina~ed to permit their widest therapeutic and diagnostic appli­catiOn.

Aclmowledgments. We are grateful for the excellent technical assist­ance of Helen Goldsman, Carol Struzziero, Amy Luttinger, and Victoria Hull. .James T. Kurnick, M.D., kindly provided the PHA blast lines.

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Received 26 June 1985. Accepted 4 September 1985.

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