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THI:: JUUI(I~Al 0

IMMUNOO Official Journal of The American

Association of Immunologists

th Anniversary

1916- 1991

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PFIZER EX. 1037 Page 1

THE JOURNAL OF IMMUNOLOGY Volume 147 /Number 4, August 15, 1991

G, A. Bishop

H. Hengel, H. Wagner, and K. Heeg

J.P. Lake, C. W. Pierce, and J. D. Kennedy

~.-M, Lee and S. Rich ·-~. Lue, R. P. Lauener, R. J. Wmchester, R. S. Geha, and D. Vercelli

I. ~elall_led, G. P. Downey, K. J ktones, and C. M. Roifrnan

· Nagamine, K. Takeda, Y. Tatsumi, M. Ogata, K. Mi­ya~e. T. Hamaoka, and H. FUJiwara

I. Nakashima, Y.-H. Zhang, s. M. J, Rahman, T. Yoshida, K.-1. Isobe, L.-N. Ding, T. Iwa­moto, M. Hamaguchi, H. Ike-

C zawa, and R. Taguchi ' M. Snapper, L. M. T. Pe­canha, A. D. Levine, and J. J. Mond

C.JM. Snapper, H. Yamada, J. · Mond, and C. H. June

H. Spits, X. Paliard, Y. Vandek­erckhove, P. van V1asselaer and J, E. de Vries '

G.D.Anderson,S.BaneDee,H. G S. Luthra, and C. S. David

·DC. Koo, C. L. Manyak, J. asch, L. Ellingsworth, and

L.D.Shultz J.,:. Lazdins, T. Klirnkait, K.

Oods-Cook, M. Walker, E. Al~eri, D. Cox, N. Cer1etti, R.

MShlpman, G. Bilbe, and G.

cMaster O.RLider, A. Milleer, S. Miron,

x' Hershkoviz, H. L. Weiner, · Zhang, and E. Heber-Katz

Contents CELLULAR IMMUNOLOGY

1107 Requirements of Class 11-Mediated B Cell Differentiation for Class II Cross­Linking and Cyclic AMP

1115 Triggering of CDS+ Cytotoxic T Lymphocytes via CD3-E Differs from Trig­gering via a/(3 T Cell Receptor: CD3-E-lnduced Cytotoxicity Occurs in the Absence of Protein Kinase C and Does not Result in Exocytosis of Serine Esterases

1121 CDS+ a/(3 or 'Y/o T Cell Receptor-Bearing T Cells from Athymic Nude Mice Are Cytolytically Active in Vivo

1127 Co-Stimulation ofT Cell Proliferation by Transforming Growth Factor-/)1 1134 Engagement of CD14 on Human Monocytes Terminates T Cell Proliferation

by Delivering a Negative Signal toT Cells

1139 Microfilament Assembly Is Required for Antigen-Receptor-Mediated Acti­vation of Human B Lymphocytes

1147 Role of a Thymic Stromal Cell Clone in Inducing the Stage-Specific Differ­entiation of Various Subpopulations of Double Negative Thymocytes

1153 Evidence of Synergy between Thy-1 and CD3/TCR Complex in Signal Deliv­ery to Murine Thymocytes for Cell Death

1163 IgE Class Switching Is Critically Dependent Upon the Nature of the B Cell Activator, in Addition to the Presence of IL-4

1171 Cross-Linkage of Ly-6A/E Induces Ca2+ Translocation in the Absence of Phosphatidylinositol Turnover and Mediates Proliferation of Normal Mu­rine B Lymphocytes

1180 Functional and Phenotypic Differences between CD4 + and CD4- T Cell Receptor-'Yo Clones from Peripheral Blood

CLINICAL IMMUNOLOGY • IMMUNOPATHOLOGY

1189 Role of Mls-1 Locus and Clonal Deletion of T Cells in Susceptibility to Collagen-Induced Arthritis in Mice

1194 Suppressive Effects of Monocytic Cells and Transforming Growth Factor-(3 on Natural Killer Cell Differentiation in Autoimmune Viable Motheaten Mutant Mice

1201 In Vitro Effect of Transforming Growth Factor-(3 on Progression of HIV-1 Infection in Primary Mononuclear Phagocytes

1208 Nonencephalitogenic CD4-cns- Va2V(3S.2+ Anti-Myelin Basic Protein Rat T Lymphocytes Inhibit Disease Induction

Continued on page 4

THE JO Presto URNAL OF IMMUNOLOGY (ISSN 0022-1767) Is published twice each month by The American Association of Immunologists, 428 East lndexe~ Street, Baltimore, MD 21202. Subscription rates $170 ($260 foreign); Institutions $300 ($390 foreign); stngle copy $10 ($15 foreign). POST by Current Contents and Index Medicus. Second class postage paid at Baltimore, MD 21202 and at additional matltng offices. Amert~ASTER: Send address changes to The Journal of Immunology at 428 East Preston Street. Baltimore, MD 21202. Copyright© 1991 by The

n Association of Immunologists.

PFIZER EX. 1037 Page 2

Continued from page 3

S. Seki, T. Abo, T. Ohteki, K. Sugiura, and K. Kumagai

D. V. Serreze and E. H. Leiter

1214

1222

. , A c Probably a Unusual H/3-T Cells Expanded in Autoimmune lpr Mice r Counterpart of Normal T Cells in the Liver , ked When

Development of Diabetogenic T Cells from NOD/Lt Marrow Is Bloc 1

in but an Allo-H-2 Haplotype Is Expressed on Cells of Hemopoietic Or g ' not on Thymic Epithelium

CYTOKINES • MEDIATORS • REGULATORY MOLECULES -J. L. Browning, M. J. Andro­

lewicz, and C. F. Ware I. K. Campbell, U. Novak, J. Ce­

bon, J. E. Layton, and J. A. Hamilton

J. A. Carman and C. E. Hayes M. R. Fung, R. M. Scearce, J. A.

Hoffman, N. J. Peffer, S. R. Hammes, J. B. Hosking, R. Schmandt, W. A. Kuzie1, B. F. Haynes, G. B. Mills, and W. C. Greene

M. K. Gariapathi, D. Rzewnicki, D. Samols, S.-L. Jiang, and I. Kushner

K. Nakata, K. Akagawa, M. Fu­kayama, Y. Hayashi, M. Ka­dokura, and T. Tokunaga

J.-H. Shieh, R. H. F. Peterson, and M. A. S. Moore

G. Strassmann, D. R. Bertolini, S. B. Kerby, and M. Fong

M. Barel, A. Gauffre, F. Lya­mani, A. Fiandino, J. Her­mann, and R. Frade

R. Busch, C. M. Hill, J. D. Hay­ball, J. R. Lamb, and J. B. Rothbard

P. E. Harris, M. C. Gutierrez, E. Reed, D. W. King, and N. Su­ciu-Foca

0. Kanagawa, Y. Utsunomiya, J. Bill, E. Palmer, M. W. Moore, and F. R. Carbone

R. W. Leu, A. Zhou, J. Rum­mage, D. J. Fast, and B. J. Shannon

S.M. Mariani, E. A. Armandola, and S. Ferrone

I. F. Mizukami, S. D. Vinjamuri, F. Perini, D. Y. Liu, and R. F. Todd III

P. A. M. Warmerdam, J. G. J. van de Winkel, A. V1ug, N. A. C. Westerdaal, and P. J. A. Capel

1230

1238

1247 1253

1261

1266

1273

1279

1286

1292

1299

1307

1315

1322

1331

1338

Lymphotoxin and an Associated 33-Glycorprotcin Arc Expressed on the Surface of an Activated Human T Cell Hybridoma tic Colony-

Human Articular Cartilage and Chondrocytes Produce Hemopole Stimulating Factors in Culture in H.esponsc to IL-l

Abnormal Regulation of IFN-y Secretion in Vitamin A Deficiencyh Human A Tyrosine Kinase Physically Associates with the {J-Subunit of t e

IL-2 Receptor

h is of serum Effect of Combinations of Cytokines and Hormones on Synt es

Amyloid A and C-Rcactivc Protein in HEP 3B Cells the prollfer­

Granulocyte-Macrophagc Colony-Stimulating Factor Promotes ation of Human Alveolar Macrophagcs In Vitro

. Vitro and IL-l Modulation of Cytokine Receptors on Bone Marrow Cells. In

in Vivo Studies oducts bY Regulation of Murine Mononuclear Phagocyte In~lammatory ~~taglandln

Macrophage Colony-Stimulating Factor: Lack of IL-l and Pr E2 Production and Generation of a Specific IL-l Inhibitor

IMMUNOCHEMISTRY -a Calcium­

Intracellular Interaction of EBV /C3d Receptor (CR2) with p6B. phocytes Binding Protein Present in Normal but Not in Transformed B Lym

(3-Chain on Effect of Natural Polymorphism at Residue 86 of the HLA-DR

Peptide Binding

. . . , DA 4 Anti-BwsynthesJs and Partial Ammo Acid Sequence of the Human N

gen: An Activation Antigen Common to BandT Cell Lineages b Mono­

Conformational Difference ofT Cell Antigen Hcccptors Revealed ~mants clonal Antibodies to Mouse V(35 T Cell Heccptor for Antigen Deter

Their Re­Reconstitution of a Deficiency of AKR Mouse Macrophages for plement

sponsc to Lipid A Activation for Tumor Cytotoxicity by corn Subcomponent Clq: Role of IFN-y Anu-I-ILA-

Diversity in the Fine Specificity and Idiotypic Profile of ~ouse ic rvtono­DR Monoclonal Antibody Elicited with the Syngeneic Anti-Idiotyp clonal Antibody F5-830 Mo3 Acti-

Purlfication, Biochemical Composition, and Biosynthesis of the Mononu· vation Antigen Expressed on the Plasma Membrane of Human clear Phagocytes man Fer

A Single Amino Acid in the Second Ig-Like Domain of the Hu Receptor II Is Critical for Human IgG2 Binding

age5 Continued on p

PFIZER EX. 1037 Page 3

Continued from page 4

L. Y. Whiteman, D. B. Purkall, 1344 Association of Activated Properdin with Complexes of Properdin with C3 andS. Ruddy

J. Hakimi, R. Chizzonite, D. R. Luke, P. C. Familletti, P. Bai­lon, J. A. Kondas, R. s. Pil­son, P. Lin, D. V. Weber, C. Spence, L. J. Mondini, W.-H. Tsien, J. L. Levin, V. H. Gal­lati, L. Korn, T. A. Wald­mann, C. Queen, and w. R. Benjamin

D.p~udig, N. J. Allison, T. M. Klckett, U. Winkler, C.-M.

am, and J. C. Powers T W K ·· ·M · U1Jpers, B. C. Hakkert,

· Hoogerwerf, J. F. M. Leeu-S ~enbe~g, and D. Roos

· chre1ber, W. F. Stenson, R. P. MacDermott, J. C. Chap­pel, S. L. Teitelbaum, and s. L. Perkins

J.i. Coutelier, J. T. M. VanDer

K ogt, and F. W. A. Heessen

. B. Madden J F U b J H . • .. r an, r., F · J. Zll~ener, J. W. Schrader,

· D. Fmkelman, and I. M. Katona

D. Muller, K. Pederson R. Mur-ra ' y, and J. A. Frelinger

J. Yagi, S. Rath J , and C. A.

aneway, Jr.

IMMUNOPHARMACOLOGY

1352 Reduced Immunogenicity and Improved Pharmacokinetics of Humanized Anti-Tac in Cynomolgus Monkeys

1360 The Function of Lymphocyte Proteases: Inhibition and Restoration of Gran­ule-Mediated Lysis with Isocoumarin Serine Protease Inhibitors

1369 Role of Endothelial Leukocyte Adhesion Molecule-! and Platelet-Activating Factor in Neutrophil Adherence to IL-1-Prestimulated Endothelial Cells: Endothelial Leukocyte Adhesion Molecule-!-Mediated CD 18 Activation

1377 Aggregated Bovine IgG Inhibits Mannose Receptor Expression of Murine Bone Marrow-Derived Macrophages via Activation

MICROBIAL IMMUNOLOGY

1383 IgG Subclass Distribution of Primary and Secondary Immune Responses Concomitant with Viral Infection

1387 Antibodies to IL-3 and IL-4 Suppress Helminth-Induced Intestinal Masto­cytosis

1392 A Single Amino Acid Substitution in an MHC Class I Molecule Allows Heteroclitic Recognition by Lymphocytic Choriomeningitis Virus-Specific Cytotoxic T Lymphocyte

1398 Control ofT Cell Responses to Staphylococcal Enterotoxins by Stimulator Cell MHC Class II Polymorphism

- MOLECULAR BIOLOGY • MOLECULAR GENETICS

D.NJ.RDecker, N. E. Boyle, and · · Klinman

R.Kay p M H • · · Rosten, and R. K.

umphries

S. Wong J leh • · D. Freeman, C. Kel-

L J er, D. Mager, and F. Takei ·~ . Zhou, D. C. Ord, A. L.

ughes, and T. F. Tedder

1406 Predominance of Nonproductive Rearrangements of V118lX Gene Segments Evidences a Dependence of B Cell Clonal Maturation on the Structure of Nascent H Chains

1412 CD24, a Signal Transducer Modulating B Cell Activation Responses, Is a Very Short Peptide with a Glycosyl Phosphatidylinositol Membrane An­chor

1417 Ly-49 Multigene Family: New Members of a Superfamily of Type II Mem­brane Proteins with Lectin-Like Domains

1424 Structure and Domain Organization of the CD 19 Antigen of Human, Mouse, and Guinea Pig B Lymphocytes: Conservation of the Extensive Cyto­plasmic Domain

Continued on page 6

PFIZER EX. 1037 Page 4

Continued] rom page 5

T. M. Blieden, A. J. McAdam, J. G. Frelinger, and E. M. Lord

J. 0. Brubaker, K. T. Chong, and R. M. Welsh

F. Novelli, M. Giovarelli, R. Re­ber-Liske, G. Virgallita, G. Garotta, and G. Forni

N. P. Restifo, F. Esquivel, A. L. Asher, H. Stotter, R. J. Barth, J. R. Bennink, J. J. Mule, J. W. Yewdell, and S. A. Rosenberg

Erratum

Announcement

Author Index

TUMOR IMMUNOLOGY

1433 Mechanism of Cytolytic T Lymphocyte Kllling of a Low Class I-Expresslng Tumor

1439 Lymphokine-Actlvated Killer Cells Are Rejected in Vivo by Activated Natural Klller Cells

1445 Blockade of Physiologically Secreted IFN-I' Inhibits Human T Lymphocyte and Natural Klller Cell Activation

1453 Defective Presentation of Endogenous Antigens by a Murine Sarcoma: Implications for the Failure of an Anti-Tumor Immune Response

1460

1461

PFIZER EX. 1037 Page 5

0022-1767/91/14 74-1352802.00/0 Tm: ,jr)tJHNr\L OF fMMUNOLOfiY

Copyrl~;ht '<c 1991 by The American Association of Immunologists

Vol. 147, 1:l52-1:EiD, No. ·1. August 15, 1991 /'rln!<'<lln U.S. i\.

REDUCED IMMUNOGENICITY AND IMPROVED PHARMACOKINETICS OF HUMANIZED ANTI-Tac IN CYNOMOLGUS MONKEYS

JOHN HAKIMI, 1 * RICHARD CHIZZONITE: DAVID R. LUKE,* PI-IILIP C. FAMILLETTI,§ PASCAL BAILON,II JO A. KONDAS,* ROBEH.T S. PILSON,* PING LIN,* DAVID V. WEBER. 11

CHERYL SPENCE, 11 LISA J. MONDINI,* WEN-HUI TSIEN,* JAMES L. LEVIN.~1 VON H. GALLATI,** LAURENCE KORN,++ THOMAS A. WALDMANN,** CARY QUEEN,++ AND WILLIAM R. BENJAMIN*

From the Departments oj *Immunopltarmacology. 'Molecular Genetics, 'Drug Metabolism. "l3ioprocess Development. and 11Protein Biochemistry. Roche Research Center, Ho.ffmann-La J~oclw. Inc .. Nutley. NJ 0711 0; 'TSI Mason Uesearclt Institute.

Worchester, MA 01608; **Central Uesearclt. F. Hc!fjmann-La Uoclte Ltd., 4002 Basel. Switzerland; "l'rotein Design Labs, Inc .. Mountain View, CA 94043; "Metabolism Branch, National Cancer Institute, National Institute q{ Hcaltlt. I3etltesda. MD 20892

The anti-Tac mAb has been shown to bind to the p55 chain of the IL-2R, block IL-2 binding and in­hibit T cell proliferation. A humanized form of anti­Tac (HAT) has been constructed that retains the binding properties of murine anti-Tac (MAT). These two mAb were evaluated in cynomolgus monkeys to compare relative immunogenicity and pharmacoki­netic properties. Monkeys treated with HAT daily for 14 days exhibited anti-HAT antibody titers which were 5- to 10-fold lower than their MAT­treated counterparts and these antibodies devel­oped later than in the MAT-treated monkeys. Two of four monkeys receiving a single injection of MAT developed anti-MAT antibodies, whereas none of four monkeys developed antibodies after a single treatment with HAT. In monkeys injected with either HAT or MAT daily for 14 days, the anti-anti­body titers induced were inversely related to the amount of anti-Tac administered. Antibodies that developed against MAT were both anti-isotypic and anti-idiotypic, whereas those developed against HAT appeared to be predominantly anti-idiotypic. The pharmacokinetic properties, that is the half-life and area under the curve values, of HAT were also significantly different from those of MAT. The area under the curve values for HAT in naive monkeys were approximately twofold more than those for MAT, and the mean serum half-life of HAT was 214 h, approximately four- to fivefold more than MAT. These pharmacokinetic values were reduced in monkeys previously sensitized with HAT or MAT suggesting that the presence of anti-antibodies al­tered these parameters.

The cellular receptor for IL-2 plays an important role in regulation of immune function (1). The IL-2R2 consists

Hecelved for publ!cation December 19, 1990. Accepted for publ!catlon May 30, 1991. The costs of publ!catlon of this article were defrayed In part hy the

payment of page charges. This article must therefore be hereby marked advertisement In accordance with 18 U.S.C. Section 1734 solely to lncl!­cate this fact.

1 Address correspondence and reprint requests to Dr. John Ilakimi, Hoffmann-LaHoche, Department of Immunophannacology, 340 Kings­land St .. Nutley. NJ 07110.

2 Abbreviations used In this paper: IL-2H, IL-2 complex; MAT, mouse anli-Tac; Tac, p55 subunit of the human IL-2H; s!L-2H, soluble r!L-2H; HAT. humanized antl-Tac: HHP, horseradish peroxidase; AUC, area under the curve.

of at least two polypeptide chains that can independently bind IL-2: the p55, IL-2R H chain, or Tac peptide (2. 3), and the more recently discovered p75 or IL-2H. {J chain (4. 5). Study of the p55 peptide was facilitated by the devel­opment of a mAb, MAT. which binds to human p55 (2). The Tac peptide is expressed on the surface of Ag- or mitogen-activated T cells but not on resting T cells. More­over, treatment of human T cells with MAT strongly inhibits their proliferative response to Ag or to IL-2 by preventing binding of IL-2 to p55 (3. 6).

High levels of p55 arc expressed on malignant cells of some lymphoid cancers such as adult T cell leukemia, cutaneous T cell lymphoma and Hodgkin's disease (1). Increased or abnormal IL-2R expression is also associated with many autoimmune conditions including rheumatoid arthritis, SLE, organ transplant rejection, and graft-vs­host disease ( 1 ). Hence, the IL-2R is a potentially useful and versatile therapeutic target. Agents that specifically eliminate Tac-cxprcssing malignant cells or activated T cells involved in an autoimmune response could be effec­tive against those disorders without harming normal Tac­negativc T cells. These agents would potentially be more selective than other immunosuppressants such as anti­bodies against the CD3 antigenic epitopc (i.e., OKT3). In the case of autoimmune conditions, it might in fact only be necessary to suppress T cell proliferation by IL-2R blockade, without destroying the T cells, to achieve ther­apeutic benefit.

Anti-IL-2H. antibodies have been effective in animal models as well as in early human trials. In vivo admin­istration of anti-IL-2R antibodies greatly prolonged sur­vival of heart allografts in mice and rats (7. 8) and alle­viated insulitis in nonobcsc diabetic mice and lupus ne­phritis in NZB X NZW mice (9). MAT Itself was highly effective in prolonging survival of allografts in cynomol­gus monkeys ( 1 0) with improved efficacy observed with HAT (11). In phase I clinical trials for kidney transplan­tation, prophylactic administration of MAT significantly reduced the incidence of rejection episodes, without as­sociated toxicity ( 12). Another anti-IL-2R antibody was also effective in this setting ( 13). Treatment with MAT induced temporary partial or complete remission in 7 of 20 patients with adult T cell leukemia ( 14) (T. A. Wald­mann, unpublished observations).

Several major problems limit the effectiveness of a murine mAh such as MAT when used in human patients.

1352

PFIZER EX. 1037 Page 6

IMMUNOGENICITY AND PHJ\H.MACOKINETICS OF HUMANIZED ANTI-TAC 1353

The mouse antibody is immunogenic in humans and provokes a neutralizing antibody response, and may not be as efficient as a human antibody at recruiting human immune effector functions. In addition, mouse antibodies have a much shorter circulating half-life in humans than do natural human antibodies ( 15).

Problems associated with the therapeutic use of murine antibodies have been partially addressed by the genetic construction of chimeric antibodies, which combine the V region binding domain of a mouse antibody with 1m­man antibody C regions ( 16). However, because chimeric antibodies retain the whole mouse V region, they may still be immunogenic. Data on the treatment of human patients with chimeric antibodies arc only beginning to accumulate ( 15, 1 7).

To further reduce the immunogenicity of murine anti­bodies, Winter and colleagues ( 18-21) constructed "lm­manizccl" antibodies, in which only the minimum neces­sary parts of the mouse antibody. the CDR. were com­bined with human V region frameworks and C regions. Based on this approach, we have recently constructed a humanized anti-Tac antibody (22). The humanized anti­Tac antibody (HAT) retains several key mouse framework residues, preclictccl by computer modeling, which arc re­quired to maintain high affinity binding for p55. In ad­clition, the humanized antibody mediates antibocly-clc­penclent cellular cytotoxicity against T cell leukemia cells (23). Previously. it was demonstrated in cynomolgus mon­keys with cardiac allografts that HAT appeared less im­munogenic than MAT (11). In this study. cynomolgus monkeys were given MAT and HAT to further evaluate the relative immunogcnicity and pharmacokinetics of the two mAb. To provide a stringent test of HAT, we applied a closing schedule of frequent injections that would reveal any immunogcnicity.

Mi\TEI{Ii\LS AND METHODS

Cells. MAT was produced in tissue culture as described previously (2). !!AT was produced from SI'2/0 cells transfccled with the genes encoding for the 1-1 and L chains of the humanized antibody (22, 23). Cells were optimized for antibody secretion by limiting dilution clon­ing. Production of IIAT was performed in a 3-liter continuous per­fusion bioreactor (£lellco Biotechnology, Vineland, NJ) equipped with a glass cylinder matrix as previously described (24. 25). The cells were grown at 37°C in Iscoves's modified Dulbecco's medium (JRI-I Bioscicnces, Lencxam. KS) supplemented with 5% FCS (JRI-1 Bios­ciences), 100 U/ml penicillin G. 100 llg/ml streptomycin, and 25 mM HEI'ES buffer. pii 6.9 to 7.0. During the production phase of the fermentation, days 9 to 83, the medium flow rate was maintained at 416 ml/h and the conditioned medium contained approximately 8 mg/litcr of !lAT.

Proteins. HAT and MAT were purified on separate IL-2R affinity chromatography columns with capacities of 125 and 300 mg. re­spectively (26). Briefly, purified recombinant s!L-2R (27) was im­mobilized on NuGcl 1'-AF l'oly-N-hydroxysucclnlmide (Separation Industries. Metuchln, NJ). Antibodies eluted and concentrated from the receptor column were further purified on two serially linked Sephacryl S-300 columns (60 X 11.3 em, Pharmacia Fine Chemicals, Piscataway, NJ) In Dulbecco's PBS (Whittaker Bloproducts. Walkers­ville, MD). All purification steps were carried out at 4°C, and buffers were prepared with ultra pure water (Hydro. Research Triangle Park, NC). The final products were sterilized through a 0.2 liM Corning filter (Corning Glass Works, Corning. NY) and found In contain less than I 0 endotoxin unlts/mg (28). Purity was determined by SDS­l'AGE under reducing and nonrcduclng conditions and found to be more than 99%.

Anti-1 !AT and anti-MAT standards were prepared by immunizing goats with the respective proteins in CF A. The goat IgG standards were Isolated on protein A-Sl'pharosc CL-4b (l'harmacla) and affinity purified on II AT or MAT AffiGcl-1 0 affinity columns (Bio-Racl. Rich­mond, CA). Purified human r!L-2 expressed in Escl!ericl!ia coli was

obtained from Dr. F. Khan, Bioprocess Development. Hoffmann-La Roche Inc .. Nutley, NJ.

HRP-labeled IL-2. IIAT and MAT were prepared using a modifi­cation of a previously described method (29). A total of 20 mg of I!RI'. grade 1 (Boehringer-Mannhcim, Indianapolis. IN) in 6 ml of distilled water was activated by adding 1.0 ml of 0.1 M Na!O_, for 20 min at room temperature (20-25°C) and subsequently quenched with 1.0 ml of 0.5 M ethylene glycol. The activated HRP was dialyzed against 5 mM sodium acetate buffer. pH 4.5. and brought up to a I ina! volume of 10 rnl. Five mg of protein were dialyzed against 0.1 M NaHCO", pll 8.0, and added to the activated HHP and diluted with 10 ml of 0.5 M sodium carbonate buffer, pH 9.5. After 2 hat room temperature. 3 ml of 0.1 M NaBI-14 were aclclecl and incubated in the dark for 4 to 6 hat 4°C. The I-IRP-conjugatecl proteins were dialyzed against 0.1 M sodium phosphate buffer. pH 6.5, and then diluted with an equal volume of 0.2 M sodium phosphate buffer, 20 mg/ml BSA, I mg/ml Thimersol, and 2 mg/ml phenol.

Monkeys and experimental protocol. Eight groups of four 4 to 6 kg cynomolgus monkeys (two males and two females; Mason Re­search Institute, Worchcstcr. MA) were treated daily on clays I through 14 (Table 1). Groups 1 and 5 received PJJS as a vehicle control. Monkeys in groups 2. 3, and 4 received HAT at closes of 0.05, 0.5. or 5.0 mg/kg, respectively, and groups 6, 7, ancl8 received MAT at doses of 0.05, 0.5, or 5.0 mg/kg. respectively. On clay 42, groups I to 4 and groups 5 to 6 received a single 5 mg/kg close of HAT or MAT, respectively. Test samples were administered via venous catheters surgically placed in the femoral vein attached to a vascular port. Samples were administered as single bolus injections within several seconds. Blood samples were obtained by venipunc­ture throughout the 55-clay study. Monkeys were tranquilized with intramuscular kctamine HCI before administration of test samples and collection of serum samples.

Immunosorbant assays. To measure the serum levels of monkey antibodies against HAT or MAT. Nunc-Immuno MaxiSorp (Nunc, Naperville. JL) wells were coated with 100 ng of either HAT or MAT in 200 Ill of PBS overnight (20-24 h) at 4°C. To each well. 100 Ill of I% fatty acid and globulin-free BSA (Sigma Chemical Co .. St. Louis, MO) In PBS were aclclecl for 1 h at room temperature. followed by washing with PBS containing 0.05% Tween 20. Wells were incu­bated with 200 Ill of goat standards or test samples, plus 50 Ill of HRI'-I-IAT or 1-IRP-MAT at a final dilution of 1/4000 overnight at 4°C. Samples were diluted in 25 mM sodium phosphate. 75 mM NaCI, 0.05% Tween 20. 0.01% BSA, 50 llg/ml phenol reel, pH 7.4. The Initial concentration of the unknowns in the assay was I /3 with subsequent threefold dilutions. The plates were washed and then developed with I mM 2,2'-azinobis (3-cthylbenzthiazoline-sulfonic acid) (Sigma) in 0.1 M citrate buffer, 0.03% H20 2 • pl-14.2. for 30 min. The absorbance at 405 nm was determined with a Vmax Kinetic Microplate reader (Molecular Devices, Menlo Park, CA). The color intensity is directly proportional to the antibody concentration in the serum samples. The relative concentrations of anti-HAT <mel anti-MAT antibodies in the monkey serum samples were calculated from a goat antibody standard curve titrated on each plate. The values expressed arc apparent antibody levels, because the detection of antibodies in this assay is dependent on concentration. affinity. and presence of blocking agents such as anti-Tac and s!L-2R. The assay primarily detects free monkey antibodies; however. some an­tibody from antibocly-anti-Tac complexes would be clctcctccl if a rccquilibrlum of the antibody interactions was established in the wells during the overnight incubation.

Serum concentrations of HAT and MAT were determined in an IL-2 immunosorbant receptor assay (27). Plates were coated with 16 ng of slL-2R in 200 Ill of PBS overnight at 4°C and then blocked with 1% BSA as clcscribecl above. Wells were washed and incubated with 200 Ill of sample overnight at 4°C. Typically. the initial serum in the assay was diluted I/ I 0 with subsequent I /2 dilutions. The initial sample concentration varied depending on which treatment group

Grmtp

I 2 3 4 5 6 7 8

TABLE I Immwwgenicily study treatment groups

Dally Dose Challenge Dose Hrsponse

Days I to 14 Day 42 ~----~--~

Vehicle control HAT, 5 ml(/kl( None HAT. 0.05 mg/kg HAT. 5 rng/kl( NonP HAT, 0.5 mg/kg HAT, 5 mg/kg None IIAT. 5.0 mg/kg Hi\ T. 5 mg/kg Anaphylaxis I /4 Vehicle control MAT, 5 mg/kl( None Mi\T, 0.05 mg/kg MAT, 5 ml(/kl( Anaphylaxis 4/4 MAT. 0.5 mg/kg None" MAT, 5.0 mg/kg None"

"Monkeys not challenged with MAT due to the anaphylaxis observed in group G.

PFIZER EX. 1037 Page 7

1354 IMMUNOGENICITY AND PHAHMACOKINETICS OF' HUMANIZED ANTI-TAC

was studied. Without washing the samples from the wells. 50 1'1 of HHP-IL-2 was added to a final dilution of 1/2000. After 3 hat room temperature. wells were washed and developed as described above. The color intensity is inversely proportional to the anti-Tac concen­tration in the samples. HAT and MAT concentrations in the serum were calculated from a standard curve of purified HAT and MAT titrated on each plate. In this assay, only the anti-Tac available to bind to the s!L-2H would be detected. As discussed above for the immunogenicity ELISA, a new equilibrium of the antibody complexes in the serum could occur in the wells during the 24 h incubation.

Pharmacokinetics. The AUC and t 112 values for HAT and M/\T were estimated to reflect the total body burden of the antibody within the intravascular pool as well as the serum die-away curve. respec­tively. Serum concentrations of the antibodies were plotted vs time on a log-linear graph and the /\UC values were calculated by trape­zoidal rule (30). The apparent elimination t 112 from a single dosing was estimated by linear regression analysis of the terminal portion of the curve from a minimum of four data points.

For multiple dose pharmacokinetics, the maximum serum concen­trations and time to reach maximum serum concentrations were obtained visually from the serum concentration-time graphs. The apparent t 112 after multiple dosing was approximated from a mini­mum of three serum concentration-time points obtained after the final dose.

RESULTS

Study design and clinical observations. A cynomol­gus monkey study was designed to evaluate the relative immunogenicity and pharmacokinetic properties of MAT and HAT. A schematic representation of the study design is shown in Figure 1 and details of the treatment groups are described in Table I. During the study, the monkeys remained behaviorally and clinically normal with the following exceptions. On day 42 one female monkey in group 4 exhibited an apparent anaphylactic response posttreatment with 5 mg/kg of HAT. This monkey was treated with epinephrine, dexamethasone, Benadryl, and was hydrated with saline. The monkey gradually im­proved and by day 44 appeared normal. All four monkeys in group 6 that received 0.05 mg/kg/day MAT initially, also exhibited an apparent anaphylactic response post­treatment with 5 mg/kg MAT. The monkeys responded to epinephrine and fluids. The animals in the MAT treat­ment groups 7 and 8 were not challenged on day 42. In various ELISA systems, no increase in total monkey IgE was observed, nor was the presence of Ag-specific anti­anti-Tac IgE detected (data not shown). The cause of this anaphylactic response remains unknown.

Immunogenicity characterization. Monkey antiglob­ulin levels (i.e., antibodies to HAT and MAT) were evalu­ated in an Ag-bridging ELISA, which can be used to detect antibodies of various species and isotypes using the same reagents. Affinity-purified goat anti-HAT and goat anti­MAT antibody standards were similarly detected in the range of 100 to 1000 ng/ml in their respective assays (data not shown).

The time-dependent development of antibodies in in­dividual monkeys is shown for MAT in Figure 2 and for

TREATMENT 14 DAYS

~~~!~~iii~!dl 10 20 30

I ; j ~ ' l BLOOD COLLECTION DAYS

CHALLENGE DAY 42

! 40 50

l!J' /""'-POST DAY 42

25,.5,1,2,4,8 12.24,36hr

Figure 1. Immunogenieity and pharmacokinetic study design for eval­uation of anti-Tac antibodies In cynomolgus monkeys. Sec Table I for additional detail.

300.-------------------------------------------,

A. Group 6 2215

1150

75 -E ....... 0

0> 2 300

B. Group 7 ._ 2211

<(

~ 150 ()

0 0 0~0 - o/:2G/ 6 >. 75

0/ ~(!) 6 -6------"0 /_¢.c:fJ~. 0 .0 0 -300 c <( c. Group 8

225

150 ~6-6

~-" 75 ~-·/'-'

A .(). ·" 0 10 115 20 21! 30 35 40 45

Time (day) Figure 2. Time-dependent development of anti-MAT antibodies In In­

dividual monkeys administered Jl. 0.05, 13, 0.50, C. 5.0 mg/kg/day MAT for 14 days. Anti-MAT concentrations were determined In an ELISA using an affinity purified goat anti-MAT antibody as a standard.

HAT in Figure 3 (note differences in the ordinate scales). Prebleed sera from all 32 monkeys and sera from day 0 to 42 from control monkeys In groups 1 and 5 showed no activity in the ELISA. In the MAT-treated groups, 9 of 12 monkeys developed antibodies during the Initial 14 day treatment period, usually by day 12. In contrast, anti­HAT antibodies In all but one of the 12 HAT-treated monkeys were not detected until at least 5 to 10 days after the final dose of HAT was administered. In addition, the HAT-treated monkeys showed dramatically lower serum antiglobulin concentrations than the MAT-treated groups.

The antibody titer developed to HAT as well as MAT was in general inversely related to the protein dose ad­ministered. In group 4 which was treated with 5 mg/kg/ day HAT, only one monkey had detectable antibodies by day 42. This was the only HAT-treated monkey that exhibited an anaphylactic response upon challenge with HAT on day 42 (Table I), even though monkeys from other groups had apparently higher serum antibody lev­els on day 42. All monkeys in group 6 that received 0.05 mg/kg/day MAT exhibited an anaphylactic response upon rechallenge on day 42. Monkeys in groups 7 and 8 were not challenged (Table 1). Thus, four monkeys treated

PFIZER EX. 1037 Page 8

IMMUNOGENICITY AND PHJ\HMACOKINETICS OF HUMANIZED ANTI-TAC 1355 80

A. Group 2 60

40

~o-----o-----o

20 OLO--0--{',---2 - /__,"~~--+-+ E

........ 0> 80

:J B. Group 3 -1- 60

----0 <( 0

I 40

0 ...... 20 10 >. o/+ "'0 _r;; ...,....-:::::: -+:--:::=:6 0

.0 ...... 80

c C. Group 4 <( 60

40

20

0 10 15 20 25 30 35 40 45

Time (day) Fiyure :3. Time-dependent development of anti-IIAT antibodies In In­

dividual monkeys administered i\, 0.05, n. 0.50, C. 5.0 mg/kg/day HAT for 14 days. Anti-IIAT concentrations were determined In an ELISA using an affinity purified goat anti-IIAT antibody as a standard.

with MAT developed an anaphylactic response at a dose I 00-fold lower than the individual high dose HAT-treated monkey.

A comparison of day 42 (prechallenge) and day 55 serum antiglobulin levels from all challenged monkeys in groups I to 6 is shown in Figure 4. A primary immune response to the single treatment with MAT was observed in two naive monkeys in group 5, although the same treatment with HAT to group I monkeys resulted in no antibodies (Fig. 4A). A secondary immune response was observed In all animals previously treated with antibody. No secondary response was observed in groups 7 and 8, because they were not challenged. The greatest second­ary responses were observed in the monkeys receiving either 0.05 mg/kg MAT or HAT (Fig. 4B).

The specificity (i.e., anti-ld or anti-isotype) of the anti­HAT and anti-MAT responses was determined in a com­petitive ELISA assay (Fig. 5). Inhibition of antibody bind­Ing in the ELISA by HAT. MAT, as well as s!L-2R indi­cates the presence of anti-CDR or anti-idiotypic antibod­ies, because these proteins specifically compete for or block recognition of the CDR regions of anti-Tac. Com­petition by irrelevant human and mouse IgG proteins indicates the presence of anti-isotypic antibodies. The

10000 A. Control B. 0.05 rng/kg C. 0.5 mg/kg D. 5.0 mg/kg

' /rJJ. '

·It ''

PI 1 fl fl (/

E 1000 .......

Ol 2 >. 100 "C 0 ..c ·.;::;

10 c ~

0.5 55 42 55 42 55 42 55

Time (day)

Fiyure 4. Primary and secondary immune responses to HAT and MAT. Anti-HAT (0) and anti-MAT (6) antibody concentrations from individual monkeys on day 42 before high-dose challenge and on day 55. Data In i\ represents the anti-MAT response on day 55 In two naive monkeys from group 5. No anli-IIAT antibodies developed In any monkeys from group I. Before challenge animals received multiple doses of B. 0.05, C. 0.50, D. 5.0 mg/kg/day of anli-Tac antibody for 14 days. Monkeys dosed with MAT in C and D were not rechallenged with MAT on day 42 (data not shown).

40 c 0

':;:; 20 .0 .s::: .E

10 100 1000 ...... c 100 Q)

B. 0 L.. Q)

75 0..

50

25

Concentration {ng/ml) Figure 5. Characterization of antl-1-JAT and anti-MAT responses In

monkeys on day 35. A shows the anti-MAT from a monkey in group G and I3 shows the anti-HAT response from a monkey in group 2. A fixed amount of antiserum was Incubated In the presence of various concen­trations of l!AT (0). MAT (6), s!L-2f{ (0), human IgG (V'). or mouse IgG (0). These data are representative of the data obtained from all of the monkeys with antiglobulin antibodies on day 35.

goat anti-HAT and anti-MAT antibodies were partially inhibited by all of the competitors (data not shown), indicating that HAT and MAT administered to goats in­duced both an anti-idiotypic and anti-isotypic response.

Serum from all MAT-treated monkeys was completely inhibited with excess MAT, demonstrating that the ELISA assay is specific for MAT (Fig. 5A). HAT and slL-2R were the next most effective inhibitors followed by mouse IgG. Thus. the monkey response to MAT was a mixture of anti-isotypic and anti-idiotypic antibodies

PFIZER EX. 1037 Page 9

1356 IMMUNOGENICITY AND PHARMACOKINETICS OF HUMANIZED ANTI-TAC

similar to the goat anti-MAT and anti-HAT responses. Complete inhibition of monkey anti-HAT antibodies was achieved with HAT, again demonstrating assay specific­ity (Fig. 5B). Human and mouse IgG had little effect indicating that the anti-HAT response in monkeys is not an anti-isotypic response. MAT and siL-2R were almost as effective as HAT in inhibiting anti-HAT binding. Thus, the monkey anti-HAT antibodies are directed toward de­terminants shared by MAT and HAT, and blocked by siL-2R i.e., toward the CDR regions. This supports our con­clusion that the anti-HAT response is anti-idiotypic in monkeys.

Pharmacokinetic characterization. The pharmacoki­netic characteristics of HAT and MAT were determined in a competitive immunosorbant receptor assay. In this assay, both proteins inhibited IL-2 binding within twofold of each other. The detection limit was in the range of 125 to 500 ng/ml. Serum concentrations of HAT and MAT were measurable only in monkeys receiving doses of 0.5 and 5 mg/kg/day of antibody (Figs. 6 and 7, respectively, note the differences of the ordinate scales). In general, HAT concentrations were increased over the dosing period, suggesting that equilibrium was not achieved with receptor sites or the extravascular space. The mean max­imum concentrations after dosing with 0.5 and 5 mg/kg/ day of HAT for 14 days were 57± 20 (mean± SD) and 726 ± 115 J.!g/ml, respectively. In contrast. maximum concentrations of 0.5 and 5.0 mg/kg/day of MAT were 26 ± 9 and 311 ± 57 J.!g/ml. respectively, but occurring at approximately 7 to 9 days after the initiation of ther­apy. The mean time course of decline or t 112 values of HAT from the serum after 14 days of dosing were highly

100r---------------------------

A. Group 7 -'E 75 ...... Cl 2 1- 50

<t: A ~

"11\~ L.. 0

1- r/1' +~,. <t:

0

I 100 - B. Group 3 0

Cl) 75

l Q) > ~

50 6'0

'0 E :J

f-c~-L.. (I)

25 C/)

0 ' 0 10 15 20 25 30 35 40 45

Time (day) Figure 6. Serum concentration profile of A. MAT or D, HAT In Individ­

ual monkeys receiving 0.50 mg/kg/day of antibodies for 14 days. Anti­MAT and anti-HAT concentrations were determined In a competitive IL-2 irnrnunosorbant receptor assay using the respective purified anti-Tac rnAb as the standard.

1000

A. Group 8 -'E 750 ....... Cl 2 1-

500

<{

~~~ ~ 250

L.. 0 J~ \r 1- 0 h-~~ <t:

. I

1000 - B. Group 4 0

Cl) 750 (I)

> 0/6 ~ • !j!_+

500 t E :J

~+ .._ (I)

250 0~ ~~--+ C/)

f 6~ o __ o <(...,___

0 10 15 20 25 30 35 40 45

Time (day) Figure 7. Serum concentration profile of A. MAT or D. HAT In Individ­

ual monkeys receiving 5.0 mg/kg/day of antibodies for 14 days. Sec Figure 6 for additional details.

variable, ranging from approximately 47 to 432 h, and independent of dose. The t 112 of MAT was not calculated due to the rapid decline of serum concentrations even during the 14-day dosing regimen.

In control naive animals, the serum concentration-time profiles of HAT were significantly different from the profiles with MAT (Table II). The Individual AUC and t1;2

values after a single I. v. dose of 5 mg/kg of HAT or MAT to control monkeys In groups 1 and 5, respectively, on day 42 are shown in Table II. The mean AUC was ap­proximately twofold more In the HAT-treated control monkeys when compared to the MAT-treated control counterparts, 26,657 ± 6237 vs 11 ,442 ± 3563 f.!g · h/ml, respectively. A four- to fivefold difference was observed in the mean t 1;2 values between HAT and MAT (213.6 ± 58.8 and 4 7.8 ± 9.04 h, respectively) (Fig. 8).

The pharmacoklnctic profiles In the multiple-dosed groups were significantly altered. Only four MAT-treated monkeys were rcchallcnged on day 42 due to the observed anaphylactic response. Three of the monkeys had no detectable serum MAT levels, whereas in the fourth mon­key, levels were detectable but not within the quantita­tion limits of our assay governed by the standard curve (data not shown). Clearly, the elimination of MAT from group 6 animals that were treated with 0.05 mg/kg/day MAT was significantly enhanced compared to group 5 animals that had not received MAT previously. Kinetic parameters of all but two of the HAT-treated monkeys in groups 2 to 4 were estimated (Table II). The AUC values in groups 2 and 3 were lower than those in naive animals (group 1). In animals treated with 5 mg/kg/day (group 4), the t 112 and AUC values were higher than the values obtained for group 2 and 3 monkeys. One monkey in group 4 had greater values than the naive group animals.

PFIZER EX. 1037 Page 10

IMMUNOGENICITY AND PHARMACOKINETICS OF HUMANIZED ANTI-TAC 1357

TABLE II

Individual AUC (J1g·l!/rnl) and(,,. (I!) values in monkeys given single i.v. dose qj' 5 rng/kg of HAT or MAT on day 42

Group 1 Group 2 Group 3 Group 4 Group 5

IIAT Controls IIAT 0.05 mg/k!( IIAT 0.5 mg/k!( HAT 5 mg/ki( MAT Controls

AUC t,

:!5,251 259 22.140 270 21.96 I 167 27.276 200

"NE. Not evaluable.

1-< J:

(/)

Qi > 10 ~

AUC t,,

8,004 19.9 NE" NE

6,705 26.9 2,848 9.7

0 50 100 150 200 250

Time (hour)

AUC

4,883 5,489 2,050

677

Figure B. I !AT (0) and MAT (0) serum concentration In naive monkeys administered a sln~le bolus of 5 m~/kg ant!-Tac. See Fi~ure 7 for addi­tional details.

This may be explained by elevated serum concentrations of HAT on day 42 from the multiple dosing regimen (Fig. 6). In addition, the t 112 values of each HAT-treated animal after rechallenge were significantly reduced compared to values after steady-state dosing (data not shown).

An association between anti-HAT serum concentra­tions and either AUC or t 112 values was observed (Fig. 9). These pharmacokinetic parameters were inversely re­lated to the serum anti-HAT concentrations, indicating that elevated antibody levels contribute to the accelerated elimination of HAT. Correlation of' the kinetic parameters and antibody levels for the HAT-treated monkeys in groups 2 and 3 appeared similar. The group 4 values appeared more typical of the parameters measured for the control group 1 monkeys suggesting that antibody effects on HAT pharmacokinetics were similar between groups. Taken together. the survival of HAT was mean­ingfully longer than that of MAT in naive monkeys. Fur­thermore, the development of antibodies to the adminis­tered mi\b was associated with a reduced serum t 112 •

DISCUSSION

In this report, we demonstrate reduced immunogenicity and improved pharmacokinetics of humanized anti-Tac relative to its murine counterpart in cynomolgus mon­keys. MAT-treated monkeys developed anti-MAT anti­bodies during the primary 14-day treatment period at all doses tested (Fig. 2). Development of anti-MAT antibodies correlated with the rapid reduction in serum MAT con­centrations with time (Fig. 7). A similar anti-MAT re­sponse was observed in cynomolgus monkeys receiving allografts and treated with MAT ( 11 ). Furthermore, a single high dose of MAT administered to the control

t., AUC t,, AUC t ••

34.5 29,485 115 9,325 53.1 26.7 99,!>70 31H H,424 38.1 18.8 6,850 28 16,388 57.6 8.9 NE NE 11,633 42.6

A

5 1001

~ :J .!. D (ij D • J: • D

10 • 0

1000001 B

'E ...... .t:: til 2 10000 • • • (.) 0 0 ::I < • 0

1000 D

500~--~----~--~----~--~--~

0 10 20 30 40 50 60

Anti-HAT (ug/mll

Figure 9. Associations between anti-HAT concentrations vs AUC (A) and half-life (B) values after a single 5 mg/kg rechallenge dose on day 42 in monkeys in group 1 (0, four animals). group 2 (0, three animals). group 3 (0. four animals), and group 4 (•. three animals). See Table II.

monkeys on day 42 was sufficient to evoke an antibody response within 13 days (Fig. 4). MAT was similar to other murine antibodies studied in primates (31, 32) in being recognized as a foreign Ag and inducing both anti­idiotypic and anti-isotypic responses.

HAT clearly proved to be less immunogenic than MAT. Anti-HAT antibody titers were 5- to 10-fold lower in their respective dosing groups, and the antibodies were not detected until several days after the dosing regimen was completed (Fig. 3). Similar results were observed with anti-Tac-treated monkeys with cardiac allografts (11). Reduced immunogenicity correlated with an improved serum-time profile (Fig. 6). Unlike MAT. a single bolus of HAT, did not induce a measurable antibody response.

Inasmuch as HAT and MAT as well as siL-2R can almost completely inhibit binding of the monkey anti­HAT antibodies to HAT (Fig. 5B). we conclude that most of the monkey response is specific to the mouse se­quences shared by the two mAb. It is feasible that a small percentage of the monkey anti-HAT response is specific to the HAT framework sequence; however, preliminary data using recombinant HAT antibodies with various

PFIZER EX. 1037 Page 11

--------------------

1358 JMMUNOGENJCITY AND i'HAHMACOKINETICS OF HUMANIZED ANTJ-TAC

combinations of CDR replacements indicate that the anti­HAT response is not against Eu myeloma or HAT frame­work regions (W. Schneider, eta!., manuscript in prepa­ration).

LoBuglio et a!. ( 15) demonstrated that a mouse/human chimeric mAb, C17-1A, was substantially less immuno­genic than its mouse counterpart in man (32, 33). In a preliminary study, a humanized anti-CAMPATH-1 anti­body administered to two patients did not induce an antiglobulin response (21). However, a direct comparison of the rodent and humanized antibodies was not reported. In rhesus monkeys, authentic human anticytomegalovi­rus antibodies evoked an antibody (anti-idiotypic) re­sponse in only one of five monkeys (34, 35). Collectively, these studies suggest that chimeric, humanized, and hu­man mAb are minimally immunogenic in primates, and that immune responses to these antibodies are typically anti-idiotypic, as was observed in this study for HAT. Whether HAT will evoke a similar anti-idiotypic response in man remains to be determined. An anti-idiotypic re­sponse might be avoided when HAT is administered with cyclosporin A as suggested by studies demonstrated in rats (36).

The monkey serum concentration-time profile for HAT was more favorable than that for MAT. Using an assay that measures biologically active HAT and MAT, the mean AUC values for HAT in naive monkeys were ap­proximately twofold more than those for MAT (Table II), and the mean t1;2 of HAT was four- to fivefold more than MAT (214 vs 48 h). In vivo survival of 125I-HAT was 2.5-fold longer than 131 I-MAT in cynomolgus monkeys (11). Thus the different framework and constant regions of the mAb appear to control the pharmacokinetics of the two mAb.

The increased elimination of MAT from the blood in all groups during treatment as well as upon rechallenge confirms the presence of anti-MAT antibodies. The serum profiles of MAT are similar to those found with other murine antibodies (32, 33, 37, 38). Distinctly, the kinetics of HAT in naive monkeys appeared comparable to human IgG (34, 39, 40) and mouse/human chimeric antibodies (15, 17, 41) that exhibited t1 12 of more than 100 h.

In this study, a multiple dosing regimen was used that should have revealed immunogenic properties of HAT in monkeys (Fig. 1 ). Immunogenicity of HAT was directly demonstrated by ELISA (Fig. 3) and independently con­firmed by the accelerated elimination of HAT from the serum of monkeys exhibiting positive anti-HAT titers (Fig. 6). The immunogenicity of HAT appeared to be inversely related to the amount of protein injected during the 14-day treatment period (Fig. 3). This observation could partly be the result of the ELISA method used to assay for anti-HAT antibodies, because elevated serum concentrations of HAT would interfere with the assay. However, the kinetics of HAT when readministered on day 42 demonstrate that monkeys with apparent de­creased serum levels of antibodies to HAT in fact exhib­ited HAT serum t 112 values closer to naive animals (Fig. 8). Hence, the reduced immunogenicity of HAT at higher doses may be due to either the induction of tolerance or to the intrinsic immunosuppressive activity of anti-Tac and its effects on B cell responses.

We have demonstrated that the humanization process

is effective in preserving the selectivity and high affinity properties of a murine mAb (22, 23) whereas producing the desirable attributes of human antibodies such as reduced and delayed immunogenicity and improved cir­culating t 112 • These results in conjunction with the pre­vious demonstration that HAT (but not MAT) manifest antibody-dependent cellular cytotoxicity with human mononuclear cells (23) and is more eiTective in primate cardiac allograft survival ( 11) support the view that hu­manized anti-Tac will be more efficacious than murine anti-Tac in future clinical applications.

Acknowledgment. We thank Nadine Tare, Maureen Griffin, Lee Carpe, Brenda Jackson, and Thorton f~eed for excellent technical assistance, and Drs. Patricia Kil­ian and William Schneider for review and suggestions concerning the manuscript, and Rosemarie Bloyer for her help in preparing the manuscript.

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