...,
`., • t
`c. 1 , _
`·~
`> .
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`t :
`J Jl J.
`
`r
`
`1 ~-
`
`'
`
`THI:: JUUI(I~Al 0
`IMMUNOO
`
`®
`
`Official Journal of The American
`Association of Immunologists
`
`th
`Anniversary
`1916- 1991
`
`-- ....
`
`' _.;
`
`· .. !.·~~­
`
`.·· •.•
`~·
`
`t->
`
`PFIZER EX. 1037
`Page 1
`
`

`

`THE JOURNAL OF IMMUNOLOGY
`
`Volume 147 /Number 4, August 15, 1991
`
`Contents
`
`CELLULAR IMMUNOLOGY
`
`1107 Requirements of Class 11-Mediated B Cell Differentiation for Class II Cross(cid:173)
`Linking and Cyclic AMP
`1115 Triggering of CDS+ Cytotoxic T Lymphocytes via CD3-E Differs from Trig(cid:173)
`gering 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 Acti(cid:173)
`vation of Human B Lymphocytes
`1147 Role of a Thymic Stromal Cell Clone in Inducing the Stage-Specific Differ(cid:173)
`entiation of Various Subpopulations of Double Negative Thymocytes
`
`1153 Evidence of Synergy between Thy-1 and CD3/TCR Complex in Signal Deliv(cid:173)
`ery 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 Mu(cid:173)
`rine 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
`In Vitro Effect of Transforming Growth Factor-(3 on Progression of HIV-1
`Infection in Primary Mononuclear Phagocytes
`
`1201
`
`1208 Nonencephalitogenic CD4-cns- Va2V(3S.2+ Anti-Myelin Basic Protein Rat
`T Lymphocytes Inhibit Disease Induction
`
`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. Mi(cid:173)
`ya~e. T. Hamaoka, and H.
`FUJiwara
`I. Nakashima, Y.-H. Zhang, s.
`M. J, Rahman, T. Yoshida,
`K.-1. Isobe, L.-N. Ding, T. Iwa(cid:173)
`moto, M. Hamaguchi, H. Ike-
`C zawa, and R. Taguchi
`' M. Snapper, L. M. T. Pe(cid:173)
`canha, A. D. Levine, and J.
`J. Mond
`C.JM. Snapper, H. Yamada, J.
`· Mond, and C. H. June
`
`H. Spits, X. Paliard, Y. Vandek(cid:173)
`erckhove, 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.
`hlpman, G. Bilbe, and G.
`cMaster
`O.RLider, A. Milleer, S. Miron,
`x' Hershkoviz, H. L. Weiner,
`· Zhang, and E. Heber-Katz
`
`MS
`
`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).
`by Current Contents and Index Medicus. Second class postage paid at Baltimore, MD 21202 and at additional matltng offices.
`POST
`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.
`
`Continued on page 4
`
`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
`ked When
`Counterpart of Normal T Cells in the Liver
`,
`Development of Diabetogenic T Cells from NOD/Lt Marrow Is Bloc
`in but
`1
`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(cid:173)
`lewicz, and C. F. Ware
`I. K. Campbell, U. Novak, J. Ce(cid:173)
`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. Fu(cid:173)
`kayama, Y. Hayashi, M. Ka(cid:173)
`dokura, 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
`
`1230
`
`1238
`
`1247
`1253
`
`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
`
`1261
`
`is of serum
`h
`Effect of Combinations of Cytokines and Hormones on Synt es
`Amyloid A and C-Rcactivc Protein in HEP 3B Cells
`
`1266
`
`Granulocyte-Macrophagc Colony-Stimulating Factor Promotes
`ation of Human Alveolar Macrophagcs In Vitro
`
`the prollfer(cid:173)
`
`1273
`
`1279
`
`. 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
`
`-a Calcium(cid:173)
`
`IMMUNOCHEMISTRY
`
`M. Barel, A. Gauffre, F. Lya(cid:173)
`mani, A. Fiandino, J. Her(cid:173)
`mann, and R. Frade
`R. Busch, C. M. Hill, J. D. Hay(cid:173)
`ball, J. R. Lamb, and J. B.
`Rothbard
`P. E. Harris, M. C. Gutierrez, E.
`Reed, D. W. King, and N. Su(cid:173)
`ciu-Foca
`0. Kanagawa, Y. Utsunomiya,
`J. Bill, E. Palmer, M. W.
`Moore, and F. R. Carbone
`R. W. Leu, A. Zhou, J. Rum(cid:173)
`mage, 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
`
`1286
`
`1292
`
`1299
`
`1307
`
`1315
`
`1322
`
`1331
`
`1338
`
`phocytes
`Intracellular Interaction of EBV /C3d Receptor (CR2) with p6B.
`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(cid:173)
`Conformational Difference ofT Cell Antigen Hcccptors Revealed ~mants
`clonal Antibodies to Mouse V(35 T Cell Heccptor for Antigen Deter
`Their Re(cid:173)
`Reconstitution 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 rvtono(cid:173)
`DR 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
`man Fer
`clear Phagocytes
`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,
`andS. Ruddy
`
`J. Hakimi, R. Chizzonite, D. R.
`Luke, P. C. Familletti, P. Bai(cid:173)
`lon, J. A. Kondas, R. s. Pil(cid:173)
`son, P. Lin, D. V. Weber, C.
`Spence, L. J. Mondini, W.-H.
`Tsien, J. L. Levin, V. H. Gal(cid:173)
`lati, L. Korn, T. A. Wald(cid:173)
`mann, 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. Chap(cid:173)
`pel, S. L. Teitelbaum, and s.
`L. Perkins
`
`J.i. Coutelier, J. T. M. VanDer
`
`ogt, and F. W. A. Heessen
`K
`. B. Madden J F U b
`J
`• . . r an,
`H
`.
`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
`, and C. A.
`J
`aneway, Jr.
`
`1344 Association of Activated Properdin with Complexes of Properdin with C3
`
`IMMUNOPHARMACOLOGY
`
`1352 Reduced Immunogenicity and Improved Pharmacokinetics of Humanized
`Anti-Tac in Cynomolgus Monkeys
`
`1360 The Function of Lymphocyte Proteases: Inhibition and Restoration of Gran(cid:173)
`ule-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 Masto(cid:173)
`cytosis
`
`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
`
`-
`
`D.NJ.RDecker, N. E. Boyle, and
`·
`· Klinman
`
`R.Kay p M
`· Rosten, and R. K.
`• ·
`H
`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
`
`MOLECULAR BIOLOGY • MOLECULAR GENETICS
`
`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 An(cid:173)
`chor
`1417 Ly-49 Multigene Family: New Members of a Superfamily of Type II Mem(cid:173)
`brane 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 Cyto(cid:173)
`plasmic 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. Re(cid:173)
`ber-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
`
`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
`
`Erratum
`
`Announcement
`
`Author Index
`
`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 in(cid:173)
`hibit T cell proliferation. A humanized form of anti(cid:173)
`Tac (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 pharmacoki(cid:173)
`netic properties. Monkeys treated with HAT daily
`for 14 days exhibited anti-HAT antibody titers
`which were 5- to 10-fold lower than their MAT(cid:173)
`treated counterparts and these antibodies devel(cid:173)
`oped 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-anti(cid:173)
`body 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 al(cid:173)
`tered these parameters.
`
`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 devel(cid:173)
`opment 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. More(cid:173)
`over, 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-vs(cid:173)
`host 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 effec(cid:173)
`tive against those disorders without harming normal Tac(cid:173)
`negativc T cells. These agents would potentially be more
`selective than other immunosuppressants such as anti(cid:173)
`bodies 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 ther(cid:173)
`apeutic benefit.
`Anti-IL-2H. antibodies have been effective in animal
`models as well as in early human trials. In vivo admin(cid:173)
`istration of anti-IL-2R antibodies greatly prolonged sur(cid:173)
`vival of heart allografts in mice and rats (7. 8) and alle(cid:173)
`viated insulitis in nonobcsc diabetic mice and lupus ne(cid:173)
`phritis in NZB X NZW mice (9). MAT Itself was highly
`effective in prolonging survival of allografts in cynomol(cid:173)
`gus monkeys ( 1 0) with improved efficacy observed with
`HAT (11). In phase I clinical trials for kidney transplan(cid:173)
`tation, prophylactic administration of MAT significantly
`reduced the incidence of rejection episodes, without as(cid:173)
`sociated 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. Wald(cid:173)
`mann, unpublished observations).
`Several major problems limit the effectiveness of a
`murine mAh such as MAT when used in human patients.
`1352
`
`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!(cid:173)
`cate this fact.
`1 Address correspondence and reprint requests to Dr. John Ilakimi,
`Hoffmann-LaHoche, Department of Immunophannacology, 340 Kings(cid:173)
`land 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.
`
`PFIZER EX. 1037
`Page 6
`
`

`

`IMMUNOGENICITY AND PHJ\H.MACOKINETICS OF HUMANIZED ANTI-TAC
`
`1353
`
`obtained from Dr. F. Khan, Bioprocess Development. Hoffmann-La
`Roche Inc .. Nutley, NJ.
`HRP-labeled IL-2. IIAT and MAT were prepared using a modifi(cid:173)
`cation 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 Re(cid:173)
`search 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 venipunc(cid:173)
`ture 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 incu(cid:173)
`bated 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 an(cid:173)
`tibody 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
`
`TABLE I
`Immwwgenicily study treatment groups
`
`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 1m(cid:173)
`man 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 anti(cid:173)
`bodies, Winter and colleagues ( 18-21) constructed "lm(cid:173)
`manizccl" antibodies, in which only the minimum neces(cid:173)
`sary parts of the mouse antibody. the CDR. were com(cid:173)
`bined with human V region frameworks and C regions.
`Based on this approach, we have recently constructed a
`humanized anti-Tac antibody (22). The humanized anti(cid:173)
`Tac antibody (HAT) retains several key mouse framework
`residues, preclictccl by computer modeling, which arc re(cid:173)
`quired to maintain high affinity binding for p55. In ad(cid:173)
`clition, the humanized antibody mediates antibocly-clc(cid:173)
`penclent cellular cytotoxicity against T cell leukemia cells
`(23). Previously. it was demonstrated in cynomolgus mon(cid:173)
`keys with cardiac allografts that HAT appeared less im(cid:173)
`munogenic 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 clon(cid:173)
`ing. Production of IIAT was performed in a 3-liter continuous per(cid:173)
`fusion 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 Bios(cid:173)
`ciences), 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. re(cid:173)
`spectively (26). Briefly, purified recombinant s!L-2R (27) was im(cid:173)
`mobilized 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. Walkers(cid:173)
`ville, 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 SDS(cid:173)
`l'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. Rich(cid:173)
`mond, CA). Purified human r!L-2 expressed in Escl!ericl!ia coli was
`
`Grmtp
`
`Dally Dose
`Challenge Dose
`Days I to 14
`Day 42
`Vehicle control
`HAT, 5 ml(/kl(
`I
`HAT. 0.05 mg/kg
`HAT. 5 rng/kl(
`2
`HAT, 0.5 mg/kg
`HAT, 5 mg/kg
`3
`IIAT. 5.0 mg/kg
`4
`Hi\ T. 5 mg/kg
`Vehicle control
`MAT, 5 mg/kl(
`5
`Mi\T, 0.05 mg/kg MAT, 5 ml(/kl(
`6
`MAT. 0.5 mg/kg
`None"
`7
`MAT, 5.0 mg/kg
`None"
`8
`"Monkeys not challenged with MAT due to the anaphylaxis observed
`in group G.
`
`Hrsponse
`
`~----~--~
`
`None
`NonP
`None
`Anaphylaxis I /4
`None
`Anaphylaxis 4/4
`
`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 concen(cid:173)
`tration 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. respec(cid:173)
`tively. Serum concentrations of the antibodies were plotted vs time
`on a log-linear graph and the /\UC values were calculated by trape(cid:173)
`zoidal 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 concen(cid:173)
`trations 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 mini(cid:173)
`mum of three serum concentration-time points obtained after the
`final dose.
`
`RESULTS
`
`Study design and clinical observations. A cynomol(cid:173)
`gus 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 im(cid:173)
`proved 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 post(cid:173)
`treatment with 5 mg/kg MAT. The monkeys responded
`to epinephrine and fluids. The animals in the MAT treat(cid:173)
`ment 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 anti(cid:173)
`anti-Tac IgE detected (data not shown). The cause of this
`anaphylactic response remains unknown.
`Immunogenicity characterization. Monkey antiglob(cid:173)
`ulin levels (i.e., antibodies to HAT and MAT) were evalu(cid:173)
`ated 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 anti(cid:173)
`MAT 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 in(cid:173)
`dividual monkeys is shown for MAT in Figure 2 and for
`
`TREATMENT
`14 DAYS
`~~~!~~iii~!dl
`10
`~
`BLOOD COLLECTION DAYS
`
`20
`
`30
`
`l
`
`I
`
`;
`
`j
`
`'
`
`CHALLENGE
`DAY 42
`
`!
`
`50
`
`40
`
`l!J'
`/""'-
`
`POST DAY 42
`25,.5,1,2,4,8
`12.24,36hr
`
`Figure 1. Immunogenieity and pharmacokinetic study design for eval(cid:173)
`uation of anti-Tac antibodies In cynomolgus monkeys. Sec Table I for
`additional detail.
`
`300.-------------------------------------------,
`
`A. Group 6
`
`2215
`
`1150
`
`0
`
`75 -E
`.......
`0>
`2 300
`._ 2211
`-
`0 -300
`
`<(
`~
`150
`
`0
`
`>. 75
`"0
`0
`.0
`c
`<(
`
`225
`
`150
`
`75
`
`B. Group 7
`
`0
`
`0~0
`o/:2G/
`0 / ~(!)
`/_¢.c:fJ~.
`
`()
`
`6
`
`6 -6 - - - - - -
`
`c. Group 8
`
`~6-6
`
`~-"
`~-·/'-'
`A
`.().
`
`·"
`40
`
`45
`
`0
`
`10
`
`115
`
`20
`
`21!
`
`30
`
`35
`
`Time (day)
`Figure 2. Time-dependent development of anti-MAT antibodies In In(cid:173)
`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, anti(cid:173)
`HAT 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 dra