`NU.l O
`• ~
`1 ~b 7
`. C1 - --- - - -- ~v: ~~~o L UUUO
`l : ~ ~0~ g ~ U1Nb~ OF TH~
`J T_ UN AL ACAb~M I 0 ~
`
`. .
`
`MAY 1987
`
`VOLUME 84
`NUMBER 10
`
`Proceedings
`
`OF THE
`
`National Academy
`of Sciences
`
`()f 'I H~~ UNI1 ED STATES OF AMERICA
`
`BIOEPIS EX. 1032
`Page 1
`
`
`
`Proceedings
`OF THE
`National Academy
`of Sciences
`OF THE UNITED STATES OF AMERICA
`
`Officers
`of the
`Academy
`
`Editorial Board
`of the
`Proceedings
`
`-----~------ ----~-- --- - ---------- -- - - - -
`
`FRANK PRESS, President
`JAMES D. EBERT, Vice President
`BRYCE CRAWFORD, JR., /lome Secretary
`WILLIAM E. GoRDON, Foreign Secretary
`ELKAN R. BLOUT, Treasurer
`
`ROBERT H. ABELES
`GORDON /\.. BA YM
`WILLIAM F. BRACE
`RONALD BRESLOW
`MICHAEL J. CHAMBERLIN
`
`MAXINE F. SINGER, Chairman
`MARY-DELL CIIILTON
`EDWARD E. DAVID, JR.
`STUART 11.. KoRNFELD
`DANIEL E. KosHLAND, JR.
`PETER D. LAX
`DANIEL NATIIANS
`
`HERBERT E. SCARF
`SoLoMoN H. SNYDER
`HAROLD V ARM US
`THOMAS /\.. WALDMANN
`FRANK II. WESTIIEIMER
`
`Mana~:ing Editor: FRANCES R. ZWANZIG
`Senior Associate Editor: GARY T. CocKs
`Associate Editor: CAY Bun.ER
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`t1ssociate Editor: MARILYN J. MASON
`A~·sociate Editor: JANET L. MoRGAN
`Associate Editor: T. PEARSON
`Associate l:..aitor: DoROTHY P. SMITH
`Associate Editor: Cou;NE WALDEN
`Assistant Managing Editor: JoANNE D'AMICO
`
`t
`(
`
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`
`BIOEPIS EX. 1032
`Page 2
`
`
`
`Proceedings
`OF THE
`National Academy
`of Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`May 1987
`
`Volume 84, Number 10
`
`pp. 3081-3536
`
`Table of Contents
`
`AUTHOR INDEX
`
`Physical Sciences
`
`CHEMISTRY
`
`Phytotoxins from the pathogenic fungi Drechslera maydis and Drechslera
`sorghicola
`
`Accessible surface areas as a measure of the thermodynamic parameters of
`hydration of peptides
`
`F. Sugawara, G. Strobel, R. N. Strange,
`J. N. Siedow, G. D. VanDuyne, and
`J. Clardy
`
`Tatsuo Ooi, Motohisa Oobatake, George
`Nemethy, and Harold A. Scheraga
`
`Discrete wave mechanics: Multidimensional systems
`
`Frederick T. Wall
`
`MATHEMATICS
`
`Algebraic K-theory of discrete subgroups of Lie groups
`
`F. T. Farrell and L. E. Jones
`
`Biological Sciences
`
`BIOCHEMISTRY
`
`Specific inhibition of Trypanosoma cruzi neuraminidase by the human plasma
`glycoprotein "cruzin"
`
`R. P. Prioli, I. Rosenberg, and M. E. A.
`Pereira
`
`Regulation of actomyosin ATPase activity by troponin-tropomyosin: Effect of
`the binding of the myosin subfragmcnt 1 (S-l)·ATP complex
`
`Lois E. Greene, David L. Williams, Jr.,
`and Evan Eisenberg
`
`vii
`
`3081
`
`3086
`
`3091
`
`3095
`
`3097
`
`3102
`
`- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`
`BIOEPIS EX. 1032
`Page 3
`
`
`
`Contents
`
`Deduced amino acid sequence of bovine retinal Goa: Similarities to other
`guanine nucleotide-binding proteins
`
`Cloning and DNA sequence of the mercuric- and organomercurial-resistance
`determinants of plasmid pDU1358
`
`Sequences from a prokaryotic genome or the mouse dihydrofolate reductase
`gene can restore the import of a truncated precursor protein into yeast
`mitochondria
`
`Thyroid hormone regulates expression of a transfected a-myosin heavy-chain
`fusion gene in fetal heart cells
`
`Properties of the duplex DNA-dependent ATPase activity of Escherichia coli
`RecA protein and its role in branch migration
`Crystal structure of a snake venom cardiotoxin
`
`Insulin stimulates phosphorylation of a 120-kDa glycoprotein substrate (pp120)
`for the receptor-associated protein kinase in intact H-35 hepatoma cells
`
`Properties of a genetically engineered G domain of elongation factor Tu
`
`Secretin stimulates cyclic AMP and inositol trisphosphate production in rat
`pancreatic acinar tissue by two fully independent mechanisms
`
`Synchrotron x-ray diffraction studies of actin structure during polymerization
`
`Cotranslational insertion of selenocysteine into formate dehydrogenase from
`Escherichia coli directed by a UGA codon
`
`Temporal and tissue-specific expression of mouse ets genes
`
`Apparent lack of discrimination in the reading of certain codons in
`Mycoplasma mycoides
`
`Krisa P. Van Meurs, C. William Angus,
`Sukadev Lavu, Hsiang-Fu Kung,
`Susanne K. Czarnecki, Joel Moss, and
`Martha Vaughan
`Hugh G. Griffin, Timothy J. Foster,
`Simon Silver, and Tapan K. Misra
`
`Alison Baker and Gottfried Schatz
`
`Thomas A. Gustafson, Bruce E.
`Markham, Joseph J. Bah!, and Eugene
`Markin
`Stephen C. Kowalczykowski, Jennifer
`Claw, and Renee A. Krupp
`B. Rees, J.P. Samama, J. C. Thierry,
`M. Gilibert, J. Fischer, H. Schweitz,
`M. Lazdunski, and D. Moras
`
`Nicola Perrotti, Domenico Accili,
`Bernice Marcus-Samuels, Robert W.
`Rees-Jones, and Simeon I. Taylor
`
`Andrea Parmeggiani, Guido W. M.
`Swart, Kim K. Mortensen, Michael
`Jensen, Brian F. C. Clark, Luciana
`Dente, and Riccardo Cortese
`
`E. R. Trimble, R. Bruzzone, T. J. Biden,
`C. J. Meehan, D. Andreu, and R. B.
`Merrifield
`Paul Matsudaira, Joan Bordas, and
`M. H. J. Koch
`F. Zinoni, A. Birkmann, W. Leinfelder,
`and A. Bock
`Narayan K. Bhat, Robert J. Fisher,
`Shigeyoshi Fujiwara, Richard Ascione,
`and Takis S. Papas
`
`Tore Samuelsson, Youssef S. Guindy,
`Florentyna Lustig, Tomas Boren, and
`Ulf Lagerkvist
`
`Aberrant splicing events that are induced by proviral integration: Implications
`for myb oncogene activation
`
`Dan Rosson, Debbie Dugan, and E.
`Premkumar Reddy
`
`Reaction of argininosuccinase with bromomesaconic acid: Role of an essential
`lysine in the active site
`
`C. J. Lusty and S. Ratner
`
`A nonsense mutation causes hereditary goitre in the Afrikander cattle and
`unmasks alternative splicing of thyroglobulin transcripts
`
`M. H. Ricketts, M. J. Simons, J. Parma,
`L. Mercken, Q. Dong, and G. Vassart
`
`Role of DNA topoisomerase I in the transcription of supercoiled rRNA gene
`
`Endonuclease IV of Escherichia coli is induced by paraquat
`
`Identification and synthesis of a recognition signal for the attachment of
`glycosaminoglycans to proteins
`
`lac repressor blocks in vivo transcription of lac control region DNA
`
`RNA from an immediate early region of the type 1 herpes simplex virus
`genome is present in the trigeminal ganglia of latently infected mice
`
`Rat ce.llular retinol-binding protein: eDNA sequence and rapid
`retmol-dependent accumulation of mRNA
`
`Purification, properties, and immunocytochemical localization of human liver
`peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase
`
`Lalit C. Garg, Susan DiAngelo, and
`Samson T. Jacob
`
`Emily Chan and Bernard Weiss
`
`Mario A. Bourdon, Tom Krusius, Steven
`Campbell, Nancy B. Schwartz, and
`Erkki Ruoslahti
`
`Marguerite A. Sellitti, Pamela A. Pavco,
`and Deborah A. Steege
`
`Anne M. Deatly, Jordan G. Spivack,
`Ehud Lavi, and Nigel W. Fraser
`
`David R. Sherman, R. Stephen Lloyd,
`and Frank Chytil
`
`M. Kumudavalli Reddy, Nobuteru
`Usuda, M. Narahari Reddy, Edward
`R. Kuczmarski, M. Sambasiva Rao,
`and Janardan K. Reddy
`
`3107
`
`3112
`
`3117
`
`3122
`
`3127
`
`3132
`
`3137
`
`3141
`
`3146
`
`3151
`
`3156
`
`3161
`
`3166
`
`3171
`
`3176
`
`3181
`
`3185
`
`3189
`
`3194
`
`3199
`
`3204
`
`3209
`
`3214
`
`Isolation of a cytochrome aa3 gene from Bradyrhizobium japonicum
`
`Mark R. O'Brian and Robert J. Maier
`
`3219
`
`ii
`
`BIOEPIS EX. 1032
`Page 4
`
`
`
`Contents
`
`Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to
`plasminogen
`
`Dan L. Eaton, Gunther M. Fless,
`William J. Kohr, John W. McLean,
`Qin-Tu Xu, Chad G. Miller, Richard
`M. Lawn, and Angelo M. Scanu
`
`3224
`
`BIOPHYSICS
`
`Single acetylcholine receptor channel currents recorded at high hydrostatic
`pressures
`
`S. H. Heinemann, W. Stiihmer, and F.
`Conti
`
`3229
`
`CELL BIOLOGY
`
`Expression of p21"" in normal and malignant human tissues: Lack of
`association with proliferation and malignancy
`
`Pilar Garin Chesa, Wolfgang J. Rettig,
`Myron R. Melamed, Lloyd J. Old, and
`Henry L. Niman
`
`The very late antigen family of heterodimers is part of a superfamily of
`molecules involved in adhesion and embryogenesis
`
`Y oshikazu Takada, Jack L. Strominger,
`and Martin E. Hemler
`
`Immunoglobulin A stimulates growth of the extrahepatic bile duct in BALB/c
`mice
`
`Cytoskeletal association of human a-interferon-receptor complexes in
`interferon-sensitive and -resistant lymphoblastoid cells
`
`Sheila Fallon-Friedlander, Jeffrey R.
`Boscamp, Rachel Morecki, Frank
`Lilly, MarshallS. Horwitz, and Joy H.
`Glaser
`
`Lawrence M. Pfeffer, Nowell Stebbing,
`and David B. Donner
`
`An insulin-like growth factor (IGF) binding protein enhances the biologic
`response to IGF-1
`
`R. G. Elgin, W. H. Busby, Jr., and
`D. R. Clemmons
`
`Periodic crosslinking of microtubules by cytoplasmic microtubule-associated
`and microtubule-corset proteins from a trypanosomatid
`
`Gregory T. Bramblett, Sulie Chang, and
`Martin Flavin
`
`Firefly luciferasc is targeted to peroxisomes in mammalian cells
`
`Functional epithelial cell line cloned from rat parathyroid glands
`
`Differential regulation of colony-stimulating factors and interleukin 2
`production by cyclosporin A
`
`Coinduction of glucose-regulated proteins and doxorubicin resistance in
`Chinese hamster cells
`
`Growth factor(s) produced during infection with an adenovirus variant
`stimulates proliferation of nonestablished epithelial cells
`
`Gilbert-Andre Keller, Stephen Gould,
`Marlene Deluca, and Suresh
`Subramani
`
`Kazushige Sakaguchi, Arthur Santora,
`Mark Zimering, Francesco Curcio,
`Gerald D. Aurbach, and Maria Luisa
`Brandi
`
`M. Bickel, H. Tsuda, P. Amstad, V.
`Evequoz, S. E. Mergenhagen, S. M.
`Wahl, and D. H. Pluznik
`
`J. Shen, C. Hughes, C. Chao, J. Cai, C.
`Bartels, T. Gessner, and J. Subjeck
`
`Margaret P. Quinlan, Neil Sullivan, and
`Terri Grodzicker
`
`Protein import into chloroplasts requires a chloroplast ATPase
`
`Debkumar Pain and Gunter Blobel
`
`A high molecular weight component of the human tumor necrosis factor
`receptor is associated with cytotoxicity
`
`Abla A. Creasey, Ralph Yamamoto, and
`Charles R. Vitt
`
`Chemical carcinogen-induced decreases in genomic 5-methyldeoxycytidine
`content of normal human bronchial epithelial cells
`
`Vincent L. Wilson, Ruth A. Smith, Jim
`Longoria, Mary Anne Liotta, Cynthia
`M. Harper, and Curtis C. Harris
`
`DEVELOPMENTAL BIOLOGY
`
`Cell patterning in pigment-chimeric eyes in Xenopus: Germinal cell transplants
`and their contributions to growth of the pigmented retinal epithelium
`
`R. Kevin Hunt, Jerry S. Cohen, and
`Barbra J. Mason
`
`EVOLUTION
`
`A molecular phylogeny of the hominoid primates as indicated by
`two-dimensional protein electrophoresis
`
`David Goldman, P. Rathna Giri, and
`Stephen J. O'Brien
`
`Geographic dialects in blind mole rats: Role of vocal communication in active
`speciation
`
`Eviatar Nevo, Giora Heth, Avigdor
`Beiles, and Eliezer Frankenberg
`
`iii
`
`3234
`
`3239
`
`3244
`
`3249
`
`3254
`
`3259
`
`3264
`
`3269
`
`3274
`
`3278
`
`3283
`
`3288
`
`3293
`
`3298
`
`3302
`
`3307
`
`3312
`
`BIOEPIS EX. 1032
`Page 5
`
`
`
`Contents
`
`GENETICS
`
`Homology between the DNA-binding domain of the GCN4 regulatory protein
`of yeast and the carboxyl-terminal region of a protein coded for by the
`oncogene jun
`
`Peter K. Vogt, Timothy J. Bos, and
`Russell F. Doolittle
`
`A family of short, interspersed repeats is associated with tandemly repetitive
`DNA in the human genome
`
`Brion Mermer, Mark Colb, and
`Theodore G. Krontiris
`
`Random cloning of genes from mouse chromosome 17
`
`Structure and expression of human dihydropteridine reductase
`
`Masanori Kasahara, Felipe Figueroa, and
`Jan Klein
`
`Jean Lockyer, Richard G. Cook, Sheldon
`Milstien, Seymour Kaufman, Savio
`L. C. Woo, and Fred D. Ledley
`
`Gene tandem-mediated selection of coliphage >..-receptive Agrobacterium,
`Pseudomonas, and Rhizobium strains
`
`Robert A. Ludwig
`
`Detection of human DNA polymorphisms with a simplified denaturing gradient
`gel electrophoresis technique
`
`Walter W. Noll and Mary Collins
`
`Expression of retrovirally transduced genes in primary cultures of adult rat
`hepatocytes
`
`Comparative anatomy of the human APRT gene and enzyme: Nucleotide
`sequence divergence and conservation of a nonrandom CpG dinucleotide
`arrangement
`
`Jon A. Wolff, Jiing-Kuan Yee, Harold F.
`Skelly, Jane C. Moores, James G.
`Respess, Theodore Friedmann, and
`Hyam Leffert
`
`Thomas P. Broderick, Dennis A. Schaff,
`Amy M. Bertino, Michael K. Dush,
`Jay A. Tischfield, and Peter J.
`Stambrook
`
`Sequence analysis of spontaneous mutations in a shuttle vector gene integrated
`into mammalian chromosomal DNA
`
`Charles R. Ashman and Richard L.
`Davidson
`
`Molecular cloning and characterization of esterase-6, a serine hydrolase of
`Drosophila
`
`Measurement of low levels of x-ray mutagenesis in relation to human disease
`(Correction)
`
`J. G. Oakeshott, C. Collet, R. W. Phillis,
`K. M. Nielsen, R. J. Russell, G. K.
`Chambers, V. Ross, and R. C.
`Richmond
`
`Charles Waldren, Laura Correll,
`Marguerite A. Sognier, and Theodore
`T. Puck
`
`IMMUNOLOGY
`
`Molec~la; cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a
`rap1d 1mmunoselection procedure
`
`Brian Seed and Alejandro Aruffo
`
`Biosynthesis: gl~cosylation, and partial N-terminal amino acid sequence of the
`T-cell-actlvatmg protein TAP
`
`Resistance of cytotoxic T lymphocytes to lysis by a clone of cytotoxic T
`lymphocytes
`
`Regulation of murine class I genes by interferons is controlled by regions
`located both 5' and 3' to the transcription initiation site
`
`1a,25-Dihydroxyvitamin D3 inhibits y-interferon synthesis by normal human
`peripheral blood lymphocytes
`
`Co~plete supp.ression of in vivo growth of human leukemia cells by specific
`1mmunotoxms: Nude mouse models
`
`Hans Reiser, John Coligan, Baruj
`Benacerraf, and Kenneth L. Rock
`
`David M. Kranz and HermanN. Eisen
`
`Bette Korber, Leroy Hood, and Iwona
`Stroynowski
`
`Helmut Reichel, H. Phillip Koeffler,
`Andreas Tobler, and Anthony W.
`Norman
`
`Hideki Hara and Ben K. Seon
`
`3316
`
`3320
`
`3325
`
`3329
`
`3334
`
`3339
`
`3344
`
`3349
`
`3354
`
`3359
`
`3364
`
`3365
`
`3370
`
`3375
`
`3380
`
`3385
`
`3390
`
`Lymphocyte major histocompatibility complex-encoded class II structures may
`act as sperm receptors
`
`Ellyn R. Ashida and Virginia L. Scofield
`
`3395
`
`Working principles in the immune system implied by the "peptidic self' model
`
`Philippe Kourilsky, Gerard Chaouat,
`Chantal Rabourdin-Combe, and
`Jean-Michel Claverie
`
`3400
`
`MEDICAL SCIENCES
`
`Interferon 'Y induces lung colonization by intravenously inoculated 816
`melanoma cells in parallel with enhanced expression of class I major
`histocompatibility complex antigens
`
`K. Taniguchi, M. Petersson, P. Hoglund,
`R. Kiessling, G. Klein, and K. Kiirre
`
`3405
`
`iv
`
`BIOEPIS EX. 1032
`Page 6
`
`
`
`Contents
`
`Recognition of tau epitopes by anti-neurofilament antibodies that bind to
`Alzheimer neurofibrillary tangles
`
`Hanna Ksiezak-Reding, Dennis W.
`Dickson, Peter Davies, and Shu-Hui
`Yen
`
`Recognition of Alzheimer paired helical filaments by monoclonal neurofilament
`antibodies is due to crossreaction with tau protein
`
`Nobuyuki Nukina, Kenneth S. Kosik,
`and Dennis J. Selkoe
`
`Purification and characterization of rat liver nuclear thyroid hormone receptors
`
`Kazuo Ichikawa and Leslie J. DeGroot
`
`Development of a monoclonal antibody specifically reactive to gastrointestinal
`goblet cells
`
`M. Vecchi, S. Sakamaki, B. Diamond,
`A. B. Novikoff, P. M. Novikoff, and
`K. M. Das
`
`Linkage of the Wiskott-Aldrich syndrome with polymorphic DNA sequences
`from the human X chromosome
`
`Monica Peacocke and Katherine A.
`Siminovitch
`
`Glucagon-like peptide I stimulates insulin gene expression and increases cyclic
`AMP levels in a rat islet cell line
`
`Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells
`
`Androstenedione may organize or activate sex-reversed traits in female spotted
`hyenas
`
`Monoclonal antibodies to the human insulin receptor that activate glucose
`transport but not insulin receptor kinase activity
`
`Epstein-Barr virus nuclear antigen 2 specifically induces expression of the
`B-cell activation antigen CD23
`
`Tis~ue distribution and clearance kinetics of non-transferrin-bound iron in the
`hypotransferrinemic mouse: A rodent model for hemochromatosis
`
`Daniel J. Drucker, Jacques Philippe,
`Svetlana Mojsov, William L. Chick,
`and Joel F. Habener
`
`Alvin Y. Liu, Randy R. Robinson, Karl
`Erik Hellstrom, E. David Murray, Jr.,
`C. Paul Chang, and lngegerd
`Hellstrom
`
`Stephen E. Glickman, Laurence G.
`Frank, Julian M. Davidson, Erla R.
`Smith, and P. K. Siiteri
`
`John R. Forsayeth, Jose F. Caro,
`Madhur K. Sinha, Betty A. Maddux,
`and Ira D. Goldfine
`
`F. Wang, C. D. Gregory, M. Rowe,
`A. B. Rickinson, D. Wang, M.
`Birkenbach, H. Kikutani, T.
`Kishimoto, and E. Kieff
`
`C. M. Craven, J. Alexander, M.
`Eldridge, J. P. Kushner, S. Bernstein,
`and J. Kaplan
`
`MICROBIOLOGY
`
`Isolation and characterization of the a-sialyl-{3-2,3-galactosyl-specific adhesin
`from fimbriated Escherichia coli
`
`Thomas Moch, Heinz Hoschiitzky, Jorg
`Hacker, Klaus-D. Kroncke, and Klaus
`Jann
`
`NEUROBIOLOGY
`
`Cholinergic phosphatidylinositol modulation of inhibitory, G protein-linked,
`neurotransmitter actions: Electrophysiological studies in rat hippocampus
`
`Neurofilament gene expression: A major determinant of axonal caliber
`
`Calcium-dependent effect of the thymic polypeptide thymopoietin on the
`desensitization of the nicotinic acetylcholine receptor
`
`Monoclonal antibody analysis of keratin expression in the central nervous
`system
`
`Multiple neuropeptides in cholinergic motor neurons of Aplysia: Evidence for
`modulation intrinsic to the motor circuit
`
`Paul F. Worley, Jay M. Baraban,
`Madeline McCarren, Solomon H.
`Snyder, and Bradley E. Alger
`
`Paul N. Hoffman, Don W. Cleveland,
`John W. Griffin, Phillip W. Landes,
`Nicholas J. Cowan, and Donald L.
`Price
`
`Frederic Revah, Christophe Mulle,
`Christian Pinset, Tapan Audhya,
`Gideon Goldstein, and Jean-Pierre
`Changeux
`
`Maryellen C. Franko, Clarence J. Gibbs,
`Jr., Dorothy A. Rhoades, and D.
`Carleton G~dusek
`
`Elizabeth C. Cropper, Philip E. Lloyd,
`William Reed, Renata Tenenbaum,
`Irving Kupfermann, and Klaudiusz R.
`Weiss
`
`Quinolinic acid phosphoribosyltransferase: Preferential glial localization in the
`rat brain visualized by immunocytochemistry
`
`Christer Kohler, Etsuo Okuno, Per R.
`Flood, and Robert Schwarcz
`
`Activation of a potassium current by rapid photochemically generated step
`increases of intracellular calcium in rat sympathetic neurons
`
`Alison M. Gurney, Roger Y. Tsien, and
`Henry A. Lester
`
`v
`
`3410
`
`3415
`
`3420
`
`3425
`
`3430
`
`3434
`
`3439
`
`3444
`
`3448
`
`3452
`
`3457
`
`3462
`
`3467
`
`3472
`
`3477
`
`3482
`
`3486
`
`3491
`
`3496
`
`BIOEPIS EX. 1032
`Page 7
`
`
`
`Contents
`
`y-Aminobutyric acid exerts a local inhibitory action on the axon terminal of
`bipolar cells: Evidence for negative feedback from amacrine cells
`
`Identification and purification of an irreversible presynaptic neurotoxin from
`the venom of the spider llololena curta
`
`Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons:
`Coexistence with vasoactive intestinal peptide and choline acetyltransferase
`Cloning and sequence analysis of eDNA for the canine
`neurotensin/neuromedin N precursor
`
`Correlation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity with
`blood-brain barrier monoamine oxidase activity
`
`Human immunodeficiency virus can productively infect cultured human glial
`cells
`
`Masao Tachibana and Akimichi Kaneko
`
`Chauncey W. Bowers, Heidi S. Phillips,
`Pamela Lee, Yuh Nung Jan, and Lily
`Y. Jan
`
`Gabrielle G. Leblanc, Barry A. Trimmer,
`and Story C. Landis
`Paul R. Dobner, Diane L. Barber, Lydia
`Villa-Komaroff, and Colleen
`McKiernan
`Rajesh N. Kalaria, Mary Jo Mitchell,
`and Sami I. Harik
`Cecilia Cheng-Mayer, James T. Rutka,
`Mark L. Rosenblum, Thomas
`McHugh, Daniel P. Stites, and Jay A.
`Levy
`
`Social Sciences
`
`PSYCHOLOGY
`
`Initial localization of the acoustic conditioned stimulus projection system to the
`cerebellum essential for classical eyelid conditioning
`
`Joseph E. Steinmetz, Christine G.
`Logan, Daniel J. Rosen, Judith K.
`Thompson, David G. Lavond, and
`Richard F. Thompson
`
`3501
`
`3506
`
`3511
`
`3516
`
`3521
`
`3526
`
`3531
`
`vi
`
`BIOEPIS EX. 1032
`Page 8
`
`
`
`Proc. Nat/. Acad. Sci. USA
`Vol. 84, pp. 3439-3443, May 1987
`Medical Sciences
`
`1 Chimeric mouse-human JgG 1 antibody that can mediate lysis of
`cancer cells,
`',
`· -
`
`/ -
`(immunoglobulin domain eDNA/DNA transfection/tumor antigen/complement-dependent cytolysis/antibody-dependent cellular
`cytotoxicity)
`
`ALVIN Y. Lru*t, RANDY R. ROBINSON*, KARL ERIK HELLSTROM:j:§, E. DAVID MURRAY, JR.*~,
`c. PAUL CHANG*, AND INGEGERD HELLSTROM:j:ll
`*International Genetic Engineering, Inc., 1545 17th Street, Santa Monica, CA 90404; toncogen, 3005 First Avenue, Seattle, WA 98121; and Departments of
`§Pathology and IIMicrobiology, University of Washington Medical School, Seattle, WA 98195
`
`Communicated by Paul D. Boyer, January 28, 1987 (received for review December 22, 1986)
`
`, A chimeric mouse-human antibody has been
`ABSTRACT
`created that recognizes an antigen found on the surface of cells
`from many carcinomas. Immunoglobulin constant (C) domains
`of the mouse monoclonal antibody L6, Cy2a and CK, were
`substituted by the human Cyt and CK by recombining eDNA
`modules encoding variable or C domains. The eDNA constructs
`were transfected into lymphoid cells for antibody production.
`The chimeric antibody and mouse L6 antibody bound to
`carcinoma cells with equal affinity and mediated complement(cid:173)
`dependent cytolysis. In the presence of human effector cells, the
`chimeric antibody gave antibody-dependent cellular cytotox(cid:173)
`icity at 100 times lower concentration than that needed for the
`mouse L6 antibody. The chimeric antibody, but not the mouse
`L6 antibody, is effective against a melanoma line expressing
`small amounts of the L6 antigen. The findings point to the
`usefulness of the chimeric antibody approach for obtaining
`agents with strong antitumor activity for possible therapeutic
`usc in man. '>
`
`The presence of tumor-associated antigens at the cell surface
`is a characteristic of many cancers. Since these antigens are
`either absent or found in much lower amounts in normal cells,
`it should be possible to use antibodies for targeting of tumors.
`A sizeable collection of relatively tumor-specific monoclonal
`antibodies (mAb) of mouse origin is available (1). Some of
`these mAb possess tumoricidal activity in the presence of
`human effector cells [antibody-dependent cellular cytotox(cid:173)
`icity (ADCC)] or serum [complement-dependent cytotoxicity
`(CDC)] (2, 3). It has been shown (4) that partial tumor
`regression can be achieved when mAb possessing such
`functional activity are given to patients. One complication
`preventing repeated use of mouse mAb in man is that they are
`immunogenic. Furthermore, mouse mAb may interact less
`efficiently with human effector cells to mediate tumor de(cid:173)
`struction.
`A method made possible by recombinant DNA technology
`was chosen to generate chimeric mouse-human antibodies. It
`entails the replacement of the mouse constant (C) domain
`regions with the corresponding human equivalents (5-7). In
`principle, antibody molecules obtained by this approach
`should retain their specificity for antigen and thus their
`usefulness for targeting, be much less immunogenic to man,
`and perhaps have increased antitumor activity.
`The mouse mAb L6 [IgG2a(K)] binds to a carbohydrate
`antigen found at the surface of cells from human carcinomas
`of the lung, breast, colon, and ovary (8). L6 can mediate CDC
`with human complement or ADCC with human effector cells
`(2). mAb L6 may thus be of use for tumor targeting, either in
`its native form or after conjugation of anticancer agents.
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`In this study we have generated a mouse-human chimeric
`L6 antibody in which the mouse constant domains Cy2a and
`C" are substituted by the human C.,1 and C". First, the cDNAs
`encoding the immunoglobulin genes were isolated. Next,
`restriction enzyme recognition sites were created in the
`eDNA sequences at the V /C junction (where V stands for
`variable) (9) by in vitro mutagenesis using oligodeoxyribo(cid:173)
`nucleotides (10). The chimeric cDNAs thus constructed were
`then introduced into lymphoid cells by DNA transfection.
`The chimeric antibody isolated from the transfectants was
`compared with the mouse L6 for effector functions.
`
`MATERIALS AND METHODS
`DNA Transfection of Mouse Sp2/0 Lymphoid Cells. Expres(cid:173)
`sion plasmid piNG2114 (50 f.Lg), linearized at a unique site
`(Aat II) in the nonessential bacterial region (see Fig. 3A), was
`transfected into 107 mouse Sp2/0 cells (CRL 1581, ATCC) by
`electroporation (11, 12). Transformants were selected in
`Dulbecco's modified Eagle's medium (DMEM) supplement(cid:173)
`ed with 10% (vol/vol) fetal bovine serum (HyClone, Log~n,
`UT) and G418 at 0.8 mg/ml (GIBCO). The transfectwn
`frequency was between w- 5 and 10-4 • Human antibody in
`the medium was detected by ELISA (13).
`Isolation of Chimeric Antibody. Antibody-producing ~ells
`were grown to a density of 106 cells per ml and then shtfted
`to serum-free DMEM 24 hr before harvest. Antibody secret(cid:173)
`ed by the cells was concentrated by ultrafiltration, then
`chromatographed on a DEAE-cellulose column equilibrated
`in 40 mM NaCl/10 mM sodium phosphate, pH 8.0. The
`antibody in the flow-through was further purified to ap~arent
`homogeneity on protein A-Sepharose (14). For production of
`ascites fluid 106 cells were injected into pristane-primed
`BALB/c mi~e. The chimeric antibody was purified by anti(cid:173)
`human IgG-Sepharose chromatography (14).
`Functional Tests of the Chimeric L6 Antibody. The follow(cid:173)
`ing tests were included: (i) measurement of antibody binding
`to target cells, either positive or negative for reactivity with
`the mouse L6; (ii) competitive inhibition of binding of L6 to
`these cells; (iii) assays for CDC and ADCC. The binding tests
`were performed using a Coulter model EPIC-C cell sorter (8).
`The assays for CDC and ADCC were carried out on 51Cr(cid:173)
`labeled target cells (2, 3) that were exposed to antibodies and
`human serum or peripheral blood leukocytes over a 4-hr
`period.
`
`Abbreviations: V, variable; C, constant; J ,joining; mAb, monoclonal
`antibody(ies); CDC, complement-dependent cytolysis; ADCC, an(cid:173)
`tibody-dependent cellular cytotoxicity; SV40, simian virus 40; H,
`heavy.
`tTo whom reprint requests should be addressed.
`~Present address: Department of Biochemistry, University of Cali(cid:173)
`fornia, Riverside, CA 92521.
`
`3439
`
`BIOEPIS EX. 1032
`Page 9
`
`
`
`3440 Medical Sciences: Liu et al.
`
`Proc. Nat!. A cad. Sci. USA 84 ( 1987)
`
`A
`
`Lb Vn
`
`met asp tro leu
`C2JAGTTTG'l'CTTAAGGCACCACTGAGCCCAAGTCTTAGACA'l'CATG GAT TGG CTG
`Aft If
`BAL31del.
`G'l'CGACTCT
`IFR1
`leader pept1de
`Sail
`trp asn leu leu phe leu met ala ala ala gln ser ala gln ala qln
`'l'GG AAC TTG CTA T'l'C C'l'G ATG GCA GC'l' GCC Cl\l, AG'l' GCC Cl1l\ GCJ\ Cl\G
`
`ile gln leu val gln s~r gYy p~o gYu l~u l~s l~s p~o gly qYu thr
`l\TC Cl\G 'I"l'G G'l'G Cl\G TCT GGA CCT GAG C'I'G 1\l\G Al1G CC'l' GG!1 GM; 1\Cl\
`
`val lys ile ser cys lys ala ser qly tyr thr rohe t~R;1c~~~~ tyr qly
`GTC 1\l\G l\TC TCC TGC AAG GCT TCT GGG TAT ACC TTC 1\C!\ J,f,C T/\T c;r;r,
`Bgl II
`Ff121
`CDR11FR2
`met asn trp val lys gln ala pro qly lys gly leu lys trp met qly
`ATG AAC TGG G'l'G AAG CAG GCT CCI\ GGJ\ 1\l\G GGT TT!1 AM; TCr; l\TG c;r;c
`Aha Ill
`
`CDR2
`trp ile asn thr tyr thr gly gln pro thr tyr ala asp asD rohe lys
`TGG ATA AAC ACC TAC ACT GGA CAG CCA ACA TAT GCT GAT GAC TTC AAG
`Nde I
`
`CDR21FR3
`gly ara phe ala Dhe ser leu glu thr ser ala tyr thr ala tyr leu
`GGA CGG TTT GCC TTC TCT TTG GAA ACC TCT GCC TAC ACT GCC TAT TTG
`
`gln ile asn asn leu lys asn glu aso met ala thr tyr ohe cys ala
`Cl,G
`l\'l'C 1\l\C 1\l\C CTC 1\l,l\ l\1\'l' GAG GAC ATG GCT
`l1Cl1 Tl\'1' TTC TG'I' CCI\
`
`CDR31FR4
`FR31CDR3
`arq phe ser tyr gly asn ser arg tyr ser asro tyr tro qly qln gly
`AGA
`'l'T'l' AGC
`'l'A'l' GG'l' !1/\C TCA CG'l' TAC
`'l'CT r;r,c 'l'AC TGG GGC Cl\!1 c;r~c
`Dsp2,2
`---------------JH2
`thr thr leu thr val ser ser ala lys thr thr ala pro ser
`l\C'l' CTC ACA G'I'C
`'l'CC
`'l'CA GCC
`l\!1!. 1\Cl'. 1\C!, GCC CC!1 TCG-------
`l\CC
`
`----------------- GC -----Ac;- G - - - - - MJH2 Apo I
`
`B
`
`L6 Vr:
`
`leader pcpt1de
`met asp phe qln val gln ile ohe ser nhe leu leu
`CgCCCCl\l\Gl\Cl\1\l\l\'l'G GAT TTT CAl\ GTG CAG ATT TTC AGC TTC CTG CTA
`---eTc------------------ s· Sol 1
`
`ile ser ala ser val ile met ser arg qlyl~ln ile val leu ser qln
`ATC AGT GCT
`'I'CA G1'C l\1'/\ ATG
`'I'CC 1\Gl\ GG!1 CM, A'l'T G'I"l' C'I'C
`'I'CC C!1C
`
`ser pro ala ile leu ser ala ser pro gly glu l~s vgl t~r l2u thr
`TCT CCI\ GCA A'l'C C'l'G
`'I'CT GCA TC'I' CCI\ GGC GAG AN; r;Tc T1Cl\
`'l"l'r; AC'I'
`
`C9,R11FR3
`~RliCD§l
`o
`o
`o
`o
`o
`o
`o
`o
`0
`cys arg ala ser ser ser val ser phe met asn trp tyr qln gln l~s
`TGC AGG GCC AGC
`'l'CA AGT G'l'A ACT TTC ATG AAC TGG TAC ~AC CAG AAG
`Kpn I
`
`P~o gly ser ser pro lys pro trp ile :;:1~0l~ thr ser asn leu ala
`CCI\ GGA
`'I'CC
`'I'CC CCC AAA CCC TGG A'I''I' TAT CCC ACA TCC AAT TTG GC'l'
`Bam HI
`
`CDR21FR3
`ser glu phe pro gly arg phe ser gly glu trn ser gly thr ser tyr
`'l'C'I' GAG TTC CCT GG'l' CGC
`'I"l'C AGT CCC GAG 1~G 'l'CT GGG ACC TCT TAC
`
`ser leu ala ile ser arg val qlu ala glu a~o ala aYa t~r t~r t;r
`'l'CT CTC GCA ATC AGC AGA G'!'G GAG GC'l' GAA GAT GCT GCC ACT
`'!'AT TAC
`
`----------------------------JK5-
`FR31CDR3
`CDR31FR4
`o
`c~s gin gln t~p asn s~r a~n pro l3u thr phe gly al