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`1'
`
`NOVEMBER 1984
`VOLUME 81
`NUMBER 21
`
`..
`
`Proceedings
`
`OF THE
`
`National Academy
`of Sciences
`
`OF THE TT~Trrrl7n ~'TATES OF AMERICA
`r
`
`(
`
`BIOLOGICAL SCIENCES
`
`PFIZER EX. 1031
`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
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`
`RoBERT A. ALBERTY
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`
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`ROBERT E. MARSHAK
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`ROBERT L. SINSIIEIMER
`DANIEL NATHANS
`PETER H. RAVEN
`SoLOMON H. SNYDER
`FRANK H. WESTHEIMER
`]OliN RoDGERS
`NEVINS. SCRIMSHAW
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`
`Managing Editor: FRANCES R. ZwANZIG
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`
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`© 1984 by THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA.
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`POSTMASTER: Send address changes to: PROCEEDINGS, 2101 Constitution Ave., Washington, DC 20418.
`
`PFIZER EX. 1031
`Page 2
`
`

`

`Proceedings
`OF THE
`National Academy
`of Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`November 1984
`
`Volume 81, Number 21
`
`pp. 6565-6908
`
`Table of Contents
`
`AUTHOR INDEX
`
`Biological Sciences
`
`BIOCHEMISTRY
`
`Isolation of a large cholecystokinin precursor from canine brain
`
`Identification and partial purification of the cardiac sodium-calcium exchange
`protein
`
`Specific in vitro adenylylation of the simian virus 40 large tumor antigen
`
`Stimulatory GTP regulatory unit N, and the catalytic unit of adenylatc cyclase
`arc tightly associated: Mechanistic consequences
`
`Single amino acid substitutions influencing the folding pathway of the phage
`P22 tail spike endorhamnosidase
`
`Triplication of a four-gene set during evolution of the goat {:l-globin locus
`produced three genes now expressed differentially during development
`Expression of human a 1-antitrypsin eDNA in the yeast Saccharomyces
`c·erevixiac
`
`F-actin is intermolecularly crosslinked by N,N' -p-phcnylenedimaleimide
`through lysinc-191 and cysteine-374
`
`Amino acid sequence of bovine heart coupling factor 6
`
`V. E. Eysselein, J. R. Reeve, Jr., J. E.
`Shively, C. Miller, and J. H. Walsh
`
`Calvin C. Hale, Robert S. Slaughter,
`Diane C. Ahrens, and John P. Reeves
`
`Margaret K. Bradley, Janet Hudson,
`Merceditas S. Villanueva, and David
`M. Livingston
`
`Hadas Arad, Jiirg P. Rosenbusch, and
`Alexander Levitzki
`
`Myeong-Hee Yu and Jonathan King
`
`Tim M. Townes, Mary C. Fitzgerald, and
`Jerry B. Lingrel
`
`Teresa Cabez6n, Michel De Wilde,
`Pascal Herion, Rosette Loriau, and
`Alex Bollen
`
`Marshall Elzinga and James J. Phelan
`
`Ji-kang Fang, John W. Jacobs, Baruch I.
`Kanner, Efraim Racker, and Ralph A.
`Bradshaw
`
`6565
`
`6569
`
`6574
`
`6579
`
`6584
`
`6589
`
`6594
`
`6599
`
`6603
`
`PFIZER EX. 1031
`Page 3
`
`

`

`Contents
`
`Detection in vivo of a new gene product (gene III) of cauliflower mosaic virus
`
`C. Xiong, G. Lebeurier, and L. Hirth
`
`Alkane biosynthesis by decarbonylation of aldehydes catalyzed by a particulate
`preparation from Pisum sativum
`DNA methylation and regulation of the human {3-globin-like genes in mouse
`erythroleukemia cells containing human chromosome 11
`
`Monomeric solubilized sarcoplasmic reticulum Ca pump protein: Demonstration
`of Ca binding and dissociation coupled to ATP hydrolysis
`
`Integration-specific retrovirus expression in embryonal carcinoma cells
`
`Calcium:binding proteins in human carcinoma cell lines
`
`Transcriptional activation of the rat liver tyrosine aminotransferase gene by
`cAMP
`
`Characterization of the genes specifying two metacyclic variable antigen types
`in Trypanosoma brucei rhodesiense
`
`Heat shock regulatory gene htpR influences rates of protein degradation and
`expression of the /on gene in Escherichia coli
`
`A viable simian virus 40 variant that carries a newly generated sequence
`reiteration in place of the normal duplicated enhancer element
`
`Nucleotide sequence of the 3' region of an infectious human T-cell leukemia
`virus type II genome
`
`Enzymes processing somatostatin precursors: An Arg-Lys esteropeptidase from
`the rat brairi cortex converting somatostatin-28 into somatostatin-14
`
`Genomic structure and possible retroviral origin of the chicken CRJ repetitive
`DNA sequence family
`
`T. M. Cheesbrough and P. E.
`Kolattukudy
`Timothy J. Ley, Yawen L. Chiang,
`Donna Haidaris, Nicholas P. Anagnou,
`Vincent L. Wilson, and W. French
`Anderson
`Dwight W. Martin, Charles Tanford, and
`Jacqueline A. Reynolds
`
`Joe Sorge, Ann E. Cutting, Valerie D.
`Erdman, and James W. Gautsch
`Gaby E. Pfyffer, Gisela Haemmerli, and
`Claus W. Heizmann
`Setsuko Hashimoto, Wolfgang Schmid,
`and Gunther Schlitz
`
`Michael J. Lenardo, Allison C. Rice(cid:173)
`Ficht, Gregory Kelly, Klaus M. Esser,
`and John E. Donelson
`
`Stephen A. Goff, Lawrence P. Casson,
`and Alfred L. Goldberg
`Candace Swimmer and Thomas Shenk
`
`Kunitada Shimotohno, William
`Wachsman, Yuri Takahashi, David W.
`Golde, Masanao Miwa, Takashi
`Sugimura, and Irvin S. Y. Chen
`
`Pablo Gluschankof, Alain Morel, Sophie
`Gomez, Pierre Nicolas, Christine
`Fahy, and Paul Cohen
`
`William E. Stumph, Clague P. Hodgson,
`Ming-Jer Tsai, and Bert W. O'Malley
`
`A purified fraction containing RNA polymerase I that can accurately transcribe
`rat ribosomal RNA gene
`
`Rabinder N. Kurl, Lawrence I.
`Rothblum, and Samson T. Jacob
`
`Identification of the tyrosine protein kinase from LSTRA cells by use of site(cid:173)
`specific antibodies
`
`Characterization of nerve growth factor receptor in neural crest tumors using
`monoclonal antibodies
`
`Functional alteration of the f3-adrenergic receptor during desensitization of
`mammalian adenylate cyclase by {3-agonists
`
`John E. Casncllic, Larry E. Gentry,
`Larry R. Rohrschneider, and Edwin G.
`Krebs
`
`Alonzo H. Ross, Peter Grob, Mark
`Bothwell, David E. Elder, Carolyn S.
`Ernst, Nadia Marano, Barbara F. D.
`Ghrist, Catherine C. Slemp, Meenhard
`Herlyn, Barbara Atkinson, and Hilary
`Koprowski
`
`Shouki Kassis and Peter H. Fishman
`
`Heat-labile alkaline phosphatase from Antarctic bacteria: Rapid 5' end-labeling
`of nucleic acids
`
`Hiromi Kobori, Cornelius W. Sullivan,
`and Hiroaki Shizuya
`
`Evidence that a major class of mouse endogenous long terminal repeats (L TRs)
`resulted from recombination between exogenous retroviral L TRs and LTR(cid:173)
`like elements (LTR-lS)
`
`Martin Schmidt, Klaus Gloggler, Thomas
`Wirth, and Ivan Horak
`
`Synthesis and glycosylation of the common a subunit of human glycoprotein
`hormones in mouse cells
`
`T.V. Ramabhadran, B. A. Reitz, and D.
`C. Tiemeier
`
`Fusion protein of the paramyxovirus simian virus 5: Nucleotide sequence of
`mRNA predicts a highly hydrophobic glycoprotein
`
`Reay G. Paterson, Timothy J. R. Harris,
`and Robert A. Lamb
`
`Subcellular distribution of DNA-binding and non-DNA-binding
`1,25-dihydroxyvitamin D receptors in chicken intestine
`
`Masaki Nakada, Robert U. Simpson, and
`Hector F. DeLuca
`
`A dual role for the Ca2+ -requiring proteinase in the degradation of hemoglobin
`by erythrocyte membrane proteinases
`
`S. Pontremoli, E. Mclloni, B. Sparatore,
`M. Michetti, and B. L. Horecker
`
`ii
`
`6608
`6613
`
`6618
`
`6623
`
`6627
`
`6632
`
`6637
`
`6642
`
`6647
`
`6652
`
`6657
`
`6662
`
`6667
`
`6672
`
`6676
`
`6681
`
`6686
`
`6691
`
`6696
`
`6701
`
`6706
`
`6711
`
`6714
`
`PFIZER EX. 1031
`Page 4
`
`

`

`Contents
`
`CELL BIOLOGY
`
`Cyclic GMP may serve as a second messenger in peptide-induced muscle
`deg.::n.::ration in an insect
`
`Lawrence M. Schwartz and James W.
`Truman
`
`High-at1inity binding of the regulatory subunit (Ru) of cAMP-dependent protein
`kinase to microtubule-associated and other cellular proteins
`
`Introductory analysis of the GTP-cap phase-change kinetics at the end of a
`microtubule
`
`Tramcriptional induction of two genes in human cells by {3 interferon
`
`Target polypeptide of a carcinogen is associated with normal mitosis and
`carcinogen-induced hyperplasias in adult hepatocytes
`
`Suzanne M. Lohmann, Pietro DeCamilli,
`lnge Einig, and Ulrich Walter
`Terrell L. Hill
`
`A. C. Larner, G. Jonak, Y.-S. E. Cheng,
`B. Korant, E. Knight, and J. E.
`Darnell, Jr.
`
`R. Philip Custer and Sam Sorof
`
`(Pro)insulin associates with Golgi membranes of pancreatic B cells
`
`L. Orci, M. Ravazzola, and A. Perrelet
`
`The translational mobility of substances within the cytoplasmic matrix
`
`Ken Jacobson and John Wojcieszyn
`
`Dilfer.::ntiation of human erythroid cells is associated with increased
`0-glycosylation of the major sialoglyeoprotcin, glycophorin A
`
`Specific binding to cultured cells of mJ.Jabeled type {3 transforming growth
`factor from human platelets
`
`Inhibition of epidermal growth factor-induced mitogenesis by amiloride and an
`analog: Evidence against a requirement for Na+ /H+ exchange
`
`Interactions between human tumor cells and fibroblasts stimulate hyaluronate
`synthesis
`
`Expression of the sis gene by endothelial cells in culture and in vivo
`
`Movement of scallop myosin on Nitella actin filaments: Regulation by calcium
`
`GENETICS
`
`Carl G. Gahmberg, Marja Ekblom, and
`Lcif C. Andersson
`
`Ronald F. Tucker, Earl L. Branum, Gary
`D. Shipley, Robert J. Ryan, and
`Harold L. Moses
`
`Jeffrey M. Besterman, Scott J. Tyrey,
`Edward J. Cragoe, Jr., and Pedro
`Cuatrecasas
`
`Warren Knudson, Chitra Biswas, and
`Bryan P. Toole
`
`Thomas B. Barrett, Corinne M.
`Gajdusek, Stephen M. Schwartz,
`James K. McDougall, and Earl P.
`Benditt
`
`Ronald D. Vale, Andrew G. Szent(cid:173)
`Gyorgyi, and Michael P. Sheetz
`
`A gene regulating the heat shock response in Escherichia coli also affects
`proteolysis
`
`The sup-7(st5) X gene of Caenorhabditis ele~:ans encodes a tRNA'UXG amber
`suppressor
`
`Tania A. Baker, Alan D. Grossman, and
`Carol A. Gross
`
`Suzanne L. Bolten, Patricia Powell-Abel,
`David A. Fischhoff, and Robert H.
`Waterston
`
`Antitermination of transcription from an Escherichia coli ribosomal RNA
`promoter
`
`William E. Holben and Edward A.
`Morgan
`
`Sequences homologous to P clements occur in Drosophila pau/istomm
`
`Activation of a translocated c-myc gene: Role of structural alterations in the
`upstream region
`
`Stephen B. Daniels, Linda D.
`Strausbaugh, Lee Ehrman, and Robert
`Armstrong
`
`Klas G. Wiman, Bayard Clarkson,
`Adrian C. Hayday, Haruo Saito,
`Susumu Tonegawa, and William S.
`Hayward
`
`Heat shock regulatory gene UapR) of Escherichia coli is required for growth at
`high temperature but is dispensable at low temperature
`
`Takashi Yura, Toru Tobe, Koreaki Ito,
`and Toshio Osawa
`
`Viral integration ncar c-myc in 10-20% of MCF 247-induced AKR lymphomas
`
`Yen Li, Christie A. Holland, Janet W.
`Hartley, and Nancy Hopkins
`
`Directional cloning of DNA fragments at a large distance from an initial probe:
`A circularization method
`
`Francis S. Collins and Sherman M.
`Weissman
`
`Spontaneous deletions and duplications of sequences in the genome of cowpox
`virus
`
`David J. Pickup, Barbara S. Ink, Barbara
`L. Parsons, Wensi Hu, and Wolfgang
`K. Joklik
`
`iii
`
`6718
`
`6723
`
`6728
`
`6733
`
`6738
`
`6743
`
`6747
`
`6752
`
`6757
`
`6762
`
`6767
`
`6772
`
`6775
`
`6779
`
`6784
`
`6789
`
`6794
`
`6798
`
`6803
`
`6808
`
`6812
`
`6817
`
`PFIZER EX. 1031
`Page 5
`
`

`

`Contents
`
`IMMUNOLOGY
`
`Isolation of eDNA clones specifying the fourth component of mouse
`complement and its isotype, sex-limited protein
`
`Monocytes are required to trigger Ca2 + uptake in the proliferative response of
`human T lymphocytes to Staphylococcus aureus protein A
`
`Antigen conformation determines processing requirements forT-cell activation
`
`Calcium dependency of antigen-specific (T3-Ti) and alternative (Tll) pathways
`of human T-cell activation
`The cromolyn binding protein constitutes the Ca2
`upon immunological stimulus
`Regulation ofT -cell function by antibodies: Enhancement of the response of
`human T-cell clones to hepatitis B surface antigen by antigen-specific
`monoclonal antibodies
`Chimeric human antibody molecules: Mouse antigen-binding domains with
`human constant region domains
`
`+ channel of basophils opening
`
`Masaru Nonaka, Morinobu Takahashi,
`Shunnosuke Natsuume-Sakai, Mayumi
`Nonaka, Shoji Tanaka, Akira Shimizu,
`and Tasuku Honjo
`
`Howard M. Lederman, Jacob W. W.
`Lee, Roy K. Cheung, Sergio Grinstein,
`and Erwin W. Gelfand
`
`Howard Z. Streicher, lra J. Berkower,
`Mark IJusch, Frank R.N. Gurd, and
`Jay A. Berzofsky
`
`Michael J. Weiss, John F. Daley, James
`C. Hodgdon, and Ellis L. Reinherz
`
`N. Mazurek, H. Schindler, Th.
`Schiirholz, and !. Pecht
`Esteban Celis, Vincent R. Zurawski, Jr.,
`and Tse Wen Chang
`
`Sherie L. Morrison, M. Jacqueline
`Johnson, Leonard A. Herzenberg, and
`Vernon T. Oi
`
`6822
`
`6827
`
`6831
`
`6836
`
`6841
`
`6846
`
`6851
`
`MEDICAL SCIENCES
`
`In vivo nuclear magnetic resonance chemical shift imaging by selective
`irradiation
`
`5a-Cholest-8(14)-en-3{:l-ol-15-one lowers serum cholesterol and induces
`profound changes in the levels of lipoprotein cholesterol and apoproteins in
`monkeys fed a diet of moderate cholesterol content
`
`P. A. Bottomley, T. H. Foster, and W.
`M. Leue
`George J. Schroepfer, Jr., Bette C.
`Sherrill, Ker-Shi Wang, William K.
`Wilson, Alemka Kisic, and Thomas B.
`Clarkson
`
`NEUROBIOLOGY
`
`Isolation and sequencing of two cerebellum-specific peptides
`
`J. Randall Slemmon, Russell Blacher,
`Waleed Danho, James L. Hempstead,
`and James I. Morgan
`
`Understanding biological computation: Reliable learning and recognition
`
`T. Hogg ami B. A. Huberman
`
`Selective association of N-methyl aspartate and quisqualate types of
`L-glutamate receptor with brain postsynaptic densities
`
`Graham E. Fagg and Andrew Matus
`
`Evolutionary conservation of key structures ami binding functions of neural cell
`adhesion molecules
`
`Stanley HotTman, Cheng-Ming Chuong,
`ami Gerald M. Edelman
`
`Differential processing of prodynorphin and proenkephalin in specific regions of
`the rat brain
`
`Nadav Zamir, Eckard Weber, Miklos
`Palkovits, and Michael 13rownstein
`
`Is ATP a central synaptic mediator for certain primary a!Ierent fibers from
`mammalian skin'?
`
`Robert E. W. Fyll'e and Edward R. Perl
`
`Prevention of some electrophysiologic and biochemical abnormalities with
`oxygen supplementation in experimental diabetic neuropathy
`
`P. A. Low, R. R. Tuck, P. J. Dyck, J.D.
`Schmelzer, and J. K. Yao
`
`6856
`
`6861
`
`6866
`
`6871
`
`6876
`
`6881
`
`PHYSIOLOGICAL SCIENCES
`
`Pharmacomechanical coupling in smooth muscle may ·involve
`phosphatidylinositol metabolism
`
`POPULATION BIOLOGY
`
`Carl B. Baron, Michael Cunningham,
`Jerome F. Strauss lll, and Ronald F.
`Coburn
`
`Inheritance of a secondary sexual character in Drosophila si/vestris
`
`Hampton L. Carson and Russell Lande
`
`iv
`
`PFIZER EX. 1031
`Page 6
`
`

`

`Proc. Nat/. Acad. Sci. USA
`Vol. 81. pp. 6851-6855, November 1984
`Immunology
`
`Chimeric human antibody molecules: Mouse antigen-binding
`domains with human constant region domains
`(transfcction/protoplast fusion/calcium phosphate transfection/intronic controlling elements/transfectoma)
`
`SHERIE L. MoRRISON*, M. JAcQUELINE JoHNSoNt, LEONARD A. HERZENBERat, AND VERNON T. 0(1=
`
`*Department of Microbiology and the Cancer Center, Institute for Cancer Research, College of Physicians and Surgeons, Columbia University,
`New York, NY 10032: tDepartmcnt of Genetics, Stanford University School of Medicine, Stanford, CA 94305: and :J:Becton-Dickinson
`Monoclonal Center, 2375 Garcia Avenue, Mountain View, CA 94043
`
`Contributed by Leonard A. 1/erzenher;:, Au;:ust I, 1984
`
`ABSTRACT We have created mouse-human antibody
`molecules of defined antigen-binding specificity by taking the
`variable region genes of a mouse antibody-producing myeloma
`cell line with known antigen-binding specificity and joining
`them to human immunoglobulin constant region genes using
`recombinant DNA techniques. Chimeric genes were construct(cid:173)
`ed that utilized the rearranged and expressed antigen-binding
`variable region exons from the myeloma cell line SI07, which
`produces an lgA (K) anti-phosphocholine antibody. The
`heavy chain variable region ex on was joined to human IgG I or
`lgG2 heavy chain constant region genes, and the light chain
`variable region ex on from the same myeloma was joined to the
`human K light chain gene. These genes were transfected into
`mouse myeloma cell lines, generating transformed cells that
`produce chimeric mouse-human IgG (K) or IgG (K) anti-phos(cid:173)
`phocholine antibodies. The transformed cell lines remained tu(cid:173)
`morigenic in mice and the chimeric molecules were present in
`the ascitic fluids and sera of tumor-bearing mice.
`
`The capability to transfer immunoglobulin genes into lym(cid:173)
`phoid cells where they produce protein in quantities suffi(cid:173)
`cient for structural studies Cl-3) provides us with the oppor(cid:173)
`tunity to generate and characterize novel immunoglobulin
`molecules. Cloned variable (V) region genes from mouse or
`rat hybridoma cell lines can be ligated to human constant (C)
`region genes and we would expect that these chimeric genes
`can be transfected into mouse myeloma cells, which then
`will produce novel human antibody molecules. We would
`thus produce antibodies that are largely human but which
`have antigen-binding specificities generated in mice. The ad(cid:173)
`ditional potential for in vitro manipulation and alteration of
`both the antigen-binding site and the structures correlated
`with biological effector functions of these antibody mole(cid:173)
`cules using recombinant DNA techniques would introduce a
`powerful approach for further understanding antibody struc(cid:173)
`ture, function, and immunogenetics.
`As we show here, both chimeric mouse heavy chain V re(cid:173)
`gion exon (VH)-human heavy chain C region genes and chi(cid:173)
`meric mouse light chain V region cxon (VK)-human K light
`chain gene constructs are expressed when transfected into
`mouse myeloma cell lines. When both chimeric heavy and
`light chain genes are transfected into the same myeloma cell,
`an intact tetrameric (H 2L 2) chimeric antibody is produced.
`In this study we used V 11 and VK exons from the mouse phos(cid:173)
`phocholine (PCho)-binding antibody-producing S107 myelo(cid:173)
`ma cell line (4, 5). Chimeric mouse-human anti-PCho anti(cid:173)
`bodies were produced in culture by appropriate transfectcd
`cell lines or by "transfcctomas" obtained when such cell
`lines arc injected into mice.
`
`MATERIALS AND METHODS
`Chimeric Genes. The cloned S107 VH and S107 VK genes
`were gifts from Matthew Scharff (Albert Einstein College of
`Medicine, Bronx, NY). The S107 VH gene was spliced to
`human IgGl and IgG2 C region genes by using Sal I linkers
`as shown in Fig. IA. Both constructs were inserted into the
`vector pSV2~H-gpt (1, 6). The S107 VK gene was spliced to
`the human K gene at a unique f/indlll site located in the large
`intron between the K light chain joining and C (JK and CK)
`region exons as shown in Fig. lB. This chimeric light chain
`gene construct was inserted into both PSV2~H-gpt and
`pSV2-neo plasmid vectors (7).
`Transfection. Protoplast fusion and calcium phosphate
`precipitation techniques (1, 8, 9) were used to transfect these
`chimeric immunoglobulin genes into the J558L myeloma cell
`line (a A. light chain-producing mouse myeloma cell line) and
`an immunoglobulin nonproducing derivative of the P3 my(cid:173)
`eloma cell line. Mycophenolic acid (GIBCO) was used for
`selection of cells transfected with pSV2~-gpt vectors as de(cid:173)
`scribed (1, 3). G418 (GIBCO) at 1.0 mg/ml was used for se(cid:173)
`lection of cells transfected ,with pSV2-neo vectors (7).
`Light and heavy chain chimeric immunoglobulin genes
`were transfected sequentially by protoplast fusion using
`G418 selection for the chimeric light chain gene vector and
`mycophenolic acid for the chimeric heavy chain gene ve'ctor.
`The protoplast fusion transfection procedure used was as de(cid:173)
`scribed (1).
`Transfection using the calcium phosphate precipitation
`procedure was done by transfecting a mixture of 40 11-g each
`of the chimeric light and chimeric heavy chain pSV2~H-gpt
`vectors into 5 x 106 cells. Mycophenolic acid was used to
`select for transformed cell lines as described (1).
`Antigen Binding. PCho binding of antibody secreted into
`the culture supernates of transfected cell lines w~s analyze?
`by using a solid-phase radioimmunoassay d~scnb~d previ(cid:173)
`ously (10). PCho-keyhole limpet hemocyamn ~ntlg~n w~s
`bound to 96-well polyvinyl plates; bindin~ of chimenc an~I­
`PCho antibodies was detected by using LSI-labeled protem
`A or 1251-labeled-anti-human lgG. PCho-binding antibodies
`in biosynthetically labeled culture supernates and c~ll .lY(cid:173)
`sates of transfected cell lines also were analyzed by bmdmg
`the biosynthetically labeled antibody to PCho-coupled ~eph­
`arose 4B (Pharmacia) and then eluting the bound antibody
`with PCho-haptcn. The bound and eluted antibody was e~­
`amined by NaDodS04/polyacrylamide gel electrophoresis
`(NaDodS04/PAGE). Biosynthetic-labeling procedures were
`as described (11).
`Idiotope Analysis. Three hybridoma anti-idiotope antibod(cid:173)
`ies, also kindly provided by Matthew Scharff and Angela
`
`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 lH U.S.C. §1734 solely to indicate this fact.
`
`Abbreviations: V, variable; C, constant; J, joining; H• heavy chain;
`L• light chain; PCho, phosphocholine; kb,. kilobase(s); NEPHGE,
`nonequilibrium pH gradient electrophoresiS.
`
`6851
`This material was copied
`
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`

`

`6852
`
`Immunology : Morri son e t a/.
`
`Proc. N at/. A cad. Sci. USA 8 1 (1 984)
`
`A
`
`1am. HI
`
`(~I) Sal I (Hind Ill)
`
`lll.m HI
`
`pSV2AH-S 107 HuG 1 t--1:H::ID---·-~~.--tH~B"i~---·
`
`Mouse VDJ
`
`Human constant region
`
`pSV2AH-S107 HuG2
`
`pBR322 ori
`
`SV40 ori
`
`B
`
`.e_m HI
`t
`t
`t Mouse VJ
`Human kappa
`pSV2AH-S107Huk ~----------------~~~--------~
`
`Hind Ill
`
`lll.m HI
`
`pBR322 ori
`
`SV40 ori
`
`Eco-gpt or
`Eco-neo
`
`FIG . 1. Schematic diagram s (not drawn to scale) of the chimeric mo use - human heavy c hain ge ne vector (A) and th e c himeri c light c ha in
`gene vectors (B). DNA fragme nt sizes are as follow s : Hindiii- BamHI human lgG l (HuG l ) o r IgG2 (HuG2) heav y c ha in ge ne , 7 kil obase s (kb):
`mou se 5107 VH gene , 4. 5 kb ; Hindlll- BamHI huma n C, ge ne (HuK), 10 kb ; and mo use 5 107 V, ge ne , 3. 5 kb . The Hindi II sit e s in the hum an
`JgG1 and IgG2 heavy chain genes were ligated to the Sa c I site of the mouse S l07 VH ge ne with S ail linke rs.
`
`Guisti (Albert Einstein College of Medicine), were usea to
`analyze the V H-V L (L is light chain) domain structure of the
`chimeric human anti-PCho antibodie s . These antibodie s
`(TC102.1.2, T139.2 , and T156.1.1), recognizing three inde(cid:173)
`pendent idiotopes (12) , were used to immunoprecipitate bio(cid:173)
`synthetically labeled material eluted with PCho from the
`PCho-Sepharose 48 matrix. Immunoprecipitates were ana(cid:173)
`lyzed by NaDodS04/ PAGE.
`Immunoglobulin Chain Composition. Monoclonal anti-hu (cid:173)
`man lgG and anti-human K antibodies (8ecton-Dickinson)
`were used to immunoprecipitate biosynth etically labeled chi(cid:173)
`meric human anti-PCho antibodie s for a nalyses using two(cid:173)
`dimensional nonequilibrium pH gradient PAGE (NEPHGE)
`(11 , 13). PCho-coupled Sepharose 48 also was used for af(cid:173)
`finity purification .
`Imrrlunoglobulin Heavy Chain Glycosylation. Tunica mycin
`(Calbiochem-8ehring) was used to inhibit asparagine-linked
`glycosylation of biosynth e tic ally labe led a ntibody from
`mou se cell lines producing mou se- human chimeric immuno(cid:173)
`globulins (11). PCho-binding antibody from tunicamycin (cid:173)
`treated cells was analyzed by NaDodS04/ PAGE . Proce-
`
`dures for tuni camyc in treatm e nt we re as desc ribed (11) .
`Chimeric Mouse-Human Antibody Production in Mice.
`Tra nsfe cted J 558 L ce lls produ c ing c hime ri c mo use-huma n
`antibody we re injected intra pe ritoneall y into 8ALB / c mi ce
`(106 ce ll s pe r mo use). Sera a nd asc iti c fluid s fro m tum or(cid:173)
`bearing mi ce were a nalyzed for huma n a nti -P C ho a ntibod y
`by a solid -phase radioimmunoa ssay (10) and by immun oe lec(cid:173)
`trophoresis using a po lyc lo nal a nti -huma n lgG anti se rum.
`
`RESULTS
`We obta ined ex press io n of c hime ri c mo use V regio n- human
`C region genes in transfec ted J558 L and the immunoglo bulin
`nonproduc ing P3 mye lo ma cell lin es . Wh en both light chain
`and heavy c ha in chim eric ge ne s (see F ig. 1) we re ex pressed
`in the same ce ll , tetra me ri c ( H 2L 2) a ntigen-binding a nti bod(cid:173)
`ies were o bt ained . Bio sy nth eti ca ll y la be led a ntibod y mole(cid:173)
`cules sec reted by J 558 L ce ll s ex pressing c him eric ge nes
`were bound a nd ha pte n-e luted fro m P C ho-Se ph arose 48 .
`Autoradi ogram s of two -dim e ns ion a l N E PHG E a na lyses
`(Fig. 2) show immun oglobulin polype ptide c hains of the ex-
`
`PFIZER EX. 1031
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`

`

`Immunology: Morrison eta/.
`
`Proc. Nat/. Acad. Sci. USA 81 (1984)
`
`6853
`
`pected c harge a nd relative molecu la r mass. Ide ntical two(cid:173)
`dimen sio nal ge l a nalysis result s were obtained with immuno(cid:173)
`precipitates by using monoclonal a nti-human K and lgG anti(cid:173)
`bodies . Simila r result s were ob tain ed when the chimeric
`lgG2 ( K) a ntibod ies produced in the transfected no nproduc(cid:173)
`in g P3 ce ll line were a nalyzed. Si nce the recipie nt P3 ce ll line
`does not produce endoge nous immunoglobu lin polypeptide
`chains, o nl y th e chimeric mou se-hu ma n heavy and light
`c hain s we re see n (data not show n). PCho binding by the chi(cid:173)
`me ri c antibody produced in the J558L cell line required the
`spec ific associati o n of both th e VH a nd VL domains of the
`S l07 myeloma protein co nnected to huma n C region poly(cid:173)
`pe pt id es . Antibody secre ted by tra nsfected J558L cell s ex(cid:173)
`press ing o nl y the c himeric heavy chain and th e endoge nou s
`J558L light chain did no t bind PCho (data not show n).
`Furthe r verifi catio n of th e app ropriate polypeptide folding
`of the mou se VH a nd V L domain s attached to human C re(cid:173)
`gio n polype ptide cha ins was done by a nalyses detecting the
`prese nce of idiotopes known to occur o n the parental Sl07
`PCI10-binding a ntibody molecule . Three monoclonal anti(cid:173)
`idiotope antibod ies- two recognizing idiotopes requiring the
`associati on of light and heavy V regio n domains (TC 102.1.2
`and Tl39 .2) a nd th e third (Tl56.1.1) recogni zing an epitope
`prese nt o n the heavy chain V regio n domain (12)-were
`found to react with the mouse-human chimeric anti-PCho
`antibodies (see Fig. 3) . This is good ev ide nce that the mou se
`Sl07 ant igen-b inding doma ins have folded into their intend(cid:173)
`ed stru ctures.
`The co nformatio n of lgG heavy chain C regions depends
`
`Basic
`
`A
`
`HuG2---
`
`B
`
`HuG1-..
`
`•
`
`HuK
`
`Acidic
`
`-67
`
`- 45
`
`- 30
`
`•
`
`-67
`
`- 45
`
`-30
`
`t
`
`J558.A.
`
`a
`
`b
`
`c
`
`d
`
`H2L2--
`
`FIG. 3. Autoradiograph of nonreduccd NaDodS04/ PAGE anal(cid:173)
`yses of PCho-binding material from a chimeric lgG2 (K) antibody(cid:173)
`producing J558 L cell line immunoprccipitated with Staphylococcus
`aureus protein A (lane a), monoclonal anti-human K antibody (lane
`b), and anti-idiotopc antibodies TC102.1.2 and Tl56 .1.1 (lanes c and
`d).
`
`on th e prese nce of the asparagine-linked carbohydrate
`moeity in the CH 2 domain of the molecule (4). The loss of
`this carboh yd rate chain affects profoundly the overall do(cid:173)
`main structure of this part of the immunoglobulin molecule .
`Concomitant with thi s structural change , the catabolism rate
`of the mol ecule is increased and biological effector function s
`suc h as complement-fixation are lost (14) . Glycosylation of
`the mouse-human chimeric antibodies in mou se myeloma
`cells was deduced by determining the molecular masses of
`antibodies sy nthe sized in the presence and absence of tuni(cid:173)
`camycin, a n antibiotic inhibitor of asparagine-linked glyco(cid:173)
`sy lation . Fig. 4 is a n autoradiogram of NaDodS04/ PAGE
`analysis of glycosylated and nonglycosy lated chimeric heavy
`and light chains produced in transfected mou se myeloma
`cells. Though we cannot be certain that in the absence of
`tunicamycin the appropriate asparagine residue in the CHz
`domain is glycosy lated , clearly, the lower molecular mass of
`the heavy chain sy nthe sized in the presence of tunicamycin
`is as expected if a single N-linked carbohydrate were absent
`
`a
`
`b
`
`-...- ._
`
`-67
`
`-45
`
`-30
`
`-20
`
`F IG . 2. Autorad iographs of two-dimensio nal NEP HG E gels of
`mouse-human c himeri c lgG2 (K) a nti-PC ant ibod y (A) and mouse(cid:173)
`human chimeric lgG 1 (K) a nti- PC a nti body (8). Anti bodies syn the(cid:173)
`~ i ze d by tr<tn'ifec tcd J558L ce ll s we re a ffinity pu rified wi th PC ho(cid:173)
`Sc pharo'ie 41:l . The e lect ropho retic mob ilit y of the J558L i-- c hain had
`bee n determined previous ly (1). Si ze marke rs arc shown in kDa.
`
`F JG . 4. Autoradiograph of reduced NaDodSO./PAGE analyses
`of ch imeric lgG2 (K) antibod y synthesized in the presence (lane a)
`and absence (lane b) of tunicamycin. Size markers are shown in
`kDa.
`
`PFIZER EX. 1031
`Page 9
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`

`

`6854
`
`Immunology: Morri son et a!.
`
`Proc. Na t!. Acod. Sci. USA 8 / (1984)
`
`anti-human lgG
`.
`
`~
`
`Immunoelectrophoresis of ascitic fluid from a BALB /c
`FIG. 5.
`mouse bearing a J558L transfectom a producing chimeric lgG2 (K)
`anti-PCho antibodies developed with a polyclonal anti-human IgG
`antiserum.
`
`from the polypeptide chain. Note that the light chain band is
`not affect ed by tunica mycin treatment. From these da ta, we
`conclude