DEPAR'I'~~lEN'I` OF HEALTH. EDI!CATlON, AND WELFARE _-.._-_-__-.-- ------ _..-..__. _,___--. -.----.--- --e-p PUBLIC HhAL'I'H SERVICE . B!L:THESL)A I.+, MD. December 8, 1960 Dr. Joshua Lederberg Department of Genetics Stanford University School of Medicine Stanford, California In reply refer to: E-3978 Dear Doctor Lederberg: .The enclosed application for a grant in aid of research will be considered soon by the Allergy and Immunology Study Section and the appropriate National Advisory Council. Before they do so the members of our reviewing groups would like to have the benefit of your ,advice, so that they may be more certain about the need for the proposed work, its feasibility, its promise and significance (among other things), and about the com- petence and past accomplishments of those who are to do the work, If you are willing to offer an opinion or comments about the pro- posal, please do so simply in a letter addressed to me, In your response, please identify the project. For your convenience a re- turn envelope which requLres no postage is enclosed. A response by December 21, 1960 would be appreciated. The application is a privileged communication, intended to be seen by reviewers only. Please be assured that any comments you offer will be similarly regarded, and that they will be made known only to the members of our advisory groups. The applicant's papers may be returned when you finish with them, or they may be destroyed, but please do not leave them where others may see them. May I thank you in advance, on behalf of our advisers? Sincerely yours, Frederick W. Appel, Ph.D. Executive Secretary Allergy and Immunology Study Section Division of Research Grants Enc. National Cancer htitutc National Hcut htitute National Inutitute uf Allergy and lnfcctiow Diiura National htitute of Arthritir md Metabolic w National lrutitutc of Dental Rcwcb National Imtitutc of Mend Heahh National Institute of Neurological Diana and Blindness The Clinical Cents Division uf Biologiu Standards Dir&m of Busioa Gpcnthu Divbion of Gcncral Medical Sricoca Diviion of Kaar& Grant, Dir&m of RCJCU& SchS R'cd date lf)-26- I --T.. ..-.L --- <. HEALTH, EDUCATION, AND WELFARE PUBLIC HEALTH SERVICE NATIONAL INSTITUTES OF HEALTH Action APPLICATION FOR RESEARCH GRANT (A PRIVILEGED COMMUNICATION) NO. ------I i-AsL~)~--- Formerly I L.--.----_------ pm: YES) Application is hereby made for a grant in the amount and for the period stated, for the purpose of con- ducting research as described herein, in accord with the Agreement signed below. A. AMOUNT REQUESTED: $--lbmn (Same as total of itemized budget, page 2, item A8,) B. PERIOD DATES- (Normally 12 months. See instructions.) ci. TITLE OF RESEARCH PROPOSAL (Do not exceed 53 typewriter spaces) .c Tnhcritzbility of Antibody Production by Cultured ;?mnali::n Cells ii E. PRINCIPAL INVESTIGATOR: Name~&~~ ?. Telephone No.~J~~~ Extensiont- Title Department or Servic Mailing address of Research oftice- `l-,l<. if-J-&f&Jj?&@r,c) `P $0, f-J&j&q..& Institution =* iT~~i~Ti ty Of Cf9lOt , tlfi PA1 C. 1 * 9 I r .ni-#..7 7,; Cl Major `"`2 , Sub Divisiow7 ": F. CO-PRINCIPAL INVESTIGATOR, if any. (Name and title only) G. David W. T&m@, 1I.D.. INSTITUTION SPONSORING REQUEST H. NAME, TITLE, AND ADDRESS OF FINANCIAL OFFICER: Name 1Tn-l `.ZCiLj k b <- `.r 0-y r* do `:I?;. ru 90 :4r. J, W. Johnston Maa ad&ess !I?{?!-, '.Ts+, 'Tjnl;h I\vw Finance Officer P'P 3CJz w-n Name 6 title of official authorized to sign application on be- i'niv. of CoILo, :kdiczl Center . . 1 half of institution Robert J. Glaser :'.D &@. w&,&-$J~ m V-jce-Prf:sj&:clt for Ijc&jeczl jf&ai-s's:lc; Manner in which check(s) should be drawn: --- Eiq~~cq $$Zficer, The Univ. 0: Cola. I. j$i&&i$pf SwqT-~f-~Ied~~~ Gi-l "r t IS un ers oo an agreed by the undersigned that`jany grant received as a result of this application is subject to the following terms: (1) Funds granted as a result of this request are' to be expended for research or related purposes as governed by Public Health Service and grantee institution policies; (2) the grant may be revoked in whole or in part at any time by the Surgeon General of the Public Health Service, provided that a revocation shall not include any amount obligated previous to the effective date of the revocation if such obligations were made solely for the purposes of research; (3) all reports of original investigations supported by the grant shall acknowledge such support; (4) if any invention arises or is developed in the course of the work aided by the grant, the undersigned will either (a) refer to the Surgeon General for determination, or (b) determine in accord- ance with grantee institution's own policies as formally stipulated in a separate supplementary agree- -----F- ment entered into between the Surgeon General and the grantee institution, whether patent protectron - on such invention shall be sought and how the rights in the invention, including rights under any patent issued thereon, shall be disposed of and administered, in order to protect the public interest. J. . PERSONAL SIGNATURES (in ink) (1) Principal Investigator____ (Same as shown in "E" ab%e) (date) - (2) Authorized official of applicant institution -.___-- (Same as shown in "G" above) Matl completed application to: Division of Research Grants National Institutes of Hew Bethesda 14. Md. (date) PHS 393 Rev. 7-59 Page 1 Form Approved Budget Bureau `No. 68.R.249.9 A. BUDGET REQUEST (for the period shown on page 1) .d (1) 1. PERSONNEL % time List all positions, including Principal and Co-investigator. Amounts requested must not exceed on this proportion of total salary computed from % of time spent. projeci ,Dar&ba I .: , `I < -.,. -- ___ 2. PERMANENT EQUIPMENT, itemize (see instructions) Kltrathin Secti0nin.F: Eicrotone and related item3 -- 014 $ spr,?re Tnver.ted Iiicrcscone CO9 Tnal&&r ave lh1hl0 3; st,i 17 er TX sec+.i ::r ?ilicrc,ccoF,e 3. CONSUMABLE SUPPLIES, itemize (see instru&onsj 5. OTHER EXPENSE, itemize (see instructions) - -I.- 6. TOTAL DIRECT COST FLEQUIFlEMENTS 7. INDIRECT COST ALLOWANCE (The administrative official signing thii application may request an amount for indirect costs. Review detailed instructions) CRound to low dollar) 8. TOTAL BUDGET (Same as amount shown in item A, page 1) L (3) Requested from PHS (omit cents) s $00 - 9 2,l.lrl B. ESTIMATE OF SUPPORT REQUESTED FOR THE YEAR FOLLOWING THE BUDGET PERIOD ITEMIZED ABOVE. Applicants for l-year grants should type the word "None" in space for TOTAL BUDGET shown below. C. ADDITIONAL YEARS OF SUPPORT, beyond the 2 years covered above, if requested. Please show the TOTAL AMOUNTS required for each such additional year, including indirect cost allowance. 3.$ s#.ooo 4. $ 5. $ 6. $ $6 7.s c.. . !: > PHS 399 , Rev. 7-59 Page 2 . EJ978 . - - - RESEKRCH SUPPORT List all other research support of the Principal Investigator, including that from own institution, and appli- cations that are pending. Use continuation page if necessary. See instructions. A. PUBLIC HEALTH SFRVICE SUPPORT: GRANT NUMBER -- TITLE OF PROJECT ___- --- approved: Entibody Production Sy Xzi2wlian Cell Clones as UsPIlS Special Postdoctoral Fello-cJ (1) Active (2) Applications submitted, awaiting decision: None B. ALL OTHER RESEARCH SUPPORT: SOURCE TITLE OF PROJECT :l) Active or appn 2) Applications Xi: Hone - Iunitted, awaiting dedsion: AMOUNT $3100 AMOUNT - -- - - - -- - - PERIOD OF SUPPORT Se-,terzber 1, 19 to Auzust 31, 1361 PERIOD OF SUPPORT PHS 399 Rev. 7-59 BIOGRAPHICAL SKETCHES E3978 - - - c Provide brief sketches for professional personnel already selected who are to be actively engaged in this project. The following format should be used for each person, with Co-investigator (if any) immediately following Principal Investigator, then other professional personnel, lettered consecutively. A. Principal Investigator: -. (Name and title) 1. Date of biih:~+' -.L Place of birth: Caldr~e-lYl . Xev Jcr~ev ---I Present nationality: U.Ki.t~~~ States -.d I&&~: Female 0. 2. Educational experience: a. Degrees conferred (Begin with baccalaureate degree. Identify honorary degrees under field.): JNSTITWTION CoNFEaRING FIELD(S) YIXR :!?.shington iJnivorsi.t,v St. Louis Jiedicine 1?57 I I I f I- I I b. Other research trcrtnmg and experience, especially that estabbshing research qualifications fn area covered by this application: The period spent at the Xational Institutes of Xealth was as a i7-esearch Associate . This program provided formal courses in basic sciences. B. David T-7. +Jk?gr:~e, Z.3,, Professor of Medicine and Mcrobiolory (Name and title) . 1. D&e of birth: Sept. 15. 1919 : Place of birth: H1.r2rxiu. Korea . Present nationality:--L!Ilit~Cd SfXtes 2. Educational experience: _ ; Malem: Female 0. a. Degrees conferred (Begin with baccalaureate degree. Identify honorary degrees under field.): INSTITUTION CONFERRING E?ELDo YRAR 10&j+ 1ph-L `- b. Other research training and experience, especially that establishing research qualiiications In area covered by thfs application: PHS 399 Rev. 7-59 Page 4 RESEARCH P&AN AND SUPPORTiNG DATA Details of the proposed plan and other necessary data should be typed (single spaced) in accord with the outline below, which is suggestive only. See instructions. Please continue numbering pages in sequence for entire application. Additional continuation sheets, if needed, may be requested from the Division of Research Grants. 1. RESEARCH PLAN A, Specific Kims - Provide a concise statement of the aims of the work immediately proposed, and relate these to your long-term goal. B. Method of Procedure - Give details of your research plan, including how results will be ana- lyzed. For each specific aim mentioned in "A" show how your plan is expected to fulfill the aim. C. Significance of this Research - Explain why the results of the proposed work may be important. D. Facilities Available - Describe the general facilities at your disposal. List the major items of permanent equipment. 2. SUPPORTING DATA A. Previous Work Done on this Project - Describe briefly any work you have done to date that is particularly pertinent, B. Results Obtained by Others - Summarize important results to date obtained by others on this problem, citing publications. Select no more than five. C. Personal Publications - Cite your most important publications on this or closely related work List no more than five. D. Justification of Budget - Defend itemized budget for the initial period (A, page 2) where you feel it necessary, and delineate reasoning basic to budget estimates for continuation years. 1. XESEKRCH PLAN A. Specific Aims - Until recently, the investi.&ion of the phenomenon of antibody synthesis had been indirect, b aaed on levels of circulating antibodTy, correlated 16th Cj~tOtx-n~phc~OgiC. changes in specific cell typ?S. To explain the mechanism of antibody synthesis three theories have been formulated. These theories differ in: 1) the role ascribed to antlcen in the variation of the antibc+y producing potential of cells and 2) the inheritability of this potential. Thus, antigen is thou&t to induce changes in antibody forming potential T:hi.ch depend on persistence of antigen and are therefore not inheritable (i!a?;roktz md ? a~l5.n~) ; antigen is thought to produce inheritable changes in antibcdy producing cells (Zurnet and Fenner, Sch-dcet. and Ozn, Sailard); or inheritable changes are thought to occw in antibody producing cells in the absence of antigen (Talmace, I3urnet and Lederberg). Since the r.iork of Xossal and Lederberg, and Cohn, Lennox and kttardi; the investigation of the mechanism of antibody synthesis has moved to a direct cellular level through the use of hikhly sensitive techniques of antibody detection and the isolation of single cells. Thus far, these t:~o,groups have found that a single differentiated cell synthesizes onl-J one (Nossal) or in some cases two (Cohn) antibodies, Althoulj;h both &I-oups qreed .that cell s varj.ed in their arrtibody Froducin; gctential, tier*e is no corlc2.lsive evi:.ience that zQtig;en playa a role in induc:iha this variat:ion, It is now povslble to isolate and cultivate sFnele mammalian cells in a plating techn3que Simikar in ?rincZ?l.e to bacterial cloniq. The rar,?:ltant clo!las I'epr~s ent a genetically identical strain of cells. Rn additio~~al untried rxq:3r%wntsl approach is the in v-iv0 cul.tilr3 tic:, of a zzqgtically uniform ml1 pq3ulstion rJith inkqcritonca 1 inillipore filkr chamber-S. The pu~'?osr? of this PBS398 Rev.743 Page 5 project is to investigate the inheritability of antibotljT ~ro&x5:~~ potenti& in cultured ma~wxilian cells. A clone of cells may be investigated in a similar manner and a deterCnation of the antiboc>J producing potential of'th' *s goilp of cells with identical inheritance made. If stimulation of cells with antigen in -Ctro does not lead to measurable -II_ antibody Troduction (the usual exoerienco of other investigators) the response of Such cells to antigen kfter transfer to a millipore chamber or an isologous tolerant host will be tested. The experirxmts outl-j.ned a'oo~2 requ;.re sensitive techniques for antibody : de-tection. Initially the labeled altigon binding techniques of Fa.rr and a modification recently developed in this l.r?boratoPy will be tried. T'nese can detect as little as 0.01 ug antibody M per ml, In order to test the antibody produced by s~nall numbers of cells, a still more sensitive technique, .that of phage neutralization, till be a?olied, As an important corollary, the persis"- +&nce of the antigen ma-y be investigated utilizing a hi,$nly sensitive technique involving electron microscopy. The protein ferritin contains up to 23;s of its weight as'iron. This molecule gives a characteristic pattern in the electron microscope and a si&le mol.?cule mav thus Be observed. .m' one intracallCk3.r presence may be seen in of the ferritin -antigen (or its absence) antibody producing cells. Significaxe of this 'i),ese,irch - particular the The nature of the antibody response and in Inheritability of the phenoxenon has -important ix?lications in our :Jmderstanding of cell genetics (somatic cell differentiation) aTlil the mechanisms of protei.n synthesis, Insight may be gained into a host of diseases presumed to be caused by an altered or misdirected antibody response. Facilities Available - The Department of Allergy and Innn~u~ology has ample fach=T to successfully carry out the outlined project. The Department of Anatomy has promised the use of a Xorelco electron'microscope. Ho:;over, vrenarative facilities for ultrat?nin section- 3 will have to be provided by us. There Is hn aAm. room and caretaker at our disposal. has been assured.'~ For cell culture, a complete laborato-ry Pa--398 REV. 7.56 PC&? -6 3 L. Su!>porting Data . A, pravicus '.Joo,nk Douc on this 3roe - During my two j/ears as a resetich associateY3Xx2 :!ai;ional Insti$utes of Health in Dethesda, I worked under the direct supervision of Dr. Harry Za$e and oollaborated as jwlior in-zestigator on tm rjf his project s in mat:r~aliarr cell physiology. In addition, I designed an operational, si,qplified rnajmalial cell chemstat md carried OU-b -experhents which described the cha~ractcristics of cell growtln under these conditions. c Se0 Publications, below), Vorking under the direction of Dr. David B, Scott and 3r. Karie U. Eylen at the ih-tional Institute of Dental Research during this nme two -year period, I completed a study of the degenerative changes occuring in' cultured nmmnali~~ cells under conditions of amino acid depriva-tion as seen with the electron microscope, (see publications, below), As a.medical student, I completed a thesis under the direction of Dr. Kelvin Cohn entitled, Vhe Production of Isotopically Labeled Antibodies to 1nsuX.n~~. B, Results Obtain The important pork in this field which has direct implications to the project has been- done by: 1. Xossal, who demonstrated that single cells synthesize one antibody, Btit. J. Exp. Tath., @:118, 1757. 2. Calm, Lennox and Attardi, w!m demnstrated that single cells synthesize one or in some cases two antibodies, Bact. Rev., 23:213, 195: . 20th of these group" J used isolated cells in nongrowing conditions from previously hyperimmunized a.rG.nm1.s. The inheritability of antibody synthesis was not investigated. 3. Algire et al (Annals N,Y, Acad. Sci. 6h:lOO7, 1757) demonstrated that intraperitoneal millipore filter chambers supyorted the growth of cells. 4. T. Kahinodan shoved that cells in these chambers produce easily detectable amounts of antibod-. (Personal communication). 5. Smith, I.?etzger et al (Proc, Sot. Zxp. Biol. 8~ Ked., 1014:336, 1760) have demcnstrated that a single ferritin molecule may be seen in the electron microscope. C* Personal Tublications - 1. A SLmplified Mamalian Cell Chemostat: Characteristics of Cell Gro;dh in Continuous Culture, E, P. Cohen and H. Eagle. Federation Proceedings, 17 :1,~6, 1960 (Part II). S~ubmitted, Journal of Experimental Xedicine, October, 1760. 2, lZ.crostruct:.~al Changes Induced in F%ammalian Cell Cultures by Omission and Replacement of a Single Essential Amino Acid, E. P. Cohen, D, 9, Scott, M.U. Xylen, %xperiaental Cell Research, In Press. * . ,,- i : PHS--398 ... REV. 724