AD-A267 126

CONTRACT NO: DAMD17-91-C-1091

TITLE: THE USE OF CD4/GP120 INTERACTIONS IN THE DEVELOPMENT OFANTI-HIV DRUGS AND VACCINES

PRINCIPAL INVESTIGATOR: Jonathan Gershoni

CONTRACTING ORGANIZATION: Department of Cell Researchand Immunology

Tel-Aviv UniversityRamat Aviv, 69978Tel-Aviv, Israel

DTICREPORT DATE: September 30, 1992 SLECTE.

TYPE OF REPORT: Annual Report A

PREPARED FOR: U.S. Army Medical Research andDevelopment Command, Fort DetrickFrederick, Maryland 21702-5012

DISTRIBUTION STATEMENT: Approved for public release;distribution unlimited

The findings in this report are not to be construed as anofficial Department of the Army position unless so designated byother authorized documents.

• • 93-16674

, , = a • I |i l d ill

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Public reporting burden for this collection of information is estimatec to average I hour per rosiporse, including tire timte for reviewing instructions. searchirng ex-st.h data soiurces.gathering and mantainitilng t he data needed. and comrpleting ano reviewing the collection of information Send comments regarding this burden estimate or anyp other aspect of -hiiscollection of information, including suggestions for reducing this burden to uivashinglon Headcl-arters Sersces, Directorate for information Operations arnd Reports. 1215 jeffersonDairis Highwiay. Suite 1204, Arlington, VA 22202-4302. and to the Office of Management and Budget. Paperwviork Reduction Projeclt (0704-0188), Washington, DC 20503

1. AGENCY USE ONLY (Leave blank) 12. REPORT DATE 13. REPORT TYPE AND DATES COVERED

T130 Sept 1993 Annual Report (9/1/91 - 8/30/92i4. TITLE AND SUBTITLE S. FUNDING NUMBERSThe Use Of CD4/GP120 Interactions in the Contract No.Development of Anti-HIV Drugs and Vaccines DAMD17-91-C-1091

6. AUTHOR(S) 6278 7A30162787A873.HC.009

Jonathan Gershoni WUDA335935

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATIONDepartment of Cell Research and Immunology REPORT NUMBERTel-Aviv UniversityRamat Aviv, 69978Tel-Aviv, Israel

9. SPONSORING/ MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING /MONITORINGAGENCY REPORT NUMBER

U.S. Army Medical Research & Development CommandFort DetrickFrederick, Maryland 21702-5012

11. SUPPLEMENTARY NOTES

12a. DISTRIBUTION /AVAILABILITY STATEMENT 1 2b. DISTRIBUTION CODE

Approved for public release; distribution unlimi tf d

13. ABSTRACT (Maximuim200 words)

The HIV envelope protein, gpl20 binds to the CD4 cell surfaceprotein and thereby the virus enters T4 helper lymphocytes. This"Orocess or- virus entry might be associat:ed wit~h the presentation ofui que epitoopes o-f the gpl2O that become revealed only as a resul:orvrsbinding zoistargec cell. The purpose of hsr eac

-as been to idniyc'tceioe hat become accessible due tonduced conformational changes that result: from t.hat complexformation of gp12O with CD4. Thus CD4/gp-12O complexes have beenused to immunize mice and monoclonal antibodies have been generated.At leas, 7 antibodies have been ident ie ashvn pr ereaffinity for the above complex. These antibodies are now beingcharacterized. They represent, 3 distinct epitopes as has beendetermined by competitive E-LIsA assays. Moreover, using a syncytiuruassay they have been evaluated for their neutralizing activity.

14. SUBJECT TERMS 15. NUMBER OF PAGESAIDS, HIVy, Epitopes, Biotechnology, Vaccines, Lab________Animals, CD4, GP 120 Epitopes, Mice, RA 1 16, PRICE CODE

17. SECURITY CLASSIFICATION 18 SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACTOF REPORT OF THIS PAGE OF ABSTRACT

[Unclassified IUnclassified I Unclassified UnlimitedNSN 7540-01 -280-SSOO Sldflda'd Ormn .98 iRev ' 2

FOREWORD

Opinions, interpretations, conclusions and recommendations are- those of the author and are not necessarily encorsed by the U.S.

(Nk Where copyrighted material is quoted, permission has beenobtained to use such material.

(6) Where material from documents designated for limiteddistribution is cuoted, permission has been obtained to use thematerial.

•A Citations of commercial organizations and trade names in:his reporz do not constitute an official Deoartment of the Ary "endorsement or approval of the products or services of theseorganizations.

(JA !n conducting research using animals, the investigator(s)adhered to the "Guide for the Care and Use of Laboratory Animals,"prepared by the Committee on Care and Use of Laboratory Animals ofthe :nstitute of Laboratory Animal Resources, National ResearchCouncil (NIK Publication No. 86-23, Revised 1985).

(v 'For the protection of human subjects, the investigator(s)have adhered to policies of applicable Federal Law 45 CFR 46.

( n conducting research utilizing recombinant DNA:ecnnology,the invescigacor(s) adhered to currenc guidelinespromulgaced by the National :nstitutes of Health.

ae................

SA•~ -i,ki .,( i or

DiA Specald

TABLE OF CONTENTS

INTRODUCTION ............................................... 1

BODY ....................................................... 2

MATERIALS ............................................. 2

METHODS ............................................... 2

V8 PROTEASE DIGESTS ............................. 2

PROTEIN BLOT ANALYSES ........................... 2

ELISA ASSAYS .................................... 2

PRODUCTION OF MONOCLONAL ANTIBODIES .............. 2

MAB PURIFICATION ................................ 3

BIOTINYLATIý-N OF IMABS ........................... 3

SYNCYTIUM ASSAY ................................. 3

RESULTS ............................................... 3

C:HARACTERIZATION OF V8 PROTEOLYZED GP120 ........ 3

PRODUCTION OF MABS AGAINST V8 FRAGMENTS OF

GP1 20 ................................. .......... 4

PRODUCTION OF CD4/GP!20 COMPLEXES ............... 5

PRODUCTION OF ANTI-COMPLEX MABS ................. 6

NEUTRALIZATION POTENTIAL OF THE MABS ............. 8

CONCLUS IONS ................................................ 3

REFERENCESS ................................................. 9

APPEN-DIX ................................................... 11

INTRODUCTION:

The development of an effective vaccine for the preventionof AIDS is a goal that needs no special justification. Anumber of approaches have been attempted over the lastdecade since the AIDS epidemic has been recognized and thevirus HIV identified as the cause of this devastatingdisease. Major. efforts have focused around the viralenvelope glycoprotein gp120 which is noncovalently attachedto the particle via the membrane-associated gp4l.

The structure of gpl20 has been extensively analyzed andthe glycomoieties, the disulfide arrangements [(] andvariable versus conserved domains have been defined [2].The most prominent region of this prote-in has been thePrimary Neutralizing Domain (PND) that consists of the V3loop (residues 266-301, [3]). Antibodies to this epitopehave been found to be neutralizing in a highly strain-selective manner [4]. Nonetheless, there are regionswithin this loop that can elicit broad spectrumneutralization [5, 6]. A second target within the gp!20has been the areas involved in CD4 recognition [7].Obviously, antibodies that bind these areas are anticipatedto interfere with the virus association to its target cellsand thus prevent infection. This epitope is believed to becomplex being constructed from discontinuous segments ofthe gp!20. in any case, a desired objective would be toproduce a subunit vaccine that would provide broad spectrumprotection against HIV infection. Unfortunately, neitherthe V3 loop nor the CD4 binding domains of gpl20 appear tosatisfy this goal.

Therefore, the search for novel more effective epitopes forthe production of subunit vaccines is still extremelyimportant and relevant. The strategy proposed in thisstudy has been to identify new aspects of the gpl20molecule that could serve this purpose. The approachadopted here is based on the assumption that gp!20 canassume a variety of conformations. in doing so gpl20 wouidthus present different aspects of itself during variousphases of the disease. For example, gp120 when associatedwith gp4l may be quite distinct from soluble gpl20 or theCD4 bound protein. The notion that the envelope undergoesconformational rearrangements is supported by the fact tnatthe N terminal domain of gp4l becomes available :ormembrane fusion only after the gpl20 attaches to CD4 [8].

Recently, this line of thought has been further elaboratedupon in studies that focus on the idea that after thebinding of gp120 to CD4 there must exist a series of eventsthat are related to viral entry rather than binding per se.Celada et al [91 and Healey et al [10] both focus on thispoint and develop mAbs against CD4 that have neutralizingactivity yet are by definition not of the classical type ofLeu3A or OKT4A namely, the new antibodies do not interferewith the complex formation.

2The major effort this first year of the present study hasbeen to produce novel mAbs that are unique in that theyhave preferred affinity for the gpl20/CD4 complex.

BODY:

Materials:

Recombinant gpl20 produced in a baculovirus expressionsystem was purchased from American Bio-Technologies.Recombinant soluble CD4 (produced in CHO cells) as well asa anti-V3 loop mAbs (NEA-9305) were obtained from DuPont.The rest of the reagents used wcre standard and ofanalytical grade.

Methods:

V8 protease digests: Recombinant gpl20 (10gg/ml) wasincubated 24-48 hours in 50mM NH4HCO 3 pH7.8/0.05% SDS/l•gStaphylococcus aureus V8 protease at 370C. Addition of 9-mercaptoethanol (5%) to the reaction generated a unique setof fragments.

Protein blot analyses: Intact or proteolysed proteinfragments were separated on SDS-polyacrylamide gels aspreviously described and transferred electrophoretically inan apparatus that generates a gradient electric field tocompensate for differences in molecular mass of thepeptides being blotted [11]. Either nitrocellulose orcharged modified nylon membranes were used as the blottingmatrix. Blots were quenched either with 1% milk solutionsin 50mM Tris buffered saline pH7.5 (TBS) or 5% hemoglobinsolutions in the same. Probes such as biotinvlated lectinsor mAbs were routinely diluted in a auenching solution andtheir presence revealed by using enzyme conjugated secondreagents and standard detection reactions (for a generalreview on the above blotting procedures see [12])

ELISA assays: Costar EIA/RIA 96 well plates (N3590) werecoated with 504! of gpl20/CD4 complex (5p g gpl20:2.5ugCD4/ml ) or gp120 (54g/ml) or CD4 (5u.g/ml) in TBS overnigh:a: 40-. The plates were then washed in TBS and blockedwith 3% BSA in TBS for 1 hour at room temperature (RT).The wells were rinsed and the mAbs at various dilutions in0.3%BSA/TBS were added to them and incubated at RT for 2-3hours. The wells were then washed with TBS and the secondantibody (alkaline phosphatase conjugated goat anti-mouseantibody (Sigma, A-01621) was added (1:1000 in 0.3%BSA/TBS)and incubated for 1 hour at RT. After washing the wellsthey were reacted with p-nitrophenyl phosphate (!mg/ml in1M diethanolamine buffer pH9.8 / 0.5mM MgCl2) and read at405nm.

Production of monoclonal antibodies: The productionof mAbs was performed using standard procedures immunizing

3the mice (Balb/C) first in complete Freund's adjuvant andboosted with in-complete adjuvant. The mice were allowedto rest 3 weeks before their i.v. boost and splenectomyfollowed three days later. NS-l myeloma cells were usedfor PEG induced fusion and HAT medium for selection.Clones were obtained by limited dilutions [13].

mAb purification: mAbs were isolated from ascitesfluids taken from pristane primed mice. The standardcaprylic acid followed by ammonium sulfate precipitationprocedure was used [13].

Biotinylation of mAbs: mAbs (-lmg/ml) were dialysedovernight against 0.1M NaHCO 3 (pH was not adjusted).Biotinamidocaproate N-hydroxysuccinimide ester (BNS, [SigmaB-2643]) was added (3.Lg/L00•±g protein) and incubated for 4hours at RT, followed by overnight dialysis against TBS.

S~ncytium assay: The formation of syncytia betweenvaccinia infected BSC1 cells and CEM cells was performedessentially as was described by Ashorn et al. [14] Inprinciple, BSCI cultures were infected with recombinantvaccinia (5pfu/ml) expressing cell surface gpl20 (VPEI6provided by Dr. Bernie Moss, NIH). These were then mixedwith CEM cells in the presence of variable amounts of mAbsand incubated for different times. The degree of syncytiaformation was monitored and thus the extent ofneutralization potential for the various mAbs wasestimated.

Results:

Characterization of V8 proteolyzed gp120: Digestionof gp120 with S. aureus V8 protease produces 5 majorfragments detectable on western blots probed with ConA (seeschematic summary in Figure 1).

90Figure 1: Schematic Diagram

S- 60 of V8 Proteolyzed gpl20.

- 50 Western blots of V8 proteclyzed gpl20

were probed with anti 72 .-nAbs, ConA orsCD4. To the left is a summazry of thebands which can be detected. Depending

! - 35 on the degree of digestion and theprobed used the presence and theintensity of each band can vary.

-- 25

4The conditions used (see Methods) were found to generatereasonably reproducible patterns although in view of thefact that the simple sum of the molecular weights of thefragments yields a total mass of over 250kDa the patternreceived can only be a set of overlapping fragments ratherthan a true terminal digest. The reasons for this areunclear. More aggressive denaturation might however, leadto oer digestion and the generation of numerous unresolvedsmall fragments. Western blots of transferred V8 digestedgp120 were analyzed with: ConA, commercial monoclonal antigpl20 antibodies, and with sCD4 itself. The signal forsCD4 overlay of proteolyzed gpl20 is weak but reproducible.in view of the fact that it is believed that the criticalCD4 binding domain is in the COOH aspect of gpl20, combinedwith the response to the V3 loop antibody, we can postulatea gross map of the gpl20 with respect to some of thefragments we resolve (Figure 2).

El~~~ -- -- I I I I

3/

V3 CD4

Figure 2: Map of gpl20 V8 Fragments.

,n the above diagram the circles indicate cys residues and thedisulfide arrangements are given. The black dots indicate theapproximate positions of the glucamic acid residues (V8 cleavagesices).

in figures 1 and 2 the 90kDa fragment contains the V3 loopbut does not bind CD4. This band is lost after reductionwith a-mecaptoethanol and thus is probably the result of anearly cleavage. The 60 kDa reacts with both anti V3 loopmAb and sCD4. The 50kDa does not bind CD4 nor anti V3 iooomAbs and is lost after reduction, indicating that it mightbe a series of small fragments held together via disulfidebridges. The 35 kDa fragment is the smallest containingthe V3 loop and the reaction with anti V3 loop mAbs seemsto intensify after reduction. The position of the 25kDafragment can only be speculated.

Production of mAbs against V8 fragments of gp120:Three mice were injected with V8 cut gpl20 and given aseries of boosts. Initially, it appeared that only a weakresponse developed and thus additionnl boosts were given.

5As can be seen in Table 1 this increased the response ofthe mice against gpl20.

TABLE 1: Analysis of Anti V8/gp12O Sera.

dilution I IIof serao•f ql20 V8/cml20 J aol20 V8/crpl20

1/50 674 - 1270 8171/100 598 - 1220 6241/200 310 - 1000 4711/400 182 - 695 3191/800 139 - 391 1681/1600 - - 229 931/3200 - 116 411/6400 -- 77 31

The above table describes the response of one mouse that wasimmunized with gpl20 V8 fragments. As can be seen, the Citer is muc.himproved in the second bleed.

The above mouse was selected and sacrificed on June 11,1992. its spleen contained 3x10- 8 cells and these werefused in three alicuots with NS-! myeloma cells and 2/3 ofthe cells were plated and processed as two separate fusionsinto a total of 13 96-well plates (the third alicuot wasfrozen down for future studies).

The hybridoma cultures were screened against gpl20 andproteolysed gpl20. Only five clones were found to bepositive and one of these was lost in the process ofcloning. The remaining four were found to be igM of verylow affinity ano low tiLer in tie ascit=• •!uids obtained.It apoears that the digested gpl20 is a poor antigen andmice should be more aggressively immunized before newfusi.ons are attempted. Unfortunately, in the mean time theremaining two mice that were immunized with the proteolyzedgpl20 have died.

Production of CD4/gpl2O complexes: CD4 and gpl20 wereused to generate their corresponding complex in a number ofprocedures. The sCD4/gpi20 complex could be demonstratedvia ELISA assays. In principle, either gp!20 or CD4 wereplated and then the corresponding counter part was used toprobe the plate. Subsequently, the plates were probed witheither anti-gpl20 or OKT4 followed by an alkalinephosphatase conjugated anti-mouse. Moreover, OKT4A wasused to ascertain that the complex that was formed was, asexpected, unable to bind this monoclonal (see Table 2).Finally, once conditions were established preparations ofcomplex were made for immunizations.

6

TABLE 2: Analyses of gpl20/CD4 Complexes.

gp 120 CD4ýta/ml OKT4A OKT4 U La/ml OKT4A OKT4

10.0 27 640 5.0 206 7295.0 23 773 2.5 135 4922.5 27 313 1.25 42 2911.25" 29 116 0.60 22 138

The above table is an example of the analysis of the formation ofgp120/CD4 complex on the ELISA plate used in this study. gp120 wasapplied to the wells using concentrations as indicated. Then CD4(5agiml) was incubated with the gp120 and the wells were subsequentlyprobed with the antibodies as indicated (numbers r~oresen. ODx LG-).The right half of :.e table 's a control in whIch CD4 was plated andthe same antibodies were tesced.

Production of anti-complex mAbs: Three mice wereoriginally immunized against the complexed CD4/gpL20.After the initial immunization two of the mice aied and itwas concluded that the MES (N-me:hylmorphiline) which isused as a preservative in the sCD4 might be toxic for themice. A second set of three mice were then immunized withcomplex that had been extensively dialyzed. These mice anda remaining original mouse developed good responses againstboth CD4 and gpi20 (see Table 3)

TABLE 3: Analysis of Anti Serum to CD4/gpl2O Complex.

dilution of gpl20 CD4 CD4/api20sera j tma/ml 5ma/ml 5ma/ml

1/50 964 803 9731/1I00 941 746 9421/200 861 696 8591/400 690 629 =51/800 571 5i6 610

1/1600 462 463 5021/3200 290 336 2841/6400 299 297 250

The above depicts the response oF a mouse thac -was :4TmunrzeQ wz:h theCD4/gp120 complex.

The mouse described in Table 3 was that with the highesttiter and therefore was given an i.v. boost and wassacrificed for splenectomy and the fusion for hybridomaproduction. The spleen was found to extremely large (>4 x108 cells were obtained) . Therefore, 4 aliquots of -108cells were taken separately. Each was fused with NS-lcells. Two fusions were processed in parallel (theremaining cells were frozen down for future studies) . Atotal of 1170 clones were obtained and after 10 days ofculture, the media were screened against CD4/gpl20 complexin an ELISA assay. 147 clones were found positive and

7these were then re-screened against CD4/gpl2O complex aswell as CD4 and gpl20 separately. Of che original clonesonly 81 continued to secrete antibodies and of these 15were selected for future characterization. Of these 13have proven to be stable and have been injected into micefor the successf, i :'.oduction of ascites fluids.

Ascites fluids it all 13 mAbs described were produced andlyophylized and sent to Captain Tom VanCott. These werereceived and analysed for their ability to bind CD4, gpl20and their complex. The mAbs were tested in ELISA assays,FAC7 analyses and BIAcore measurements. A collection often mAbs were identified as being interesting for furtheranalyses (see Table 4).

Table 4:mAbs Produced Against gpl20/CD4 Complex.

I lmAb subclass gpl20/CD4 .gp!20 [CD41 JG-!. 3 IgGi -..-2 JG-4.5 igGl +.+ +.+ -

3 JG-7.19 :gGl +. . - + / -4 JG-8.l igGl -.- .I5 JG-9.5 igGl +++ - ++6 JG-10J IgGl +++ -

7 JG-25.3 _gGl +++ - ++8 JG-30.5 .gG+ - ++9 JG-40.l -gGl +.+ . -10 JG-76.5 -gG- ++ - ++

Results obtained in Tel Aviv corresoonded well wit:h toseat Walter Reed except for :hree -hbs (J-1.3, JG-7.1 JG8.1) which appeared at Walter Reedc to have a--nOtv :or CD4as well as the comolex, :hus these :t".bs wilL have :o oe_-_rther Tarac:er::ed. The ant:hcdIes were cio:nvy _oand conoet:-_i EL:SA assays were -er :•-r7ne 7:: -eoit ope overlaps. :n :his manner s_:x epo:opes were -. aefi'ned(see Table 5)

Table 5: Epitope Classification by Competitive ELISA.

4 +. 1- 1- - + + . .....-

4 - + ... .....- -

7 + - + + . . . . ..8 + - + + . . . . . .9 . . . . + + - -10 - - - - + . . . .25 - - - - + - + + - -30 - - - - + - + + - -40 . ..- - - +76 . . . . . . . .. +

8

Neutralization potential of the mAbs: The variousmAbs were tested for their neutralizing activity using thesyncytium assay described in the Methods. In thisprocedure BSCI ceils are infected with VPE16 recombinantvaccinia virus so that they now express cell surface gpl20.These cells are mixed with CEM cells and can therefore formsyncytia. Syncytia formation is inhibited by theneutrAlizing antibodies. At least seven antibodies havebeen. found to be neutralizing to various degrees. Ofparticular interest is the fact that the mAb JG-10.1 whichis complex specific by all the assays -- rformed thus far inboth labs, at Tel Aviv and at Walter Reed, is neutralizing.As these results are not rigorously quantified as yet theymust be further developed before the efficiency of thesystem can be fullv aopreciated.

CONCLUSIONS:

The major target of this project focuses on the postulatethat gp!20 changes its conformation upon its associationwith CD4. The molecular ramification of such inducedconformational change would be the appearance of structuresthat are otherwise hidden, i.e. crvptic epi topes. Onewould hope that antibodies directed towards these uniquestructures might interfere with the biology of the gpi20.

The experiments performed here illustrate that suchepitoces are a reality. The closest results published thusfar are chose of Celada [9] where he and his coll-aguesisolated one m.Ab that could correspond to any one of the:nree CD4 eoitopes that we have identif'ed (this isassuming that clones JG-l.3, JG-7.19 & JG-3.! indeed bindCD4) . Moreover, clone JG-O0.l is genuinely unique with nooarallel reported thus far. This clone is the first andmost direct example of a comolex dependent mAb as -t4 has

_<celient a::.=Znty for the complex and no detec:ableinng t-o C04 or gplo2. ?:ortunate.., it also is able to

inhibit in the svncycium assay described in this report.

A major task that has been more fully appreciated in thecourse of this work has been the problem or quantitativelycharacterizing the mAbs that we have produced andqualitatively mapping their ecitoces. The coLlaborationwith Capt. VanCott has already proven to be extremelyuseful for the Tel Aviv laboratony and is sought to beintensified. Towards this goal we are scalina uo theproduction of purified mAbs to supply the U.S. lab so tocoordinate an effective research plan. Moreover, newexperiments are being designed so to increase ourunderstanding of the biology and the effects of theseunique reagents.

During the course of the next year the mAbs will be used toevaluate the range of their specificity, their ability toneutralize field isolates and serological studies of bloods

9from Israeli cohorts as well as other HIV infectedindividuals will he conducted. Such studies have alreadybeen initiated in collaboration witb the Israelihemophiliac center at the Chaim Sheba Medical Center at TelHashomer.

Additional fusions will be performed using the mice thathave already been immunized with the CD4/gpl2O complex aswell as novel complexes and protocols that will be tested.

Finally we believe that the application of the PharmaciaBlAcore technology is extremely exciting and very importantto the progress and insurance of high quality work for thisstudy in particular. We therefore request to purchase aBlAcore for the Israeli laboracory. This will in no wayhinder the extent of the collaboration with the Walter Reedbut quite the contrary. it is our belief that having aBiAcore system functioning in both labs will enable a morefruitful dialogue between the two groups. In view of thiswe are submitting a request to this point as an Appendix tothis report.

REFERENCES:

1. Leonard, C. K., Spellman, M. W., Riddle, L., Harris, R. J.,Thomas, J. N. and Gregory, T.J. (1990) Assignment ofintrachain disulfide bonds and characterization of potentialglycosylation sites of the type 1 recombinant humanimmunodeficiency viruis envelope glycoprotein (gpl20) expressedin Chinese hamster ovary cells. J. Biol. Chem. 265:10373-10382.

2. Starcich, B.R., Hahn, B.H., Shaw, G.M., McNeely, P.D., Modrow,S., Wdolf, H., Parks, -. S., Parks, W.P., Josephs, S.F., Gallo,R.C. and Wong-Staal, F. (1986) Identification andcharacterization of conserved and variable regions in theenvelope gene of HTLV-III/LAV, the retrovirus of AIDS. Cell45:637-848.

3. Javaherian, K., Langlois, A.J., McDanal, C., Ross, K.L.,Eckler, L.I., Jellis, C.L., Profy, A.T., Rusche, J.R.,Bolognesi, D.P., Putney, S.D. and Matthews, T.J. (1989)Principal neutralizing domain of the human immunodeficiencyvirus type L envelope protein. Proc. Nacl. Acad. Sci. USA86:6768-6772.

4. Profy, A.T., Salinas, P.A., Eckler, L.I., Dunlop, N.M., Nara,P.L. and Putney, S.D. (1990) Epitopes recognized by theneutralizing antibodies of an HIV-l infected individual. J.:nm. unol. 144:4641-4647.

S. Ohno, T., Terada, M., Yoneda, Y., She,, K.W., Chambers, R.F.,Stroka, D.M., Nakamura, M. and Kufe, D.W. (1991) A broadlyneutralizing monoclonal antibody that recognizes the V3 regionof human immunodeficiency virus type 1 glycoprotein gpl20.Proc. Nacl. Acad. Sci. USA 88:10726-10729.

6. Ho, D.D., McKeating, J.A., Li, X.L., Moudgil, T., Daar, E.S.,Sun, N-C. and Robinson, J.E. (1991) Conformational epitope on

10gpl20 important in CD4 binding and human immunodeficiency virustype 1 neutralization identified by a human monoclonalantibody. J. Virol. 65:489-493.

7. Syu, W-J., Huang, J-H., Essex, M. and Lee, T-H (1990) The N-terminal region of the human immunodeficiency virus envelopeglycoprotein gpl20 contains potential binding sites for CD4.Proc. Nacl. Acad. Sci. USA 87:3695-3699.

8. Bosch, M.L., Earl, P.L., Fargnoli, K., Picciafuoco, S.,"Giombini, F., Wong-Staal, F. and Franchini, G. (1989)identification of the fusion peptide of primateimmunodeficiency viruses. Science 244:694-697.

9. Celada, F., Cambiaggi, C., Maccari, J., Burastero, S., Gregory,T., Patzer, E., Porter, J., McDanal, C. and Matthews, T.(1990) Antibody raised against soluble CD4-rgpl2O complexrecognizes the CD4 moiety and blocks membrane fusion withoutinhibiting CD4-gpl20 binding. J. Exp. Med. 172:1143-1150.

10. Healey, D., Dianda, L., Moore, J.P., McDougal, J.S., Moore,M.J., Estess, P., Buck, D., Kwong, P.D., Beverley, P.C.L. andSattentau, Q.J. (1990) Novel anti-CD4 monoclonal antibodiesseparate human immunodeficiency virus infection and fusion ofCD4+ cells from virus binding. J. Exp. Med. 172:1233-1242.

ii. Gershoni, J. M.,and Palade, G. E. (1983) Protein blotting inuniform or gradient electric fields. Anal. Biochem. 144:32-40.

12. Gershoni, J. M. (1988) Protein blotting: a manual. in:Methods of Biochemical Analysis (ed. Glick, 0.) John Wiley &Sons , New York. vol. 33, pp. 1-55.

13. Harlow, H., and Lane, D.. (1988) Antibodies: a laboratorymanual. Cold Spring Harbor Laboratory.

14. Ashorn, P.A., Berger, E.A. and Moss, B. (1990) Humanimmunodeficiency virus envelope glvcoprotein/CD4-mediatedfusion of nonprimate cells with human cells. J. Virol.64:2149-2156.

APPENDIX:

Request for Purchase of a

Pharmacia BIAcore System

Justification:

During the course of the first year of this project we havehad to analyze thousands of hybridomas in order to focus ona collection of unique monoclonal antibodies. Onceidentified, these mAbs are now being characterized;determining their affinities, mapping their epitopes andcomparing their epitope similarities or differences withother mAbs, establishing the extent of antigenic crossreactivity and of course evaluating their potential asneutralizing agents of HIV infection and potential use asim•nunotherapeutics. These tasks require an extensiveamount of work and should be performed quantitatively in amost rigorous fashion in order to produce meaningful anduseful information of the highest quality.

As more mAbs are to be produced in this project thelogistics become compounded in their complexity as new mAbsobviously must be compared with those alreadycharacterized.

When this project proposal was initially submitted theBIAcore was barely in its infancy. Last year when theproject was approved i had the opportunity of visiting theBIAcore facility headed by Captain VanCott at the WalterReed / Rockville, MD and was very impressed with whatappeared to be the ideal system for the efficient analysisof the mAbs _ hoped to produce. During the course of this!as- year i have discussed the progress of B3Acoretechnology and applications with Capt. VanCott and at theKeystone meeting. last March was delighted and excited tolearn that this system is in fact much more effective thati had ever imagined.

:n order to evaluate the utility of this system in theanalysis and characterization of the specific Ahbs that wehave already successfully isolated, the Tel Aviv laboratoryhas initiated a genuine collaboration with Capt. VanCottand sent him samples of ascites fluids of 13 mAbs andseveral samples of purified mAbs, all these to be tested inthe BlAcore system at Rockville. In August this year Ispent a few days with Capt. VanCott in Rockville havingtherefore the opportunity to appreciate the power of theBlAcore "on hand".

It is clear that not only are many of the tasks I am in theprocess of conducting at Tel Aviv amenable to BlAcoreanalyses and far more quantitative using this system, there

12are numerous parameters that would be impossible or highlyimpractical to obtain otherwise, with out this system. Inview of these developments and the success we have alreadyhad in generating what we believe to be extremely uniqueand useful mAbs, we are convinced that purchase of aBIAcore for the Tel Aviv laboratory would be exceedinglyuseful and therefore essential and would enable a quantumstep ahead for generating the high quality reagents wedesire to produce.

In view of the importance we foresee in this acquisition ofthe BlAcore I have initiated discussions with bothPharmacia in Israel and their headquarters in Germany aswell as with a number of local funds to obtain some supportfor this purchase. In summary, I believe that I couldobtain at least S50,000 towards the purchase of a BlAcorefrom local sources. Moreover there is a chance that Icould come to some agreement with Pharmacia to obtainspecial conditions for this purchase as this would be thefirst BlAcore in this area of the world.

As is indicated, the price that we have been quoted for thecomplete system is $156,000.- (see cuote attached). Wewould anticipate that running costs for chips, pumps etc.would be $10,000.-/year although this estimate might needadjustment.

Therefore, we request $116,000.- for the purchase of aBlAcore system a soon as possible (Pharmacia Germany haspromised immediate delivery). We would ask for anadditional $10,000 for next year's (year 3 of the contract)budget for the running costs of the BlAcore.

We sincerely hope that the funds can be found to assist usin the purchase of this essential piece of equipment.

13HIV Omnan Ius n

GAMIDOR Ltd.310 Medlcatl Serces & Supplies

Ordering nr. article price in US $

BR-1000-01 BIAcoreTm System

biosensor-based, automated system for real-time B.iospccificInteraction Analysis (BIA) without using any label.Qualitative as quantitative data (e.g. association rate anddissociation rate constants, affinity constants etc.) areavailable within short timcý(scconds to minutes). BlAcorcerwas developed for research purposes and pilot-steps ofroutine assays (e.g. development of ELISAs, receptor/ligandassays, process control, method development for affinitychromatography etc.) In combination with the SensorChipCMS, BIAcoreJ' shows an extemly high reproducibility ofthe results (CVdose normally < 5%).

Quantity delivcred :

- BIAcoreTu processing unit- one and one sample racks type A and B (type A for 57

sample vials with 7, 9 u. 16 mm o o.d. and type B for 60sample vials with 9 mm o o.d.

- Integrated ý.-fluidic cartidge (IFC)- BIAloguc~m systcmsoftware for controlling BIAcorcrm

Gamidor Ltd. P.O. Box 120 Savyon, 56915 .. 56915 lI'30 120 .7.171 n11)"3 '1171t"NAcd: 54 Harei Yehuda SL Givat Sayon fl"3o flU2 ,54 MliT, "1ii .1Trrl :nalnll

-l: 03-5351505, 5351205 Fax. 03-346573 03-346573 :ORD 03-5351505 . 5351205 :713

1 -"1L- 1"117 1 i "

GAMIDOR Ltd. A3;o Medical Serces & Su~pIies

- controlling instrument Compaq 386/20e witre 4 MB RAM,40 MB hard-disk, VGA coulored monitor, 80387 Co-processor

- keyboard (engL), MS DOS version 3.31(engL), MS -- Windows version 3.0, MS Excel version 1.0- Laser printer, type I-T Laserjet HI

Initial issue of consumables:

4 x Sensor Chip CM5 (3 pcs/package each)Amine Coupling Kit for 50 immobilisationsSurfacrant P 20Autosampler vial kit

all cables, wires and tubings necessary for the installation

US $ 156.000.-

Specifications:

Processing Unit:

Programmable parameters:timed events : transfer, mix, inject, report pointsFlow rate : 1 - 100 .l/minFlow cell selection (1 to 4) and automatic sample loop (S and 450.d selection on the IFC; integrated microvalves are controlled bypressurized airrequired sample volumes 30 p.1 for 1-4 p.l- injections

75 p. for 5-50 J±l-injectionsDead volume : < 0.5 41Reproducibility: >95 %, C.V. < 5%max sample positions 2 x 96

Mains supply 100-120 V or 220-240 V, + 10 %Power consumption 580 VA (max)Weight : 45 kgDimensions: 76 x 35 x 60 cm (I x w x h)

Sensor Chip CMS:

Gamidor Ltd. P.O. Box 120 Savyon, 56915 56915 W20 120 .-Trf .n" 'J2 -Tl'r-N

:d: 54 Harei Yehuda St. Givat Savyon "0'2U nfl:hJ .54 n'7"in1f l 21T• 1 :mnllJ

-: 03-5351505,5351205 Fax. 03-346573 03-346573 :0M9 03-5351505 ,5351205 :'21

T2"33: 111T12NI. ~AU"•l1S1-1'2 I•'flT'Ifl ,l'Ii•ll

GAMIDOR Ltd.31O mectcoi Sertces & SuoplieS

The sensor chip is a three layer composition of glass, goldand carboxymethylated dextran, optimized for concentratingand immobilising of biomolecules. The four possible,parallel channels (2.1 x 0.55 x 0.05 mm) will be formed bydocking the Sensor Chip to the integrated g-fluidic cartridge.Each channel can be used several times (life-time depends onthe stability of the immobilised ligand), up to 50 to 100times. The sensor chip is fixed on to a polystyrene supportframe and protected from dust and mechanical damage by aplastic sheath. Each sensor chip is packaged in ahermetically sealed packet under nitrogen atmosphere.

Total: US $ 156.000.-

This price information includes packing charges andshipment, installation and introduction to BIAcore'm intheory and practice

Gamidor Ltd. P.O. Box 120 Savyon, 56915 56915 1ia0 120 .1.2 l Tn wa T

Acd: 54 Harei Yehuda St. Givat Savyon 11"20 nDJ3, .54 ,-rv'n I •, f111 :.nf nr

-el: 03-5351505,.5351205 Fax. 03-346573 03-346573 :0P9 03-5351505. 5351205:3

nnm irNic

GAMIDOR Ltd.M3'o Mealcal Services & suloolles

1992 'iiua 23 i,vtao nyaa

lonon ~ 2'2 ipinriiniN

773-nra , au-: n~ r'r j N

Pharmacia Biosysterns :p a3 0 n

.t"r ');i ii'N -vinini .2.nva 6 :lpfloN Inr .3

'Zamidor Ltd. P.O. Box 120 Savyon, 56915 56915 1'L20 120 T~n ,I-1):l 1TT'nNAdd: 54 Harel Yehuda St. Givat Savyon fl13U nl2 .54 171,11 'lil 21111 fl:lflfl

el: 03-5351505,5351205 Fax. 03-346573 0)3-346573 :M29 03-5,351505,5351205 :'2D