CA2064710A1 - Recombinant protein which binds to a complex viral antigen of hiv-1 - Google Patents

Abstract

Abstract The invention relates to a recombinant protein which binds to a complex viral antigen of HIV-1. The manufacture, production, purification and application of this protein are described. The protein contains the variable regions of a human anti-HIV-1 antibody connected by a linker. The production is carried out in various prokaryotic or eukaryotic systems. Biochemical chromatographic methods are used for the purification.
The described recombinant protein can be used for detecting, quantifying and purifying HIV-1 antigen.

Description

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COMPLEX VIRAL AN~IGEN OF RECOMBINANT PROTEIN
BINDING HIV-l H~man monoclonal antibo~ies (mAk) c~n b~ produced thereby that B-lymphocytes are obt~ined from humans which, due for example to illness~ show an immune reaction against an antigen and that these B-lymphocytes are imm~rtalized t~rough fusion with suitable cell lines, in particular with myeloma cell lines. Hybrid c211 lines, so-called hybridom~s, ob~ained in this way, serve as,production vehicle for m~Xs~ They can be utilized in vitro in the ~orm of cell cultures and can be cultivated on the required scale (1).
The substance produced in the process represents as a rule a complete mAk characterized by 2 heavy and 2 light chains, whîch are linked with eazh other through disulf ide bridges and through non-covalent bon~s, and ~hich ~orm the specifically binding~ antibody (2).
The structure of such an antibody can be divided into a constant region responsible for the so-called effector functions, such as for exalmple complement activatlon, and into a variable reS~ion which brings about the specific binding of the particular antigen.
Biochemical methods can be used to split a~tibodies ~:enæy~atically~ For exampl2 ~sing papain or pep~in, a por~ion of the constant region can be spli~ of~. The Fab' or (Fab' )2 fragments are able in a manner a~alogous to that of the original antibody, to bind the particular antigen (2). Also de~cribed~was the proteolytic ~plitting of the complete constant regions resulting in a so-called Fv f~ag~ent. However, the reproducibility of this procedure is not nearly as good as the papin or pepsin splitting o~ antibodies described above (3,4).

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With methods of gene technology it is, however, possibl~
to produce Fv fragments reproduciblyO The prerequisites necessary to achieve this as well as the methods utilized will be described in the following.
With the aid of standard methods a cDNA bank of a hybridoma cell line producing mAk is produced. Total RNA
is isolated from mAk-producing hybridomas. Apart from ribosomal ~NA, this RNA comprises the totality of the transcripts of the cells. Present are incompletely processed, nuclear transcripts as well as mature cytoplamatic transcripts, the so-called messenger RNSa.
These are characterized by a poly-adenosine tail at the 3' end. This poly-a region can be used to isolate the mature mRNAs through affinity chromatography with oligo-dT-cellulose. With the aid of the enzyme "reverse transcriptase" the mRNA can be rewritten into a so-called cDNA. By using suitable vectors, the obtained mixture of cDNAs can be cloned which results in a so-called cDNA
bank (51. Immunoglobuline-speci~ic hybridisation probes permit the identification and isolation of clones which comprise the desired sequences. By se~uencing the DNA of these clones and hy comparing the sequences with known i~munoglobuline genes (E~BL Nucleotide Sequence Data Library, Heidelberg, Germany) certainty can be gained about the identi~y of the clones (5). In this way, clones can be isolated for example which carry the sequences of the light or the heavy chain of an mAk.
Through sequence analysis of the immunoglobuline cDNAs obtained in this way, the individual domains of the heavy or the light chain can be identified by comparison with known immunoglobuline sequence~: it is possible to identify the variable and the constant region and, for example, to identify within the variable region the so-. ~

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- 2a -called "hypervariable" or 'Icomplementarity determining"
regions which, in fac~, ar2 re~ponsible for the specific antigen hinding (6).
Antibody genes cloned in this way can be brought to expresslon in different systems. On the one hand, animal cell cultures can be used, such as for example...

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m~eloma cells if suitable expression ~ec~ors are used (7). Ihe use of yeast (8) or of bacterial cells (g) as exp ~ ion vehicle for ccmplete anti ~ es is problematic, since such cells are apparently not able to synthesize correc~ly the for them - very larg~ molecules ~udh as an~ibodies represent.
Suc~ess in this direction took shape when at ~ were made to bring to expression subfragm~n~s of an~ibodies in lower eukaryokes or in prokaryotes.
In the followin~ four different m~thods will be described which ~ t the expression of FV or Fab fragments in Escherichia coli:
Skerra and Pl~ck~hun (1988 (10)) insertsd thQ gene for the variable regions o~ a murLn~ an~i ~ rylcholine - antibo~y (McPC603) adjo m m g the lac pnc=otcr~cper~tor region follow~d by one backerial leader se~uence ea~h which served for the tra~sport of the prodNc~s intn the periplasmic spa~e of the bac~eria. This is the leader of the ~uter membrane protein A (opm~) as 1 as ~he alkaline ph~spha~ase (pho~1. After txansfecti~n of this pl ~ d int~ Escherichla coli, the expression of functional i.e. antigen-bi protein in the periplasmic space of the bacteria was prcven.
Be~ter et al. (1988, (11~) pro~uc~d ~he Fab fragment of a chimeric murme-h~e antibody which recognizes a ganglioside antigen such as is four~ fre~ently on the surface of h~ carcin~ cells. Ihe herein applied plasm~d consb:-uction ~rises the Sal~nella tyFihi~ium araB pr~moter as well as t~he pelB leader se~enca in eac~h instan~e before t:he seq~enc~ codirP3 for the particulæ c~in. ~igen~bixxling Fab ~ 3nts were c~ta~n~d fram e culbJre = ~ rr ~ of the transfonn~d bacteria.
Interestingly, S ~ as well as Pl ~ (1988, (10) and Better et al.
(1988j (11)) used sc-called dicistrcnic c;nstructi~ns, i.e. such, m which in a single mes~2nger RN~ m~lecule ~he in~ormation for ~he two chains ~o be eKpres~e1 separately is present. The ~uthcrs state that thereby the spacial closeness o~ the ~orm mg pclypeptide chains is e ~ which represen~s a prer0guisit for the correct orientation of the ~ ia~le r~gion of the heavy (VH) with ~ha~ of the ligh~ chain (VI~.
Attempks ko solve preciæly this prnblem, namely the formation of the FV
pe~tide hetemdin~r (in nature not cavalently bo~d) w2re made by Hustan et al. (1988, (12)) an~ by Bird e~ al. (1988, (13~) in diffe~nt ways, n~nely ~rou~h covale~t lirdcage o~ the chain v~a an am~no acid lir~ sequence suc~h as does not occur in na~ure. Ihis lir~cer se~uence is disti~gui~ed there~y - . . .

~ . , ~at i~ cc~rises a particular r~e~ a~l s~e of ~nino acids so ~hat it can bridge the s~ce ~idh e~s ~ l~e na~ral aonforma~ion of an a~tibod~
be~an the regions to be k~l, wit~out intro~c~ l~r~ssary ~ss ir~to ~he ccrlormatio~:
~ StoT~ ~t al. (1988, (12)) li~ced ~ variable region~ of a n~r~ne anti-digoxin anti~r via a lir~ of 15 amino acids havin~ t:he s~ence _B. Ihe selected or~er was: VH - lir~r - VL. mis so-called sequence urx3er the ~trol of the ~akic trp prc~r~ator in ffle farm of ~l~le ~nclusi~ bodies. Aft~ ~heir solution in 6 M guanidine ~Cl and after ~ of ~e leader ~ acidic ~y~rolysis b~ ~e an~no acids Asp an~ Pro as w~ll as sc~ne ~natograEihic st~ps, active antig~n-bin~i:r~ sir~le ~hain Fv fragmer~ was ~btain~.
A basically analogous a~proa~ih was ~hosen b~ Bird et al. (1988, (13) ) for ~he constructical of a m~rine aT~gen-bin~ling pr~ein ~ich bin~s fluorescine specifically. I~s gro~, hawev~r, used a lir~ of 18 a~uno acids with ~e se~e ~S~SID. qhis li~ is a part of ~e se~ence of t~he human "ca~o~ic ar~y~rasa" an~l MaS selecl:ed frc~ the E~oo~ha~ protein stmcture data baæ as a loap structure which spacially fits pr~cisely c>n ~e position of ~e ~ acids of the Fv fragm~nt to be ba~d wi~ eatih ath~r. The ord~ of ~he in~liYidual regions here was differ~nt than with ~st~ et al. (1988, (12) ), r~nely VL - l~n~ - V~.
q~he abav~ described gene co~stxu~tion for the pro~uction of antibo~T
fra~ts in Esc~l~hia coli refers to nDur~ne ~en~es or ~n one case to a ~rine-human chi~. No c~esE~ ex~#rimer~ts wi~h h~man s~nces hav2 b~
Fab, (Fab )2 a~ FV fra~ts off~r differ~t a~antages ~han do car~lete antibodies. Due to ~eir small siz~ in oparison to oa~lete ant~ies, ~y ~ dif~use easier ar~l fast~r, in vitro as well as ~n p~ial in vivo applications. For ~i~ reason ~hey are g~ly easier to work with and in m~st cases in ~c~ he i~ctions of the constar~ 310ns (for exa~le effector ~ctions, bi~ to cell rec~tors, bin~i~ to other molecules) æe not re~ired or evel~ pres~t di~ ges, are eguivaler~t to cc~lete antibodies arxl possibly are even to be preferred. For e~le, in t~nor imaging ~en usin~ car~lete antibodies, pr~ibl~ fre~ently arise ,~ ~ , . . : ........................ :
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thm~ ~rvor~ ~ignals, ~ich are caused 1~ n~n~ecific bin~i~ o~

antibodies. It is lmac~ ~hat ~ us~ Fab fra~ts su~h pr~blems can be re~ced. Accordingly it is to be es~ ~at ~he use of EV fragmerxts or of single chain FV fxa~ts will o~fer fur~ i~pravem~ts in this re~ect (13, 12).
I~til naw ar~tibodies of ml~rine origin ha~re been w~rked wit~ ~ich bir~
to ~oall wall des~ibed allti~ens su~h as fluorescine or diyMc~n. me er~tire gen~ s~on hlilds on the fact that a law m~lecular eul~tan~ (~ >
1000) is ba~ as antigen. Ihe antigellic s~bstance~ occurrin~ m~st fre~tly in nature are p~ptides, peptidog1~, prateins ar~.
po1yseodharides, and. as su~ are high n~1~1ar.
A~rdin~ to t~e ir~ention ~he pr~tein of the abov~ stated type c~prise~ ~e antiger~ inding regic~s of an ~ s~mnir~ frcan the cell line 3~ ssia~ No. 87110301, ~:S, Port3n r~n, I~K (1, 14, 15, 16).
lher~ith for the first t~ne a pra~ein of h w ~ origin is ~tained ~ ich has the desired b~nding characteristics and which can also be expxessel in unicellul r micrco~ such as yeast or bacteria.
EurthermDre, accordin~ to the pre~ent in~ention the produstion of a sing1e cha m construc*ion, derived fram a human antibody is described. This sing1e cha m construction binds to an high mo1ecu1ar comp1ex vira1 antigen, in con~rast to sm311 we11 defined antigens.
It cou1d not have been predieted tha~ ~he oorresponding methods for eonstruet mg the single eha1n fragments w~N1d a1so 1ead in the case of okher than the pNb1ished antibodiçs, in partieu1ar with human an~ibodi~, to funetio ~ , i.e. antigen~binding mo1~eN1es.
It is further also not obvicus, tha~ ~o~pleK antigens such as for e ~ le an~ig~ns on the surface o~ vlrus, m which accD ~ to experience a g~eater number of am mo acids participate m the antigen-antibody formation than with small antigens, tolerate Ln the same way manipulation in the area of the variable regions of the oorrespondlng binding antibodies.
start mg with the cell line 3D6 which produces a human monoclon~l an~ibody of type IgGl k~ppa which react6 specifically with HIV-l ~p41 and shows a w~ak crcssreaction with HIV-l gp120 (3D6; (1, 14, 15, 16), total RN~
was isolated. IhQreLn ~he method of the guam dine isothiocyanate extraction ' ~ ~
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an~ ultracentrif~i~ via a c~hic~ of 5.7 M CsCl was used (5).
mr~ adso~ption on oligo~cellulose, ~e poly A ~ 30 frat::tion, cc~e~tly t~ n~, was isola~ed fram ~e tc~ RN~, (niE~ P~rification Kit, E~an~cia, Swed~).
Ihe ~ serves as a substra~e for ~e synthesis of clX~ (cl~ Syn~sis Kit, l~ha~cia, Swed~).
Ihe cl~ of the cl~ bar~c too~ plac:e in ~e plasmid vector plJ~l9.
lhe re~ibinant plasm~d was t~sformed in Es~i~hia coli, strain HB101 an~l cultivat~d in l;B medi~n (5).
Positive clo~ were identi~ied thr~ hybridization wil~h specific oligonucleotide pr~bes. Ihe ~ ~or ~e prcibes w~re tal~en fram the EME~ ~ ses3~ data base fmm consta~ regiorls of h~an Igt:l heavy or kappa light ~hains.
Irhe clone~ identified ~ h positi~e hy3:xridiza~ion signals were fur~her c~aracterized by restriction analysis a~d tbose clones which carry the plasmlds with the 1ongest inser~s w~re idbnti~ied.
ThrcNgh ~equenoe analysis of these clones c~e clone each with the complete ooding region for the hea~y or for the light chain of the antibody was identified. These clones carry th~ identifi.cation PUC3D6~C (SEQ ID No:
1) or pUC3D6LC (SEQ ID NO:2).

Exampl~ 1 In ~he sequence of the ~ of ~he clones pUC3D6HC (SEQ ID NO:l) or pUC3D6LC (SEQ ID NO: 2) the transition ~it~s ~ the region of the leader peptide and ~he variable region as well ~9 between the variable region ~nd the oonstant region was identified. lhrcugh oligonucleckide-direcbed mutagenesis (in vitro muta~enesis system, Amersham, UK) ~he follcwing mN~ations were carried cut at ~hese transiti~n sites (see also A-D):
1) RRcognition sequences for particNl æ r~striction en~ymes were mutated in. With ~he aid of the~e res~riction sites the variable regions of the heavy or the light chain of ~he ankibcdy 3D6 were cut out of the particul æ
plasmids.
2) ~he start and st~p ccdons r~uired for subseguent expression were muta~ed in.

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Irl order to be able to li~c the vari~le regiorls of ~he an~ibod~ 3D6 wi~ a link~, ~o synt~hetic oligo~cl~tides were pro~ced ~i~ih form the t~Fo ~ strands of the lir~. T~e ~wo olig~cle~tides were selected so if they hy~ridize wi~h orle an~her, a dauble ~trand is formed at ~ose ends overhar~ing single ~tra~ ~ regi~s are pres~t whic~h co~respond precisely to those averhang~ ends ~ich are generated ~erl cutt~ with the correE:p~iDg restriction ~3zy~ at the above stated mutated-~n restriction sites. mis permits t~he liga~i~ o~ ~he variable r~gions isolated wi~ he aid ~ ~is ~icticn enzyme wi~ ~e syr~ic oligormcleotides of the lir~.
~>LE~ER I VP~LE REGION >
-~X G V Q C I E V Q L V ~>
141 A~rG 170 wild t~pe ** * ** ***
E~iRI NcDI
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A: Mxtatic~n at the transition b2tw~en ~e leader region an~l the variable region of ~he heavy dhain of ~e an~ody 3D6 (S~3Q ID NO:l). ~ta~ed bases are denoted by "*". me coded anono ac:ids in ~ w~d ~pe ~ an3 given, ~d = t:he Tmltation~nerat~l restrictio~ ~ites Ec~ and NcoI as well ~he ~ codon A~G.

--> variable region I ~onstant region -->
--> V T V S S I A S T K G -->
519 ~C ACC GT~ T~r 1~ GOC ~C ACC A~G GGC 548 wild ~pe **
Ba~

B: ~tation at l:he transition betwe~ ~he variable region and the corlstant region ~ ~he heavy cihair~ o~ ~e antibody 3D6 (SE52 ID NO: 1).
2~tated baæ ~ are d ~ d ~ *~. me ccded amino acids in the wild t~pe ~N~ are given, Durthcr~are the restriction site ~amHI generated b; che .; .. ~ ' . . .

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~tation.

--~ leader r~ion I variable region ~
~> P G A. K C I D I Q M T ~>
79 ac~ G~ GC~ AaA T~T Si~C Arc ~: ~ ~CC 108 ***
SalI

C: ~tation at ~he transiti~ be~ ~e leade;r r~iorl and. ~e variable region of ~he light dh~in o~ ff~e a~ti3~ 3D6 (S~Q ID NO: 2j. ~:a~l bases are d~ted bs! "*". ~he coded amino acids ~n the wild type 1~ are give~, R~re the restriction site SalI g~rseratçd by t~he muta~ion.

~> variable region I ~ region >
--~ V D I K R I T V A A P --~
397 ~G ~1 ~C A~ ACr ~ GCI~ GCA ~ 426 *** ** *
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~ S~p D- ~tation at t~e transition ~ ~e variable regia~ and t~e : ~nst~c regicn of ff~e light ~in of the antiba3~ 3D6 (SEQ ID ~O; 2).
2~ ba~ are d~ted ~y "*". ~e cod~ amin~ acids in ~he wild ~ype are giv~n, ~ ~ ~ictical site HindIII g~era~ he ~tation as well as ~ s~: codon q~A.
-- ': - - - -1~ ligatic~ of ~e 3 aF~riately pretrea~ fragm~nts (VH,lir~, VL) wit~ one an~er a gen~ btain~ h at the transition sit~ bet~reen the vari~le regione and th~ lix~ still carried t~he mutated-~n re~triction site, ~idh ~rstains rmcl~okides nat: c=rresp~ng to the nucleotides nat~rally occurrir~ at ~hese sites. Ih~ also a char~ed amino ,, , ~

' ~3~ ~) acid se~el~ce (ses E ar~ F) resulted.
In order to res~re ~he original ~o acid seg~nce at the sta1:ed transitic~ sites, the desired 1~ se~e wa~ prod~ed (see E ar~ F) throu~h a r~pea~ed mutatiorl process.

~ I 1~ ~>
I * **
380 ~C A~ aC GGr GGC T~G GG~ 412 ~> V :r v s s G G G G S G ->

E: D~ sequellce of the l~cage site ~I li~cer before and after the bac:k naltation for ~e restora~ion of the natl~ral am~no acid ~ce in the area of ~e VEI region ~SEQ ID N0: 3). ~tated b~æs are densted by "*". The final am~no ac:id ~e is giv~n.
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422 T~G G~ ~GC G~;C GG~ C A~ C~ A~ 451 - ~ S G G G G S D I Q M ~>

F~ Se~w3 of t~e lirdcage si~ - VL before ar~ af~ ~e ~tation badk for th~ r~storation o~ the na~ural amino acid seg~ ce in ~he area of th0 VI re~ion (SEQ 121:) N0: 3) . ~tated bases are deIIoted ~ "*".
Ihe final ~nino acid s~e is giv~.

C~tly, with the aid of this me~ods a yene was ~tmcted hav~n~
the st~ re VH - 1~ - VL. lhis c~onst~ct is des~ibed as sc3D6 ~si~gle cha~n 3D6) a~ was inæ~t~ in ~he cloni~ vector p~J~l9 (S~Q ID N0: 3). Ihe resulting vect~r c~ries t~he identificatic~ lXJCW3D6.
'rhe sc3D6 gene wa~ art out of ~e plasmid pUCsc3D6 ~ restriction :., $ ~

enz~ and ir~d into ~he bact~ial ~n3ssio~ vector p~223-3 (~armacia) ~ic:h cc~rises ~he tac ~3rc~er in~cible wi1~h is~pr~l beta-~hiogalas::toside (IPTG). The result~ vector cæries t;he identification p~:3D6 and w transfor~d ir~ ~e E. coli st:~in JM105.

Culti~ration o~ t~he bact~ia me transformed bacteria were cultivated in a labora~ory f~ up to an OD600 f 2.0 in T~3 culture medi~ (5). g,~bykly, the in~tion of ~xpressi~ tocac place thr~ the ~dditio~ of iscprc~Tlthiogalactoside (IPr&). Ihe bacteria were conti~ed ~o be cultivated for 3 h~rs in the p~ce of IPl'G, s~er~tl~ harvested ~ cer~trifu~ation and stored at -80C. Subsy~tly the protein was e~tracted and purifi~.

E~on asld purification For eacih ~?eri~ental preparatian 10 g bican~ss (wet weigh~) were used.
Ihe cells were dis~ ~ mez~s of ly~ozyme in ca~ination wi1~h ~tic shock and =a~tl~ froze~ at -20C. me ffflz~n E. coli p~ste is br~en into small pieces and 2 1096 s~ion is p~ wit~ E buffe~ (10 ~5 Iris, 100 n~f NaCl, 1 niM EDq~, ~H 8.0). q~ this ~sior~ a ly~is co~ctail ca~rising rlucleases, lys;:zy~s and ir~ibitors is ad~ed (se~ Table 1).
Ihis E. coli su~erlsion is in~ated for 15 mi~ at 42C. mraugh the addition of triton-X-100 (~i~l ~tion 0.5%) and a re~
incu:~tlorl of 5 mi~te~ at 42C ~ cells are ly#d.

Ihe sedir~ is :es~ in SqE buf~ and sti~ed for 8 h~rs at 4C. The inclusion bodies are c~ted ~ c~t~ifugation. To this e~d a glycerine c~ion (50% glycerine in piho~ate buff~d saline (PBS) is placed into centrifuge b~bes, a layer of an eq~al vol~m~ of s~e~sion is placed aver it ar~d the ~bes ar~ cer~tri~uged (30 mi~utes, 6000 rpm, 4C, JA-20 rotor, J2-21 centrifuge"ra~: B~nan).

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The cc~t~ated i~lusion bodies are dissolv~d in 6 M GliHCl (~dine hydm~loride) ~n P~S, pH 813 ~er s~rrir~ at 4C (12 hr~). Subse~ently th~ p~n conl:ent is de~ermined ~trically.

Refol~

The pn~tein dlssolv~ l is refolded in the pres~oe of Ioreig~
proteins. First a pm1:ein de~minatiol~ tahes place. me dissolved inclusion ~es are dilut~ with refol~ buffer (GuHC~ 1 M, gluta~ia2l re~es 30 r~, g:Lutath~ oxidize~ 3 nM, E~ 100 ,uM"n PBS, ~iH 8.3) in suc~ a way that the fi~l c~nc~tratior~ is ~0 m~ prc~tein~l.
Dilutir~ 1~he dissc~lved inclusiall ~odies ta~s place ~ a laboratory scale us~n~ a ~ te ky slawly dripping ~ pratein solution into the ~e~oldirq }~uf~ is b~t t~ work a~ 37~.
me refolding was purs~ 1~ means o~ er~d phase ~LC. To ~his ~nd sa~les w~re t~3n, ~ H value set to 5.5, in orde~ ~ pr~N~nt fL~
refol ~ , ~e san~les were centri~u~ed (Millipore table c ~tri~uge, 4700 rpm, room tenperature), f~ltared steril (pore size 0.22 ~Im, lcw protein binding),:and, if ne~essary, ccnc~ntrated (Millipore table centrifuge, 4700 rpm, 20C) and in eadh ~ e 250 ~1 ~n~ly~cl by m~ans of reversed phase chrc=atc3r~phy HELC (nucleoeil 300, 5~lm, 4x125 mm, m~nufactur~r Vogel, Ger~2qy. A linear gradient 0.1% T~ / acetonitril 10 - 60 % was appliel on the ~olumn within 40 minutes).
The fd ded sc3D6 ~ ultrafil ~ . A 10000 Dalton cutoff polysulphone me~brane:was used. T~e ultrafiltered protein solution is placed on an anion exnhanger and cub~eqyntly eluted from ~he aolumn with 100 mM NaCl.
The sc3D~ is desalina~ed with Sephadex G-25 ~ ~ acturer: Fha~macia, Swedbn) gel filtra~ion an~ conjuga~ed with alkalina phGsphatase aocording to the method by Nakane et al. (17).
The purified sc3D6 prote m was exam med ~ S~S PAGE (Illustration 7). For demons~ration o~ ~e functionality of the sc3DK a Western 81ct Test with HIV-l test striF~ (BioRad, U~S~) was carried out. AS positive control an analogous test wa~ carried out with the natNral antibodies isolated from ., .:
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ani~al cells. As n~t~ve co~rol ~ved a pr~ation of tot~l pr~cein frc~m E. s:oli. Ihs result of l~ est was poæitive an~l is ~ha~ in Illustra~ion 8.

Purification of sc3D~ protein ~y ~ of affinity ~natoqraFihy With ~e apprc~pria~ly purified sc3D6 prcrtein a ~it s~n was p~ und~ s~ ~itions wil~ mple~e Freund'~ A~uvans. With ~e aid of ~q-Se~hæoæ Fa~ F1aw Chr~atogra~y (Il~:~a~ ~harmacia, S~rede31) :t~he IgG fracti~ wa8 ~ain~d frl the ~it s~rum. Ihe speci:Eicity of ~e ar~ti~odies wa~ de~nined ~y means o~ ELrSA. Wi~ he aid of a pe~ptide syn~esizer the 15 aINno acid lon~ li~ p~ptide ~Seq~: G~GGSGG~S) ~tion wit:h bavine se~n al~ain (~) in a mo1æ ratio of 6:1. With t~his conjugat~ ~c~tit~r pla~ were ~ted. The s~n s~le was incubated m ~ c~ated microtite~ plat~s and the b~l ant:ib~r was dGx~t~ wit~h a percxidase-markf3d goat anti rabbit Ig~. Ihe ar~i sc3D6 pro~ed an~l ch~ed was ~ to a BrCN-activated S~r~ 4B (~mfa~urer: E~a~acia, Sweden). Ihe n~ ma~erial wa~ w~d out. A pr~purified ~cb:act of sc3D6 protein, ~idh was refolded and desal~rlat~l with Se~hadex G-25 (~nuf~: E~armacia, Swe~) as des~i~ al~ave wa~ placed or~to the anti-sc3~6 ~1un~. The n~d material was wa~ out ar~ the ~acifical1y baund sc3D6 protein wa~ elutad with a 0.1 M gl~c:ine HC1 l~fer, pH 2.5. Ihe eluate was ~ly neutralize~l wit~h 1 M l~is ~uffer arld sc3D6 protein as ~des~ib~ charact~ized ~ means of SDS elecb~horesis and ex~nined for i~unctior3alit~ means of West~n Blot.
Ano~ me~hod for ~ ~e affin~ty ~hrarnatogra~ic purification of sc3D6 p~e~n is as follc~
With the above des~ib~d ~-cou~l~d 1~ p~ptide a ra~it s~m was produced with the a~d of ~le~ adjuvans. The Ig~ fractian was ~in~d ~ C~ffe~ Fast Flow ~atograFihy (mar~ufact~:
~harr~cia, Sweden) ar~ ~ur~r p~ri~ied via a BSA-Se~e 4~3 col~
(monu~: l~a~nacia, Swederl) in oxd~ to rema~e t~e anti-~A antibodies.
I&le so obkair~d anti lirdc~ IgG was co~ le~ to an BrCN-activated SeF~arose 4B
(manu~acturer: Pharmacia, Sweden). A prepurified extract of sc3D6 p~otein ., - . ............ . .
.

~r.~

~3 ~ic~h wa~ refolded ar~d desalina~ed wil~h SeE~hade~ G-25 (maTna~acturer:
~cia, Sw~) as des~ibed above was placed orrto the arrti-lir~ colu~.

3~n was eluted wi~ arl 0.1 M glyc~ H~ ~r, EiH 3.0~ Ihe eluata was s~2rrtly neutralized with 1 M Tris buffer ar~ the sc3D6 prote~n was, as descr;hPd, ~haracterized ~ m3ans of SDS el~oresis and examir~ for functionalit:y ~ n~ans of West~:n Blo~.

~nunoaffinity chr~natoqra,rihic ~urificatiaQl of ~:V-l crp150 For ~e pmduction of an sc:3D6 i~finit~ col~ ~e puri~ied sc3D6 prote~n was ~2d to a 1 nl ~ col~ (~ ha~nacia, Sw~
(acc. to pr~ol of 1 harmacia) .
m e prepurification of the gp160 (of the coat p mtein o~ l) ~ ich is spe¢ifically bcund by the antibody 3D6 as w~11 as by the sc3D6 prckein) was carried out according to ~ tt et al. (18).
Ihe pr~purified material compris m g the reco~binant gp160 was con=cntraeel by ultrafiltration and con~itio~2d for ~he ~c3~6 immLnoaffInity chroma~oyraphy. ~his ~anditioned m~terial was placed onto ~he sc3D6 lr~unDaf~inity column. A9 equilibration buffer a 100 mM Tris buffer, pH 7.4 wi~ 0.1% ~ 20 was used. Ihe recombina~t ar~:igen was elu~ed with 3 M
rhodanide. Ihe yields of the individual skages are summarized in Table 2.

E~ampl~ 2 A furth~r clomng of the sc3D6, m whlch the sc3D6 gene w~s fused with ~he gene for alkalLne phosph~ase ( ~ A~ isolated fm m Escherichia coli, ~lS carried out as folIows:
The sc3D6 gen2 was cut from the plasmid pUCsc3D6 ~ restriction enzymes and inserted in ~hQ vector pEc~hoAMut3 (19). The result mg vector carries the identification E~ D6. qhe vector ~EcFho~Mu~3 contains the gene for alkaline phsKphatase (20) isolated frcm Escheri~hia coli in which a restriction site was mutatEd in through oligo~u~leotidc-dircctel mutagenesis at the 3' end of the coding regio~, which permits the fusion of the ~cphGA
gene with other genes. In this way thrnugh expressian of a ~usion gene . . ~ ;, :.

Z~?~
1~
fusion pr~te~ns are produced i~e. pr~teins in ~ich t~e particulæ codir~
regions are lir~d ~ough pe~tide b~s via am~no acids.
~ he EcE~ sc3D6 ~usion gelle was cut ~ran p~::3D6 with ~striction enzymes ar~d ins~ ~o the baat~ial e~pression vector p~23-3 (manufact~: l~ar~cia, Sweden). me resultin~ plasm~d is id~ified as ~3D6 .
me plasmid p~aPsc3D6 was transformed irr~o ~he ~ihEæi~hia coli (5). Aft~ ir~ction wi~ l~rG active Ec~ - sc3D6 fusion pr~tein ~n ~he periplaE;m~c spa~:e of the bact~ia ~8 purified as folla~s ~e l~actexia wer~ harves~d t~ c~tri~atl~n and waqhed in 10 nM
u*fer, ~I 7.5 to which had ~ ad~d 30 ~I NaCl. Ihe wa~hed bacteria were resasp~2d in 33 nM l~ris buffer, pH 7.5 ar~ mi~d with an eq~al vol~
of 40% sac~har~ solu~ion in (33 nM Tris ~suffer) and E~ was added to final concentratioal of 0.1 n~. A~ter inc~atio~ for 10 mirn~ at man ~ahn~e the bact~#ria w~re c~ntri:eug~d ofî and placed into 0.5 nM MgC12 solutiorl. Aft~ an incubation time of 10 mi~ute3 at oC a protease ir~ibiti~n co~tail c~r:Lsin~ ~F and Etæ~ was ad~d and ~he bacteria centrifuged off. me supexr~t is brough~ wi~ 1 M Tris soluti~, pH 7.5 to a ~inal concentrat~on of 25 n~ Tris. 1~ this procedu~e the peripl~c spaoe of the E. ~li csells is ~.
Thr~ugh cent~i~ugation a~ 12000g t~e protein solution is clarified ar~d s~se~ue~tly oonc~ntra~l ~r~ ultrafiltratio:n.
The EcEhoA - sc3D6 pmte~n is ~ purified wi~h h5~droFhcibic ~nteraction ~na~y. A l~lse~harose Fast Fl~w (l~armacia, Sweden) was equilibrated with 60~6 sa~urated amm~nium sulfate solution in 25 n~ ~rris buffer, pH 7.5. ~ p~otein solution was placed on~o ~he col~an alternating with the e~il~ ion buffer. me sc3D6 is eluted with a linear gradi~nt of 60% ~noni~n s~lfate to 0% ~um ~ulfa~ rhe fractions ~idh conta~n t~e Ec~ioA ~ sc3D6 protein are desal~ated ~rou~h g21 filtra~ion.
After proving the f~nc~ic~alit~y of ~he Ec~A - sc3D6 protein, a Westexn Blot wi~h ~V-l test s~ (Bi~Rad, USA) was carried out. AS a control, an analogous test was carried out wit~h ~he nat~ ar~cibo~ isolate~ fr~m ani~
cells. As negative corl~ol s2n~ a pr~paration of total protein fmm E.
coli. ~rhe result of thi5 tes~ was positive and is sha~n in Illustration 8.

.. . ..
. ~ :
- ~
, ~ , .

.:
..

x~

Di~t proQf of ~ 1 anti~ o~ EIir~

A gp120~ ic mor~oclonal antibo~T ~Cloale 25 C2, A~cession No.
89120601, ~D:S, Porton D~n, ~ was coat~d onto m~c:roti~er plates (grade I, ~c, D~nark). HIV-l ccntaini~ cu:l~e s~tant (16) was placed onto ~:he coated micn~ er plates. P~ibinarrt gp160 (18) was used as ~tanda~.

a~plied and in~ated. lhe no~ mat~rial was aga~n rins~d and the baur~
E~hoa-sc3D6 prutein was d~nstrated Ebot=~t~ically a~ 602 ~n wit~
~te. In Illustxatio~ 9 ff~e s~ curve and differ~nt sa~le~ of Hl~ l positiv~ cult~r su~natarst ars depict3d.

Microkite~ plates (grade I, Nl~nc, D~mark) w~re coa~ed wit~h a solutiorl of 10 m~/~ reo~t ~p160 (18). h~ly the plates we3~e wa~ed with PE~S + Ool~ 20 + 196 ~
A solution of 5 llg/ml Ec~a~ - sc3D6 ~usi~ pr~ein was mixed at a ra~io of 1:1 wi~h HIV-l positive or HIV-l negative s~n and placed o~ ha coat~
plates. As corl~ol Ec~oA ~ sc3D6 fusion protein d.with dilution buffer was aF3?lied an~ inc~a~ed at 37C for ~0 m~utes. ~e nonbaur~ mate~ial was ~y ~ins~. ' ~ ~e additicn oP p-n~tr~lE~te the fraction of b~
Ec~aA - sc3D6 pr~ein was d~ted. me resultin~ color was q~antified Eh~ri~ly at 602 r~n. me ir~ibitior~ of the s~ra was det~rm~ned as p~t of the e~ tion of l~;cPhoA - sc3D6 pr~te~n with~rt ær~n. As depicted in Illus~ ion 10, all ~V-l p~sitive sera ird~ibit the bir~ir~ OI
Ecl~ - sc3D6 ~usion p~ote~n to gp160. All HIV-l nega~iv~ sera E~hawed less ir~ibition than t~e HIV-l positive sera.

Ar~o~ e~pre~si~n type for the sG3D6 pr~tein, in w~ch m~use ~el~ma cells we~e used as host cells, was carried out as follaw~:

. ., ~:
"
,~: . . . , ' , . ' . ~ ., i, :' 2~

The 3' portic~ of ~e sc3D6 gene was isola~ed fmm the plasmid pUCsc3D6 (SI~Q ID N~: 3~ ~ partial Ec~r di~estio~ as well as thra~ can~lete Hi~II dige~ioll (le~ of ~he fragmE~: 401 kp). E~n ~e plasl[id p~JC3~IC (SEQ IF N0: 1) the 3 ' porti~ of ~he gene for the heavy ~ihain was r~ved ~ c~ng wil~h Ecc~V an~l Hi~III. IrTto ~e n3mainir~ vector ~e 401 l~p frzlgmen~ of ~he sc3D6 gene isola~ throu~ agaro~ gel electr~phoresis and purified ~ i~ed~ l~e g~ne ~ined in ~ way conse~e~ly ca~ri~es the ~ ~or the leader p~de of ~he heavy ~hain of the a~tibod~ 3D6 follawed }:~y the seq~ of t~he sc3D6 g}~ne. 'rhe plasnid carries 'che identi~ ion pl~;c3D6. mis c~sb~uction p2rm~ts ~e ~ar~ort of ~he sc3D6 prc~tein in ~ cells. me c~ing c,~ne was iRolated frc~n p~c3D6 with t~he ~ NcoI ar~l Hin~l:II, ~he overhar~ ~s were filled with Xlenaw polym~ase an~ cloned into the SmaI si~e of the ex~ession vector ~RSV (~vitrogen, ~) su~table for animal cells. Ihe ~I site of ~is e3~?ression v~r lies be~e~n the lc~ ~min~ re~a~ of RSV, c~tly o~iginally fmm bovine gr~ hormo31. ~ ins~tion i~o ~is restrictia~ si~ i~ is possible to brir~ ary str~ re gene irrto a molecular ~vir~t ~ich permits ff~ ression of ffie genes in animal cells.
~, t~he vec~or ~ additionally has a ~lec ticm marlcer "ne~in resistance" w~ch ~ts ffie selecti~ of suacessfully t;ransfonn~d animal cells in t;he cul~re.
The plasm:Ld constm~ted in ~his way carries the ir~tification ,~iRdRSVI~~ It was t~fec~d in~o m~se ~yelama cells of line P3-X63-Ag8.653 (21~. A t~al of 5 clo~ w~re selected after selection of ~formed cells wi~h the antibiatic ne~in in 2 cl~ ar~d ~reenin~
~ i~h e~ressed the sc3D6 gene. ~he ~ressi~ level of ~e in~ividu~l cl~es were ~ ans of anti~ pcific E~irsA. ~h~ yields of the individual purification stages w~rd2 det~:mined k~ means of anltige~-Ihe culture superr~t containing the sc3D6 pn~tein of the t~ansfectedm~use ~elama cells was clarified by cer~ ugation at 5000 g in a bu~et centrifw~e. Ihe clæified s~rnatant was ~at~ by the 10-fold thr~ ultrafiltration (Minit~, ~rGC, c~ off 10000 Dalto~, mar~facturer Millipore) and diafilt~xd with a 50 nM Tris buffer, F~ 7.2 with the fiver 2~6 L~O

fold ~ol~.
Ihe d:iafilt~red pmkein solutior~ ~ purifi~d wit~ S~raF~o~;e Fast Flaw (m~ufactur~æ: ~rTnacia, Sw~d~) (eq~ilibration ~suffer 50 ~I ~ris l~uffe~, p~ 7.2). me elution ~ ~e s~3D6 protein took place with 150 ~M
NaCl. me purified protem wa~ ed ~ means of ar~tigen-~pecific ELISA.
Ihe yields of t~he indivi~ual purification stages are sha~ in Table 3.

E~le 4 Ihe plasmid p~VIsc3IX was transfected ir~to ~e ha~is~ cvary (CEIO) ~lls. In analog~s ma~ to ~t des~ in Exa~le 3, tx~sformed cells w~re selected and s~eenPd arx~ t~he sa3D6 pr~tein fram the cell culture supernatallt was purified. Testing of ~e expres~ levels ~ ~ of antigell~aific ELCSA brought val.~ 5 yg/ml antibodies.

E~l~ 5 Iq~e sc3D6 g~ne was cut fram t~he pla~mid pUCsc3D6 ~:ou~ restriction enxyme~ and ~ ~nto ~e yea~t ~58i~1 ~Tector pGl (Clonte~ :
~boratories ~c., Palo Alto, USA). In ~is con~truction the sc3D6 gene was placel un~ ~e r~gulatioal of the &~1 pr~er ind~lcible thr~ galactose.
qhe ~tr~ct was transfected into ~e sa~a~y~ c~visiae st~ain ~n~2 (t~pl) and ~e:L~ in ~o~di.mn withaut trypt~ for can~l~atlon oî ~e tr~ne a~y. Positive transformar~ts were isolated and utilized for the ~ti~ of sc3D~ pr~tein. q~e cc~itiors for the cultiva~icn of ~he ~3:ctic~ sl:rain as well as f~r the isolatial, pr~para~ion and puriflca~ion of the pro~ct were carried cut a~ ing to sta~æd protocols (22).

E~mp1~ 6 ~ e ~c3~6 gene was cut fmm the plasmid pUCsc3D6 th~ re~triction y ar~ ins~ted into th~ vactor p~:373 (23). mis reo~inant pla~nid was trans~ected together wi~ rN~ of the baculo~irus autogra~ha californica rmalear polyhedrosis virus ~A~lPV) into tl~e cell line Sf9 s~mni~ fram , , :: ' ' ~' . .

~podcptera frugiper~. me cultiva~ion of ~he Sf9 cells t~ place accc~rding to the staT~ar~ method desc~ibed in the Catalogue of the P~ican Type Culture ~llection. 3 to 5 days af~ the tr~sfection plague~ of reccm~ainant v~ were m~crosc~ically i~tifi~ an~ ~olated. In order to be s~re that ~he isolai:ed rec~inant virus were not cc~tamina~ed with wild t~pe v~, three f~e~ plaq!le purification processes follaw~d. Infeccior of Sf9 cells wit~h recarb~ virus a~ter 3 to 5 days lead to lysis of the infected cells arx~, c:o~nitan~ly wi~ , to ~e pm~ction of sc3D6 pn:~ein in ~he sul?ernatant of the c~ll lysate. Ihe sa3D6 pratein was, as des~ibed analogws~y ~n Ex~mple 3, pl~rified and analyse~ this way ~e functionali~y of ~his rec~inar~ p~ ~uld be d~.

The se~nce (2~) coding for the pr~ln avidin was p~ced as synthetic g~ne ~uqh syn~hetic oligarn~cleotides and E;pecifically so fflat additionally at the 5' d of t~e gene ~e seql~ence of the lead~ peptide for E. coli a~ine ~tase (20) and at ~e 3 ' d a polylir~er region for in~ing o~ g~nes is pres~t. Genes ins~d in ~is polylird~ gion are expre~ssed ~ suitable cor~itions as i~ian proteins with avi~ as fusion partner. With the aid of the 1~ loca~d a~ ~e 5' ~1, these fusion proteins were t~ans~rted in active ~orm into ~he periplasmic space of Es~erichia coli. mis constr~t was ~ i~to a 6u~able re~triction ~ite of the ~ac~i?l ~pression ~lector pEr-3a (25) ~ilih ~nt~ns Ior th~
~p~sion of cl~ geI~es the bact~i~hage T7-~10 ~ramoter as well æ the ~t~tor. me resultin~ vector carri~ ~e iderrtification pEr-3a~
Ihe ba~icEihage q~ o pr~ has the ch~tic of nG~ beir~
t~anscribed in E. coli cells in ~ absence of the bactericFiha~ T7 RN~
poly=e~s~. If, hawever, for es~ple a ~ely gr~n E. coli culture is infected wit~h a F~age vector ~ic~ carries ~he gene~ic inforr~tion for ffle T7 polym~ase, ~e t~her~ pro~ced r7 pol~ leads to the e~ sion of genes, ~i~h for ~le in vectors su~ as ~se des~ibed above are present in cloned fo~m. I~s ~acteristic is ~ery important for ~e e~pression of avidin and avidin fusion proteins in E. coli since the avidin is taxic for g~ing E. ~::oli cultures.

2~?~'.q ~

Ihe so3D6 g~ne wa~ aIt fram t~e vector pUCsc3D6 ~ restriction enzyn~ ar~l ins~ into ~he polyli~ region of the vector pEr~3a-Av. m~
result~ vector carries the iderltification pl~3a-Av-sc3D6. Suitable E.
coli host cells (for exa~le ~;174~ were transformed with this vec~or arx3 cultivat~d. AS soan as the culb~re had rea~hed an OD600 f 0.6, infectian wae carried out with the bacteri~Eihage CE6 (lalbbda cIts857Sam7) (25) whicih carries the bac~eriophage T7 genel. m e ~ y formed T7 polymrase led to the expression of the avidin-sc3D6 fusion prokein in ~he periplasmic space of the E. coli. AS suon ~s the expre~sicn had reached its maximum (dependlng on the culture condi~ions be*ween 3 and 12 hcurs after inection with the phage), the recrmtlnant prctein was, analogtusly to the manner described in Example 2, freed anl conc~ntrated ~hraugh ~ ~ filtration.
The oonoantratel prctein sDlution was fur~her purified cver Sephacryl S
20~ (Pharmacia) and ca~centrlte~ for a second time by m~ans of ultra~iltration. This solutio~ is plao~d onto a biotin column. The correspondlng fusicn proteLn avidln~sc3D6 rema ms specifically bound. The ~ ities are rinsed cut. The affinity column p ~ in this way w used for the purification of reccmbinant gpl60 analogously to Example 1, i.e. the prote m sDl~tion prepurified ac~oxding to Barret.t et al. (18) was placed onto the affinity column and after rinsing out the no~baund material the recombLnant gp160 was eluted wlth 3 ~ rhodanide. The yields achieved thereby e~re analog~us to the results ahQWn in Table 2.

, . , .. : -,- , ,, , .: , . :, : : I

, -"

2~

S~Q ID NO: 1 Iype of Sequer~ rmcle~tides wi~ ~ espor~ng pr~*e~n I~th o~ sequeIxe: 1548 base pairs S~ ~orm: single strarld Type o~ cule: plaE~m~d 1~ with i~ OI ~nan cDt~
Origin:
Organ~sm: human ~nedia~e ~rin~t~ origin:
Name o~ cell l~ne: 31~6 ~c~stics:
~ram1 to36 ~p plasmid p~JCl9 polylir~cer 37 1527 insert h~av~ c~hain of ar~tibo~ 3D6 37 98 5' n~translated r~i~
99 1526 codi~ ~ion 99 155 signal ~ide 15~15~6 mature pel?tide 156 533 variable regi~
156 245 fr~rX 1 246 260 ca~l~i~y det~mi~ r~gic~ 1 261 302 ~r~rk 2 303 353 campl~it~y determirl~r~ r~giorl 2 354 449 frz~k 3 450 500 o~l~it~ det~mining re~ion 3 501 533 frz~rk 4 534 1526 : ~ons~ r~gi~
~ 1527 1547 plam~id Euc 19 pol~li~?~
Characteristics: c~ clone o~ the heavy ~hain of t~e antibo~r 3D6 ~nserted into the plasmid pUCls.

G~C G~ ~aOE~ G~a~ ~r 60 MEr Glu Leu Gly Ieu Ser l~rp Ile E~e L~u Leu Ala Ile Ieu Lys ,:

Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu : ~ , ; , ,~ :, , :

Val Gln ~ro G~y Arg Ser Ieu Arg Ieu Ser ~s Ala Ala S~ Gly ITC ~CC TrT ~ GP~r ~ G~C ~G C~C ~G G~C ~G ~A G~ 278 l~he qtrr ~ A~3n ~p Tyr Ala MEr Elis Trp Val Arg Gln Ala l~ro 3~ 35 40 Gly Lys Gly Le~l Glu q~:p Val S~r Gly Ile Ser Trp Asp S~ Se~
45 5~ 55 A~ GGC ~ GCG G~C TCr ~G AAG GGC C~ TrC ~CC ~C ICC 368 Se~ Ile Gly Tyr Ala Asp S~r Val Ly~ Gly Arg l~e Ihr Ile Ser AGh G~ A~ ~ ~ A~C TCC C~rG ~r cr& C~A A rG ~ A~r C~rG 413 Arg ~p Asn Ala LYB Asn 5e~ ~u l~yr Ieu ~:ln MEr i9æn Sar Ieu Arg :Ala Glu Asp MEr ~a ~u Tyr Tyr Cyæ Val Lys Gly Arg Asp :~:
~C ~ &~ A~ G~ G~ q~ q~ AaG GTr GC~r m G~ ~ ~GG 503 q~yr Tyr Asp Ser Gly Gly Tyr E~e q hr Val Ala E~e Asp Ile Trp 1~5 110 1~5 ~120 ~GC ~A G~ AC~ ArG ¢rc AC~ GrC TCr TC~ G~ TC~ ~CC A~G GGC 548 Gly Gln Gly ~r MEr Val ~hr Val Ser Ser Ala Ser q5~ Lys Gly CC~ TCG GTC ITC COC CrG GC2. ~C l~CC ~C A~G AGC ACC I'cr GGG 593 Pro Ser Val Eh~ Pro Leu Ala Pro Ser Ser Ly~ Se~ ~ Ser Gly ...-.. .. .
, . ", ........
.,, . ......

2~

GGC AC~ GOC CrG GGC TGC CrG ~ A~k: G~C ~C Tr~ A 638 Gly Thr Ala Ala Leu Gly ~ ~eu Val Lys A~ Tyr ~e ~o ~lu 155 1~0 165 ~ro Val 'rhr Val Ser l~ ;n Se~ ~;ly Ala Leu ~hr Ser Gly Val CAC Aac Tr~ C~ GCr ~rc ~ C~G TaC ~ ~ cr~ I~C T~C cr~ 728 ~His Thr ~!he l~ro Ala Val leu Gln Ser Ser ~i:ly L~u Tyr Ser Ieu AGC AGC GTG GrG ACC QTG ~C TCC AGC AGC l'rG GGC ACC ~ ACC 773 Se~ Ser Val Val Thr Va} :E~ro S~r 5er Ser leu Gly mr Gln ~hr q~C ArC IGC A~C ~rG ~ C~C AAG ~ A~:C AAC AOC i9a~ Gr~ G~C 818 Tyr Ile C~ys Asn Val Asn His Lys ~ro Ser ~n ~rr :Lys Val Asp :

Lys Lys Val Glu Pro Lys Ser ~ys AsE~ Lys mr Hi~ '~r Cys Pro : :
.

Pro t~ys Pro Ala l~ro Glu Ieu Leu Gly t~ ro Se~ Val ~e ~u 245 250 ~ 255 T~ C~ CC~ A~ CCC AA~ G~C ~CC ~C ~ A~rC TCC CGG ACC CC2 953 A~ rG ~C ~ C C~C t~A ~C ccr G~ 998 Glu Val ~r Cys Val Val Val A~;p Val Se~ ~i6 Glu ~p Pro Glu GrC ~ TrC A~C TGG ~C ~ G~C GGC ~ ~ GT~ C~ A~ GC~C 1043 Val Lys ~e A~ qrp Tyr ~7al Asp &ly Val Glu Val His Asn Ala 290 ~35 300 A~: AC~ A~G COG a3G ~ G~: C~ ~C AaC TCC A~ C COE GrG 1088 Lys Thr Lyæ l~ro Arg Glu Glu ~ yr Asn Ser l'hr Tyr Arg Val ~C AGC G~rC ~rc ACC ~rc ~G C~C ~ GAC T~: C~G A~ GGC AAG 1133 V~ Ser Val Leu mr Val Leu His Gln Asp Trp Leu A~n Gly Lys Glu Tyr Lys ~ys Lys Val Se~ AS~ hys Ala leu l~ro .~a l~ro Ile ~ A~ A~ A~rC ~C A~ GOC AA~ GGG ~: COC ~ W. ~ C~: 1223 Glu Lys ~hr Ile ~e~ Lys Ala Ly~ Gl~ Gln ~ro Arg GlU ~ro Gln GCG~l~C A~C C~; ~ CC~ ~ OGG ~ G~G ~: A~ ~AG Ai~C C~G 1268 Val T~ Ieu Pro ~ Se~ ~ ~p Glu Ieu mr Lys Asn Gln 365 370 ~ 375 OEC ~GC C~G A~C ~C CIG GrC A~ GGC TrC ~r CCC AGC GAC A~rC 1313 Val Ser leu Ihr Cys ~eu Val Lys Gly E~e Tyr ~ro Ser ~p Ile GCC GrG GAG TGG G~: A~C ~ GGG ~G CCG GAG ~C A~C ~C A~G 1358 Ala Val Glu Trp Glu Ser Asn Gly Gln ~ro Glu Asn A~ Tyr Lys ZC?~

ACC ACG C~ OOC ~IIG CI~ (~S:~ IY~C GAC t~C q~:!C 1~ ~ c!IC l!~C 1403 Ihr Ih:r ~o Pro Val Ieu Asp Ser A~p Gly S~ ~ ~he I~u Tyr AGC ~ crc ACC ~lÇ: G~ A~i: ~GC AGG IY;G C~G C~: GGG ~C (~C 1448 Se~ Ly8 leu Thr Val AE;P LYE; Se:r Arg Trp Glll Gln Gly Asr~

ITC 1~ 1~ l~C Gl'G A~ C AAC C~C IAC AC~ 1493 ~he Se~ Cyi3 Ser Val MhT Hi8 Glu A1a Leu Hi8 A~l Hi5 Tyr Ihr CP.G A~G AGC C!IC I~C Cl~ 1~ Ct~G GGr AAA q~ 1526 G1I1 Ly~ Ser leu Ser Leu Ser ~ix) Gly L~3 5tc~p ~: 455 460 G;~GG ~1~ T 1547 :

.. - . .. .

:

2~

SEQ ID NO: 2 Type of Sequ~:e: ~cleatides wi~h corresponding prat~3in h of se~ce: 9d,5 ~ase pa~:s S~ ~orm: s~le st~ar~
q~pol~gy: c ~ ar q~pe o~ lecule: p:La~nid ~ with in~t of h~nan c~
Orig~n:
Organism: h~nan ~ate experim~tal orig~n:
Name o~ cell li~: 3D6 Q~is~
~f:= ~1 to 21 l:~p plas~ d pUCl9 polylir~r 22 732 iT~S~t light o~ antibod~ 3D6 22 27 5' n~lated region 28 732 c~di~ :regiCltl 28 93 Sil3rlal pep~:ide 94 732 mat~ tide 94 40~ variable regl~n 94 162 f~rk 1 163 195 can~l~tari~ dst~minir~ region 1 196 240 ~rk 2 24~ 261 a~ple~t~ determi~ ion 2 262 357 fr~rk 3 ~358 378 ca~lementari~ determ~gion 3 379 408 fr~rk 4 409 732 ~ regio~
:: :733 :905 3' nontrarLsla~ed region : : ; 906 : 945 ~ plasmid plJC 19 polylir~
:
.

c:~arac~istics: ~ ~ c~ c~ e o~ ~he ligh~ ch~ir~ of ~he ar~tibo~ 3D~ ins~ted into~ ~pla~mid pUCl9.

MEr Asp ~ Arg Val ~ro Ala Gln ~eu ~eu G1~ ell Leu -18 -13 ~ -8 ~p Leu l~ro Gly Ala ~ys ~ys A3p Ile ~ln IlEr Ihr Gln S~r l~ro , - :, ~ .
, ~ .
, , . . .

- 2~?~7~

T~C ~CC CrG TC~ GCaA ICr G~ GG~ G~C A~ ~C Aac ~rc ACr ~rGC 162 Ser qhr Leu Ser Ala Ser Val Gly ~sp ~ Val qhr Ile Ihr ~ys Arg Ala Ser Gln Ser Ile Ser Ar~ l'rp Ieu Ala Trp I~r G~n Gln AAA CC~ GGG AAP. GT~ Ct2 AA~ cr~ C$G A~ ~r A~G GCa. ~r A~r 252 ro Gly Lys Val Pro Ly~ Leu I~U Ile l~r LYB Ala Ser Se~

q~ G~ r GGG ~rc ccP~ TC~ A~G ~ AG~ GGC ~r GGa. T~r GGG 297 ~Leu Glu Ser Gly Val Pro Ser Arg l~ha Ser Gl~ S~ ~ly S~r Gly ~CA G~ TTC A~ CI~C ACC A~ AGC A~ ~rG C~; CX~ Ga!r ~r m 342 : ~hr Glu ~he q~r Leu Thr Ile Ser Ser T~- Glll l~o A~p Agp ~ :' Ala ~r~ yr Cys Cln Gln Tyr A~n S~ r Se~ Phe Gly l~ro , GGG AC~ AAA GIG G~r A~C A~ 0~ ~r GrG G~r GCZ~ cr Gr~ 432 Gly mr L~; Val Asp Ile LYB ~rg Ihr Va1 Ala Ala Pr~ S~ Val 103 108 ~ : 113 ' ITC ArC ITC ca~ rcr GAr ~; CihG ~TG AAA ~r GGA ACT G~C 477 l~he Ile ~he Pro Pro Ser A~p Glu Gln Ieu Lys Ser Gly Ihr Ala . . . - .
, ~ .
' : . , .' .. . .
' . ' ' ' - : . : `
' ~?;~

Ser Val Val Cys Leu Ieu Asn A~ ~he Iyr l~ro Arg Glu Ala ~ys ~ C~G qGG Aa~ ~G G~r Ah~ GOC CTC C~ IY~ C TCC C~ 567 Val Gln Irp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 1~8 153 158 G~G A~r ~C AC~ i; ~C A~ G G~C ~ P.DC ~C ~S~ 612 Glu Ser Val Ihr G~u Gln AE~P Ser Ly~ Asp Ser Ihr q~r Ser Leu ~GC ACC CrG AQ; C~rG W A~A GC~ G~C 12~C G~G AAA QC AA~ 657 Ser Ser lhr Leu lhr Leu Ser Lys Ala A~p Tyr Glu Lys His Lys ' Gl'C ~C GCC TGC G~ rC AaC c~ G GGC cr~ C~ GTC 702 Val Tyr Ala Cys Glu Ual ~r His Gln Gly I~u S~r Ser E~ Val 193 198 ~03 .
ACA A~ A~ AAC P~GG GGh G~G ~GT q~ 732 ~hr Lys Ser Phe Asn Ar~ Gly Glu C~ys Stcp CAC~ T~ C~GAaXX~ TI~IT T~ ~Im~ 792 AC~C TAa~T G~altX~CC W~IT ~CC t~:C 852 TCC~3FIT TAT~IGCT ~G~ GeG~AT AP~ A~3!C 912 C~C Gt~a:~ C}~ IY~G ~ 945 .. ... ..
.
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.

;~r~6~

S~Q ID N0: 3 th of se~ 776 }~ase Strand f~ s~le strand Topology: c~ar Type of r~lecule: plasmid ~ with ins~t of ~ine~ed human cl~
orig~n:
organism: h~nan ~liate ~per~ntal origin:
Name of cell line: 3D6 ~cteri~tics:
fr~n1 to13 l~p pla~;mid pUC19 polylird~
14 760 ir~t so3D66 14 16 start cod~
14 394 variable regio~ vy chain 17 106 f~rk 1 heavy ~ihain 107 121 ~plemersl:arity d~n~ region 1 heavy ~ihain 122 163 framework 2 heavy ~
164 214 ca~plemer~il:y det~ni~ regi~ 2 heavy cihain 215 310 ir~rk 3 heavy cihain 311 361 ~l~arit~ det~n~ n~i~n 3 heavy dhain 362 394 f~r~c 4 hea~ ~hain 395 440 lir~
441 760 variable r0gi~ ligh~ ~ain 441 508 f~ewo~k 1 l~gh~ dhain 509 542 cc~le~nl:arit~y deter~ regic3n 1 light cihain 543 5S8 f~work 2 li~h~ dhain ~89 607 ~ple~n~arit~ det~lni~ re~ioll 2 li~ dhain 608 703 fr~work 3 li~ht ~
704 724 ca~le~it~r det~ini~r7 r~gic~ 3 li~ht ~hain 725 757 framework 4 li~ht ~n 758 750 st~ codc~
761 776 pldd pUC ~9 polyli~
Characte;ristics: Clone o~ ~e ~ine~d s~n~le~ha:ln Fv fra~ement of the antibody 3D6 i~ ~rlto the pla~;mid ~UCl9.

~1~ CCC 13 ATG ~A Gr~ C~ c~r& GTG ~: ICr GGG GG~ GGC ~ GrP~ C~G ~ 58 MEr Glu Val Gln I~u Val Glu Se~ Gly Gly Gly 12u Val Glrl E~

Gly Arg Ser I~u Arg Ieu S~ s Ala Ala ~e~ Gly ~he Ihr ~e . .

2~

Asn A~p Tyr Ala MEr His I~p Val An3 Gln Ala Pro Gly Lys Gly Ieu Glu Trp Val Sç~r Gly Ile Se~ Trp A~p 5e~ Ser S~ Ile Gly 6û

lY~ GOG G~C T~ ~ Aal; G5C ~ ~ AOC ~ ~roc AG~ C 238 l~r Ala Asp Ser Val Lys Gly Arg ~e mr Ile Ser ~rg ABP Asn GC~C AA~ ~C TCC ~& ~ ~ CaA Am A~C A~ C~G A~ GCl ~G 283 Ala L~s Asn Ser Leu Tyr Leu Gln I~ Asn Se~ I~U Arg Ala G1U
~0 85 90 GI~C ~G GOC q~ q~ q~ 1~ ~ A~ A~ G~ q!~ ~I' GP~ 328 A~p MEr Ala Leu Tyr Tyr C~ys Val Lys Gly Arg Asp q~ I~ Asp 95 100 lû5 At~ GG~ GOE qP~ 11~ AaG OE~ GCr 1~ G~ A~ T~ A GGG 373 Ser (:ly Gly q~r ~he mr Val Ala ~e Asp I1Q ~p Gly Gln Gly 110 115 12~

AC~ ~GrC AaC ~C ~ TC~ r GGC TaG GGC Gt~ r 418 Ihr MEr Val mr Val Sex Se~ Gly Gly Gly Gly Se~ Gl~ Gly Gly 1~5 130 135 Gly Ser Gly Gly Gly Gly Ser AEP Ile Gln 2~ Thr G~n Ser Pro .

;~'5~

T~C ACC CrG TCr GC~ I~r (~ ~ G~C A~ GrC ACC ArC A~ I~C 508 Ser Thr I~u Ser Ala S~r Val Gly Asp Ar~ Val ~hr Ile l~r (~ys 155 16~ 165 A~g Ala Ser Gln Sex Ile Ser Arg Trp Ieu Ala Trp ~yr Gln Gln Lys ~ Gly Lys Val Pro I,ys ~eu ~u Ile Tyr Lys ala Ser Ser Leu Glll Ser Gly ~al Pro S~ Ar~ ~he Ser Gly Ser ~ly S3r Gly AC~ GA~ 1~ A~T CIC AaC AlC ~C AGC ~ cx~r G~ GAl' ~ 688 mr Glu l~he Thr Lell Ihr Ile Se:r S~r Leu Gln E~ro A~p A3p ~e GC~ ~cr qY~r I~C T~ C~ ~T A~ A~ ~r 1~ Il~ Gæ a~ 733 Ala Ihr Tyr Tyr ~s Gln Gln 1~ Asn S r 1~ Ser E~e Gly l~ro GGG ~ GA~r A~C A~A C~ q~aA 760 Gly Thr Lys Val As~p Ile Ly~ ~ St~

GCll~C CP~CI~ : 776 . ' ' '" '.' .
' ' ' ' 7~

Ta~les ly~ozS~n~3 0.2 m~/ml ~Naæ 15 U/ml 15 U/ml E~ 100 ~le 1: Fi~al oa~tra~ of ~e lysis ~mi~als in ~e cell su~sia~

Stage ~olume ~ml) }~:e~n (3~)gpl60 (~) Yield (g6) E~ cti~ 7~00 38,200600 lO0 Lerrtil S~ar~se 520lO00 372 62 sc3D6 af~in~l~y 130 148 144 24 ~ ~o Table 2: Yield ~ ~e ~dividual stages of 1~e ~noaffinit~
: c~natogra~ihic p~rific~ati~ o~ ~i~arlt ~160 wi1~h ~c3D6 as afflnity li~

S~a~e Voïum~ (m~) ~te:Ln (nq) Titre ~ltare ~enat~t3500 7600 1:25~
~afiI~:t:io3l 350 5300 1:2048 Q-S~ 50 72 1:10000 :
~3: Yie1d of ~e i$~iv.i~tual stages of the p~rificatioql of sc3D6 prate~n fram ths cul~ure sup~natarrt o~ o~d n~use ~elama ce11s.

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Claims (12)

WE CLAIM:

1. A recombinant protein binding to a complex viral antigen of HIV-1, characterized in that it comprises the variable regions of an antibody derived from the cell line 3D6.

2. Recombinant protein as stated in Claim 1, characterized in that it comprises the variable region of the heavy chain according to SEQ ID NO: 1.

3. Recombinant protein as stated in Claim 1 or 2, characterized in that it comprises the variable region of the light chain according to SE ID NO: 2.

4. Recombinant protein as stated in one of Claims 1 to 3, characterized in that it is constructed according to SEQ ID NO: 3 wherein the variable region of the heavy chain is connected with the variable region of the light chain through a linker.

5. Process for the production of a recombinant protein as stated in one of Claims 1 to 4, characterized in that a DNA insertion sc3D6 or a sequence hybridized with this insertion or a sequence derived through degeneration from the expressed protein is introduced into a plasmid, transforms a host with this plasmid, and that the construct is expressed.

6. Process for the production of a recombinant protein as stated in Claim 5, characterized in that it is expressed as a fusion protein, in particular together with alkaline phosphatase or together with avidin.

7. Insertion for use in the process as stated in Claim 5, characterized in that the insertion sc3D6 has the nucleotide sequence given in SEQ ID NO: 3.

8. Process for purifying the recombinant protein as stated in one of Claims 1 to 4, characterized in that specific antibodies against the protein and/or against the linker are emplaced between the two variable portions.

9. Process as stated in Claim 8, characterized in that the antibodies used for the purification are immobilized on a carrier.

10. Process for the isolation and/or purification of HIV-1 antigen, characterized in that the isolation and/or purification takes place through affinity chromatography, wherein, if necessary after suitable prepurification, the sc3D6 protein or the avidin sc3D6 protein is used as ligand for the affinity chromatography.

11. Process for the direct proof of HIV-1 antigen, characterized in that a fusion protein comprising EcphoA
sc3D6 protein is utilized as combined detection and signal protein.

12. Process for the proof of HIV-1 positive sera in competitive immune assays, characterized in that a fusion protein is utilized which comprises EcphoA so3D6 protein as combined detection and signal protein.