CA2078545A1 - Hiv envelope polypeptides - Google Patents
Hiv envelope polypeptidesInfo
- Publication number
- CA2078545A1 CA2078545A1 CA002078545A CA2078545A CA2078545A1 CA 2078545 A1 CA2078545 A1 CA 2078545A1 CA 002078545 A CA002078545 A CA 002078545A CA 2078545 A CA2078545 A CA 2078545A CA 2078545 A1 CA2078545 A1 CA 2078545A1
- Authority
- CA
- Canada
- Prior art keywords
- residues
- amino acid
- antibody
- thr
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
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- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Communicable Diseases (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Novel isolated physiologically active polypeptides are provided, as well as antibodies directed against the isolated polypeptides.
Methods for the preparation and pharmaceutical use of the polypeptides and antibodies are also provided.
Methods for the preparation and pharmaceutical use of the polypeptides and antibodies are also provided.
Description
wo 91 / 1551 2 PCI /US91 t~21 66 HIV ENVELOPE POLYPEPTIDES
Field c~ th~ Invçn~iQn This invention is concerned with anti~ens of ths HIV virus, and to novel physiolo~icallyactive polypeptides found in the HIV env olycoprotein.
5 ~ackaround of the Invention Acquired immunodeficiency syndrome ~AIDS~ is caused by a retrovirus identified as the human immunodeficiency virus IHIV). A number of imrnunolo5~ic abnormalities have been described in AIDS includin~ abnormalities in B-cell function, abnormal antibodv response, defective monocyte cell function, impaired cytokine production, depressed natural killer and 10 cytotoxic cell function, and defective ability of Iymphocytes to reco~ni2e and respond to soluble anti~ens. Other immunologic abnormali~ies associated with AIDS hav~ been reported.
Amon~ the more important immunolo~ic defects in patients with AIDS is tha deplelion of the T4 helper/inducer Iymphocvte population.
In spite of ~he profound immunodeficiency observed in AIDS, the mechanismls) 15 responsible for immunodeficiency are no~ clearly understood. Several postulates exist. One accepted view is that defects in immune responsiveness are due to selective infection of helper T cells by HIV resultin~ in impairment of helper T-cell function and eventual depletion of ceils necessary for a normal immune response. /n vitro and in vivo studies showed that HIV can also infect monocytes which are known to play an essential rolq as accessory cells 20 in the immune response. HIV may also result in immunodeficiency by interferin~ with normal cytokine production in an infected cell resultin~ in secondary immunodeficiency as for example, IL-1 and IL-2 deficiency. An additional means of HlV-induced immunodeficiency consists of the production of factors which are capable of supprassin~ the immune response None of these models resolves the question of whether a component of HIV per se, rather 25 than infection by replicative virus, is responsible for the immunolo3ic abnormalities associated with AIDS.
The HIV env protein has been extensively described, and the amino acid and RNA
sequences encodin~ HIV env from a number of HIV strains are known (Modrow, S. et dl~ J.
Viro/oDy ~1~21: 570 (19871. The HIV virion is covered by a membrane or envelope derived 30 from the outer membrane of host cells. The membranq contains a population of envelope ~Iycoproteins l~p 160~ anchored in the membrane bilayqr at their carboxyl tr~rminal re~ion.
Each ~Iycoprotein contains two se~ments. The N-terminal se~ment, called ~p120 by virtue of its relative molecular wei~ht of about 120kD, protrudes into the aqueous environment surroundin~ the virion. The C-terminal seqment, called ~p41, spans the membrane. qpl20 35 and ~p 41 are linked by a peptide bond that is particularly susceptible to proteolytic cleava~e, see e.~. McCune et al., fPOApplication No. 0 335 635, priority 28 March 88 and references cited therein.
wo 91tlS~ PCI~tUS91/02166 Th~ major envel~pe olyc~prot~in (~p120) of HIV-1 has been the object of intensive investi~ation since the initial identification of itlV-1 as the etiological aUent of AIDS
IBarre-Sinoussi et a/., 1983). The ~p120 molecule is of interest as a vaccine candidate li3erman et ~/., 1988; Arthur et al., 1987), as the mediator of viral attachment via the virus receptor CD4 IDalgleish et al., 1984; Klat~man er al., 1984) and the spread of the virus by cell-to-cell fusion (syncytia formationl, and as an aoent with immunosuppressive effects of its own ~Shalaby et ol., 1987; Diamond et ~1., 1988). It is aiso a potential mediator of the patho~enesis of HIV-1 in AIDS ~Siliciano et ~/., 1988; Sodroski et al., 19861 and has been su~gesled to be the viral protein most accessible to immune attack.
Currently, gp120 is considered to be the best randidate for a subunit vaccine, because:
li) 0p120 is known to possess the CD4 bindin~ domain by which HIV attaches to its tar~et cells, ~iil i-ilV infectivity can be neutralized in vitro by antibodies to gp 120, liii~ the majority of the /n vi~ro neutralizin~ activity present in the serum of HIV infected individuals can be removed with a ~p120 affinity column, and liv) ~he gp120/~p41 complex appears to be essential for the transmission of HIV by cell-to-celt fusion. See, e.g. Hu et ~1., N~ture 328:721-724 ~1987~ ~vaccinia virus-HlV ~ recombinant vaccine~; Arthur et a/., J. Viro/.
63~121: 5046-5053 ~1989) lpurified gp120~; and Berman et a/., Proc. Natl. Ac~d. Sc;. USA
85:5200-5204 11988) ~racombinant envelope ~Iycoprotein gp120).
The gp120 molecule is synthesized as pan of a membrane-bound ~Iycoprotein, gp160~AIIan et a/., 1985~. Via a host-cell mediated process, gp160 is cleaved to form gp120 and the inte~ral membrane protein ~p41 ~Robey et ~/., 1985h Together gp120 and gp41 form the spikes observed on the surface of newly released HIV-1 virions ~5elderblom et a/., 1987).
As there is no covalent attachment between ~p120 and gp41, free gp120 is released from the surface of virions and infected cells tGelderblom et a/., 19851.
The ~p120 molecule consists of a polypeptide core of 60,000 daltons; extensive modification by N-linked glycosylation increases the apparent molecular weight of the molecule to 120,000 tLaskY et a/., Science, 233:209-212 ~1986)). The amino acid sequence of ~pl20 contains five relatively conserved domains interspersed with five hypervariable domains ~Modrow et al., J. V/rolo~y 6112~:570 ~1987~; Willey et al., ~roc. N~tl. Acad. Sci.
USA 83:5038-5042 ~1986~. The hypervariable domains contain extensive amino acid substitutions, insenions and deletions. Sequence variations in these domains result in up to 25% overall sequence variabilitv between ~pl20 molecules from the various viral isolates.
Despite this variation, several structural and functional elements of gp120 are highly conserved. Amono these are the ability of gp120 to bind to the viral receptor CD4, the ability of ~p120 to interact with ~p41 to induce fusion of the viral and host cell membranes, the positions of the 18 cysteine residues in the gp120 primary sequence, and the positions of 13 of the approximately 24 N-linked glycosylation sites in the ~pl 20 sequence.
Field c~ th~ Invçn~iQn This invention is concerned with anti~ens of ths HIV virus, and to novel physiolo~icallyactive polypeptides found in the HIV env olycoprotein.
5 ~ackaround of the Invention Acquired immunodeficiency syndrome ~AIDS~ is caused by a retrovirus identified as the human immunodeficiency virus IHIV). A number of imrnunolo5~ic abnormalities have been described in AIDS includin~ abnormalities in B-cell function, abnormal antibodv response, defective monocyte cell function, impaired cytokine production, depressed natural killer and 10 cytotoxic cell function, and defective ability of Iymphocytes to reco~ni2e and respond to soluble anti~ens. Other immunologic abnormali~ies associated with AIDS hav~ been reported.
Amon~ the more important immunolo~ic defects in patients with AIDS is tha deplelion of the T4 helper/inducer Iymphocvte population.
In spite of ~he profound immunodeficiency observed in AIDS, the mechanismls) 15 responsible for immunodeficiency are no~ clearly understood. Several postulates exist. One accepted view is that defects in immune responsiveness are due to selective infection of helper T cells by HIV resultin~ in impairment of helper T-cell function and eventual depletion of ceils necessary for a normal immune response. /n vitro and in vivo studies showed that HIV can also infect monocytes which are known to play an essential rolq as accessory cells 20 in the immune response. HIV may also result in immunodeficiency by interferin~ with normal cytokine production in an infected cell resultin~ in secondary immunodeficiency as for example, IL-1 and IL-2 deficiency. An additional means of HlV-induced immunodeficiency consists of the production of factors which are capable of supprassin~ the immune response None of these models resolves the question of whether a component of HIV per se, rather 25 than infection by replicative virus, is responsible for the immunolo3ic abnormalities associated with AIDS.
The HIV env protein has been extensively described, and the amino acid and RNA
sequences encodin~ HIV env from a number of HIV strains are known (Modrow, S. et dl~ J.
Viro/oDy ~1~21: 570 (19871. The HIV virion is covered by a membrane or envelope derived 30 from the outer membrane of host cells. The membranq contains a population of envelope ~Iycoproteins l~p 160~ anchored in the membrane bilayqr at their carboxyl tr~rminal re~ion.
Each ~Iycoprotein contains two se~ments. The N-terminal se~ment, called ~p120 by virtue of its relative molecular wei~ht of about 120kD, protrudes into the aqueous environment surroundin~ the virion. The C-terminal seqment, called ~p41, spans the membrane. qpl20 35 and ~p 41 are linked by a peptide bond that is particularly susceptible to proteolytic cleava~e, see e.~. McCune et al., fPOApplication No. 0 335 635, priority 28 March 88 and references cited therein.
wo 91tlS~ PCI~tUS91/02166 Th~ major envel~pe olyc~prot~in (~p120) of HIV-1 has been the object of intensive investi~ation since the initial identification of itlV-1 as the etiological aUent of AIDS
IBarre-Sinoussi et a/., 1983). The ~p120 molecule is of interest as a vaccine candidate li3erman et ~/., 1988; Arthur et al., 1987), as the mediator of viral attachment via the virus receptor CD4 IDalgleish et al., 1984; Klat~man er al., 1984) and the spread of the virus by cell-to-cell fusion (syncytia formationl, and as an aoent with immunosuppressive effects of its own ~Shalaby et ol., 1987; Diamond et ~1., 1988). It is aiso a potential mediator of the patho~enesis of HIV-1 in AIDS ~Siliciano et ~/., 1988; Sodroski et al., 19861 and has been su~gesled to be the viral protein most accessible to immune attack.
Currently, gp120 is considered to be the best randidate for a subunit vaccine, because:
li) 0p120 is known to possess the CD4 bindin~ domain by which HIV attaches to its tar~et cells, ~iil i-ilV infectivity can be neutralized in vitro by antibodies to gp 120, liii~ the majority of the /n vi~ro neutralizin~ activity present in the serum of HIV infected individuals can be removed with a ~p120 affinity column, and liv) ~he gp120/~p41 complex appears to be essential for the transmission of HIV by cell-to-celt fusion. See, e.g. Hu et ~1., N~ture 328:721-724 ~1987~ ~vaccinia virus-HlV ~ recombinant vaccine~; Arthur et a/., J. Viro/.
63~121: 5046-5053 ~1989) lpurified gp120~; and Berman et a/., Proc. Natl. Ac~d. Sc;. USA
85:5200-5204 11988) ~racombinant envelope ~Iycoprotein gp120).
The gp120 molecule is synthesized as pan of a membrane-bound ~Iycoprotein, gp160~AIIan et a/., 1985~. Via a host-cell mediated process, gp160 is cleaved to form gp120 and the inte~ral membrane protein ~p41 ~Robey et ~/., 1985h Together gp120 and gp41 form the spikes observed on the surface of newly released HIV-1 virions ~5elderblom et a/., 1987).
As there is no covalent attachment between ~p120 and gp41, free gp120 is released from the surface of virions and infected cells tGelderblom et a/., 19851.
The ~p120 molecule consists of a polypeptide core of 60,000 daltons; extensive modification by N-linked glycosylation increases the apparent molecular weight of the molecule to 120,000 tLaskY et a/., Science, 233:209-212 ~1986)). The amino acid sequence of ~pl20 contains five relatively conserved domains interspersed with five hypervariable domains ~Modrow et al., J. V/rolo~y 6112~:570 ~1987~; Willey et al., ~roc. N~tl. Acad. Sci.
USA 83:5038-5042 ~1986~. The hypervariable domains contain extensive amino acid substitutions, insenions and deletions. Sequence variations in these domains result in up to 25% overall sequence variabilitv between ~pl20 molecules from the various viral isolates.
Despite this variation, several structural and functional elements of gp120 are highly conserved. Amono these are the ability of gp120 to bind to the viral receptor CD4, the ability of ~p120 to interact with ~p41 to induce fusion of the viral and host cell membranes, the positions of the 18 cysteine residues in the gp120 primary sequence, and the positions of 13 of the approximately 24 N-linked glycosylation sites in the ~pl 20 sequence.
3~ A ~ ~:
Many work~rs in the fi~ld hav~ prspared mutagenic and fraçlment variants of ~p120.
Sae, e.~.: Matsushita er al., _'. Vitolo~y 62:~107-2114 (19881; Rusche et al., Proc. N~rl.
Ac~d. Sci. USA 85:3198-3202 11988); Goudsmitet~/.,AlDS2:157-164 11988);.Javaherian et ~I., Proc. N~tl. Ac~d. Sci. USA 86:6768~6772 ~19891; Lasky et al., Cel/ 50:975-985 (1987); Kowalski et al., Science 237:1351-1355 (19871; Willey et at., Proc. Natl. Ac~d. Sci. .
USA 83;5038-5042 (19861; Modrow et ~/., J. Virolooy 61:570-578 (1987).
The disulfide bondin~ pattern within gpl 20 and the positions of actual oli~osaccharide moieties on the molscule would be useful inforrnation for directin~ muta0enesis and fragmentation studies aimed at defining the func~ional domains of ~p120 and sites for :
potential pharmacolo~ical interruption of its functions (e.~., type-cammon neutralizin~
epi~opes). This information has been difficult to obtain due to the small amounts of ~pl 20 available from natural sources, the complexity of the disulfide bondin~ and oli~osaccharide structures in Qp 120, and uncertainty re~arding the functionality or structural relevance (Moore e~ al., in press) of r~pl 20 produced in non-mammalian systems. ~ -The invent~rs herein have surprisin~ly discovered that certain regions of native gp120 exist in specif;c three-dimensional conformation, ~,vhich conformation is conserved over isotype and strain. `It is an object of this invention to provide novel polypeptides which are useful as diagnostic tools for assayin~ biolo~ical samples for evidence of HIV infection.
It is a further object of this invention to provide novel polypeptides which are usable for vaccines, and for pharmacolo~ic interruption of the course of HIV infection.It is a further object of this invention to provide methods for preparin~ such polypeptides, and antibodies directed to such polypeptides.
Other objects, features, and characteristics of the present in~/ention will become ~ -apparent upon consideration of the following description and the appended claims.
Summarv of the Invention The objects of this invention are accomplished by the preparation and administration of compositions compr;sin~ isolated cyclized polypeptides which are suitable foradministration to a human or non-human patient having or at risk of having HIV infection.
These cyclized polypeptides are selected from the following:
a) C V K L T P L C C N T S V I T a A C ISEC~. ID NO~ 11 and containin~ less than about 28 amino acid residues;
bl P I H Y C A P A G F A I L K C N N K T F N G T G P C T N V S T V Q C T H G
I R P lSEO. ID NO. 21 and containin~ less than about 45 amino acid residues;
c~ C N N K T F N G T G P C lSEO. ID NO. 3] and containin~ less than about 22 amino acid residues:
dl CAPAGFAlLKCCTNVSTVQClSEO.lDNO.41andcontainingless than about 30 amino acid residues;
~,:
WO 9t/1531~ PCI/IJS91/0216 el P I H Y C C T H G I R P ISEQ. ID NO. 51 and containin~ less than about 22 amino acid residues;
f) G G D P E I V T H S F N C G G E F F Y C N S L P C R I K Cl F I N M W Q E V G
K A M Y A P P I S G Q I R C S S N I T G ISEQ. ID. NO. 61 and containin~ less than about 65 amino ac;d residues;
~) C G G E F F Y C C R I K Q F I N M W Q E V G K A M Y A P P I S G Q I R C
ISEQ. ID NO. 71 and containino less than about 45 amino acid residues;
h) CASDAKAYDTEVHNVWATHAClSEQ.lDNO.81andcontainin~
less than about 30 amino acid residues; and i) T T T L F C A S D A K A Y D T E V H N V W A T H A C V P T D P N [SEQ. lt) ;
NO. 91 and containing less than about 50 amino acid residues.
Addiionall~, this invention is also directed to compositions comprising an isolated ~ :
polypeptide havin~ an antigenic determinant or determinants immunolo~ically cross-reactive with a determinant of the HIV env polypeptide of strain HTLV-IIIB havin~ an amino acid 15 sequence selected from the ~roup consistin~ of a) residues 1-80;
b) residues 8-180; . :~
c) residues 165-260;
dl residues 160-260;
e) residues 260-310; and f) residues 320-479.
This invention is particularly directed to vaccines comprising the compositions of this invention. The compositions of this invention, including variant analo~ues thereof, are also useful in dia~nostic assays for HIV neutralizing antibody in patient samples.
Monoclonal antibodies directed to the isolated polypeptides of this invention are provided, characteri7ed by their affinity for li~and, epitope binding, and ability to a) block CD4/~p120 bindin~, bl neutralize HIV virions, c~ reduce reverse transcriptase activity in vitro, and d) inhibit syncytia formation. .
These antibodies are useful as dia~nostics for the presence of HIV infection in a patient or patient sample, and for affinity purification of HIV env. Thesn antibodies are also useful in passively immunizin~ patients infected with I IIV. In certain embodiments, antibodies are provided which are conju~ated to a cytotoxin, a water-insoluble matrix, or to a detectable marker~
Antibodies directed to HIV env epitopes have been described in the literature; however, it should be noted that, due to the variety and confusion amon~ authors currently as to numbarina systems for HIV env sequences, not all antibodies described in the literature as directed to certain re~ions will actually the same residue numbers as defined herein ~see e.g. ~ :
Matsushitaeta/.,J. Vlfol 62:2107~2114(1988);EPOApplicalionNo.EP339504; Rusche "~
~:
W~ 91/15~12 PCI`/US91/02166 5 ~,78~4~ :~
er a/.. Proc. N~t~. Acdd. Sci. USA, 85:3198-3202 ~19881; Looney et al., Science 241:357- ~ :
359 (19881;
Brief DescriDtion of the Drawin~s FIGURE 1 provides the amino acid sequences of la) the mature envelope ~Iycoprotein (~p1201 from the IIIB isolate of HIV-1 lSEQ. ID N0. 101, and ~b) the N-terminal sequence portion of the recombinant fusion glycoproteins (9AA [SEQ. ID N0. 1 1 ] or CL44 ISEQ. ID N0.
121) from the herpes simplex gD1. Fusion sites between the ~D1 and ~p120 se~ments in the 9AA and CL44 constructions are marked with (-I and (~-~, respectively. The letter T refers ~
to observed tryptic cleava~a of the ~p120 se~ment, and ~he peptides are ordered sequentially ~:
starting at the N-terminus of the molecule. Lower case letters following the T number indicate other unexpected proteolytic cleava~es. The letter H refers to the observed tryptic cleava~e of the herpes simplex ~D1 protein portion of CL44. Peptide T2' contains the fusion site in CL44. The cysteine residues of gp120 are shaded, and potential N-olycosylation sites are indicated with a dot above the correspondin~ asparagine residue.
~5 FIGURE 2 shows a reversed-phase HPLC tryptic map of RCM CL44. This chromato~ram was ~enerated with 7.5 nmol of trvpsin-di~ested RCM CL44. Chromato~raphy conditions were as described in Experimental Procedures. Peaks were collected and identified by AAA and in some cases confirmed by N-terminal sequence analysis ~Table 1). Identified peaks are labelled accordin~ to the nomenclature ~iven in Fi~ure 1. Peptides containino poten~ial tryptic sites that were not hydrolyzed ara desi~nated by two T numbers separat0d by a comma.
FIGURE 3 shows a reversed-phase HPLC tryptic map of 9AA. This chromatogram was ~enerated with 6.8 nmol of sample. Chromato~raphy conditions were as described in the Example herein. Peaks containin~ cysteine residues were identified bv N-terminal sequence ; -analysis. These identifications are summarized in Table ll.
FIGURE 4 shows the results of further manipulations of tryptic peptides from the map of 9AA to isolate individual disulfides. The chromatograms are details of microbore 30 reversed-phase HPLC separations of peptides rèsultin~ from: ~a) treatment of peptides T12, T13, and T14 (Peak C, Fi~ure 3~ with PNGase F follûwed by endoproteinase Asp-N, ~b1 treatment of peptides T3, T4, and T11 ~F'eak F, Fi~ure 3) with PNGase F followed by endoproteinase Asp-N, and Ic) treatment of peptides T28 and T31 (Peak D, Figure 31 with S. aureus V8 protease. Chromato~raphy conditions were as described in the Example herein.
35 Peak identifications were determined by N-terminal sequence analysis and are ~iven in Table 111. :
FIGURE 5 shows reverse-phase HPLC tryptic maps of endo~lycosidase treated RCM
CL44. The chromato3rams are tryptic maps of: ~a) untreated RCM CL44, (b~ PNGase : - . .. . . ..
: . : , ~, , .. , .. ,~ , ... . ..
W(~91/15:~12 . l~ J ' PCT/US9~/02166 F-treated RCM CL~4, and lc~ endo H-treated RCM CL44. Each tryptir, map was ~enerated with 7.5 nmol of sample. Chromato~raphy conditions were as described in Experimental Procedures. Peaks were collected and identified by AAA Idata not shown). Glycopeptide peaks are labelled accordin~ to the nomenclature in Fi~ure 1.
FIGURE 6 is a schematic representation of gp 120 of the Illa isolate of HIV-1 showin~
disulfides and ~Iycosylation sites, with the amino acids represented in single-letter code lSEQ.
ID N0. 101. Roman numerals label the five disulfide bonded domains. The five hypervariable re~ions of Modrow e~ a/., J. Virol. 61 :570-S78 11987~ are enclosed in boxes and labelled V1- :
V5. Glycosylation sites containin~ hi~h mannose-type and/or hybrid-type oli~osaccharide t 0 structures are indicated by a branchin~-Y symbol, and glycosylation sites containing complex-type oli~osaccharide structures are indicated by a V-shaped symbol.
FIGURE 7 shows a schematic representation of the HIV env 31ycoprotein ~pl 20 of HIV-2, showing disulfides and potential glycosylation sites ~SEQ. ID N0. 131. Glycosylation sites are indicated by a shaded box around a N residue. i~oman numerals label five disulfide-bonded domains.
Detailed DescriDtion of the Invention .
HIV env is de~ined herein as the envelope polypeptide of Human Immunodeficiency Virus as described above, together with its amino acid sequence variants and derivatives produced by covalent modification of HIV env or its variants in vitro, as discussed herein~
As used herein, the term "HIV env" encompasses all forms of Dpl20 and/or 160, e.~.
including fra~ments, fusions of op160/120 or their fragments with other peptides, and variantly ~Iycosylatad or un~lycosylated HIV env. The HIV env of this invention is recovered free of active virus.
HIV env and its variants are conventionally prepared in recombinant cell culture. For example, see EP publication No. 187041. Henceforth, gp120 prepared in recombinant cell culture is referred to as r~pl 20. Recombinant s~nthesis is preferred for reasons of safety and economy, but it is known to prepare peptides by chemical synthesis and to purify HIV env from viral culture; such env preparations are included within the definition of HIV env herein.
Genes encoding HIV env are obtained from the genomic cDNA of an HIV strain or from available subDenomic clones containinD the Dene encodinD HIV env.
This invention Is dirflcted to isolated polypeptides. Certain of thflse isolatedpolypeptides are defined as cyclized polypeptides comprisin~ a particular amino acid sequence, and certain isolated polypeptides are described by reference to specific amino acid residue numbers. The amino acid numbering reflects the mature HIV-1 ~pl20 amino acid sequence as shown by Fi~. 6. and Fiq. lA ISEQ. ID N0. 10], not counting any signal sequence or other upstream re~ions, and is used throu~hout this description to conveniently connote the intended residues. however it is understood that this invention is not limited to .
~' ~
those specific residue numbers. For gp120 sequences which includa the native HIV-IIIB N-tsrminal si~nal sequence, numberin~ may differ. The same nuclsotide and amino acid residue numbers may not be applicable in other strains where upstream deletions or insertions chan~e the len~th of the viral ~enome and HIV env, but the re~ion encodin~ this portion of ~p120 5 is readily identified by reference to the teachin~s herein. Also, variant si~nal sequences (such as those resultin~ from a fusion with a fra~mented or heterolooous si~nal sequence as discussed below may lead to a sli~ntly different numberin~, however the precise amino acid sequences are discerned for all embodiments by reference to Fi~. 6 and/or Fi~ 1A iSEQ. ID
N0. 101.
1~ Included within the scope of the isolated polypeptides of this invention, as those terms are used herein are polypeptides havin~ spacified amino acid sequences, de~lycosylated or unglycosylated derivatives, homolo~ous amino acid sequence variants, and homolo~ous in vitro-~enerated variants and derivatives, and which variants are capable of exhibiting a biological activity in common with the HlV env of Fi~. 6 or Fig. 7.
Isolated polypeptide biological activity is defined as either 11 immunolo~ical cross-reactivitV with at least one isolated polypeptide, or 2) the possession of at least one adhesive or effector function qualitatively in common with the isolated polypeptide. Examples of the qualitative biolo~ical activities of an isolated polypeptide include the ability to bind to the viral receptor CD4 or known monoclonal antibodies, and the ability of ~pl 20 ta interact with ~p41 20 to induce fusion of the viral and host cell membranes.
Immunolo~ically cross-reactive as used herein means that the candidate polypeptide is capable of competitively inhibitin~ the qualitative biolo~ical activity of an isolated polypeptide havin~ this activity with polycional antisera raised a~ainst the known active analo~ue. Such antisera are prepared in conventional fashion by injectinU qoats or rabbits, 25 for example, subcutaneously with the known ac~ive analo~ue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds.
The ordinarily skilled worker may use the disulfide bondin~ pattern within qp120 and the positions of actual oli~osaccharide moieties on the molecule as described herein for directing mutagenesis and fragmentation variants of the claimed isolatnd polypeptides. It i5 30 intended that the variants of this invention include isolated polypeptides in which one or more residues have been substituted, deletions of one or more residues, and insartions of one or more amlno acid residues.
This inventlon also contemplates amino acid sequence variants of the isolated polypeptides. Amino acid sequence variants are prepared with various objectives in mind, 35 includin~ increasin~ the affinity of the isolated polypeptide for a liaand or antibody, facilitatin~
the stability, purification and preparation of the isolated polypeptide, modifyin~ its plasma half life, improvin~ therapeutic efficacy, and lessening the severity or occurrence of side effects durin~ therapeutic use of the isolated polypeptide. In the discussion below, amino acid . ` , ;.
WO ~ 512 PCl/US91/021~ -sequenc~,~nants of the isolated polypep~ide are provided, exemplary of the variants that may be selected.
Amino acid sequence variants of isolated polypeptide fall into one or more of three classes: Insertional, substitutional, or deletional variants. These variants ordinarily are 5 prepared by site-specific muta~enesis of nucleotides in the DNA encodin~ the isolated polypeptide, by which DNA encodin~ the variant is obtained, and thereafter expressin~ the DNA in recombinant cell culture. However, fra~ments havin~ up to about 100-150 amino acid residues are prspared conveniently bV in vitro synthesis. The followin~ discussion applies to any isolated polypeptide to the exten~ it is applicabie to its structure or function.
The amino acid sequence variants of the isolated polypeptide are predetermined variants not found in nature or naturally occurrin~ alleles. The isolated polypeptide variants typically exhibit the same qualitative biolo~ical--for example, antibody binding--activity as the naturally occurrin~ isola~ed polypeptide or isolated polypeptide analogue. However, isolated polypeptide variants and derivatives that are not capable of bindin~ to antibodies are useful 15 nonetheless (a) as a rea~ent in dia~nostic assays for isolated polypeptide or antibodies to the isolated polypeptide, ~b) when insolubilized in accord with known methods, as a~ents for purifyin~ anti-isola~ed polypeptide antibodies from antisera or hybridoma culture supernatants, and ~c) as immunogens for raisin~ antibodies to isolated polypeptide or as immunoassay kit components llabelled, as a competitive rea~ent for the native isolated 20 polvpeptide or unlabelled as a standard for isolated polypeptide assay) so long as at least one isolated polypeptide epitope remains active.
While the site for introducin~ an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize theperformance of a mutation at a given site, random or saturation mutagenesis ~where all 20 25 possible residues are inserted) is conducted at the tar~et codon and the expressed isolated polypeptide variant is screened for the optimal combination of desired activities. Such screenin~ is within the ordinary skill in the art.
Amino acid insertions usually will be on the order of about from 1 to 10 arnino acid residues; substitutions are typically introduced for sin~le residues; and deletions will ran~e .
30 about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. It will be amply apparent from the following discussion that substitutions, deletions, insertions or any combination thereof are introduced or combined to arrive at a final construct. Insertional amino acid sequence variants of the isolated polypeptide are those in which one or more amino acid residues i~
35 extraneous to the isolated polypeptide are introduced into a predetermined site in the tar~et isolated polypeptide and which displace the preexistinp residues.
Commonly, insertional variants are fusions of heterologous proteins or polypeptides to the amino or carboxyl terminus of the isolated polypeptide. Such variants are referred to as : . . .. , .; , .:
WO 91/15~12 ~ 8 ~ 4 ~D~/US91/02166 fusions of the isolated poivpeptide and a polyp~ptide containin~ a sequance which is other than that which is normally found in the isolated polypeptida at the inserted position. Several ~roups of fusions are contemplated herein.
The novel isolated polypeptides of this invention are useful in dia~nostics or in purification of the antibodies or li~ands by known immunoaffinity techniques.
Desirable fusions of the isolated polypeptide, which may or may not also be immunolo~ically active, include fusions of the mature isolated polypeptide sequence with a si~nal se~uence heterolooous to a native isolated polypeptide as mentioned above. Si~nal sequence fusions ars employed in order to more expeditiously direct the secretion of the isolated polypep~ide. The heterolo~ous siynal replaces tha native isolated polypep1ide signal, and wh0n the resultin~ fusion is reco~nized, i.e. processed and cleaved by the host cell, the isolated polypeptide is secreted. Si~nals are selected based on the intEnded host cell, and ~
may include bacterial yeast, mammalian and viral sequences. The native HIV env signal or the herpes gD ~Iycoprotein si~nal is suitable for use in mammalian expression systems.
C-terminal or N-terminal fusions of the isolated polypeptide or isolated polypeptide fra~ment with an immuno~enic haptan or heterologous polypeptide are useful as vaccine components for the immunization of patients against HIV infection. Fusions of the hapten or heterologous polypeptide with isolated polypeptide or its active fragments which retain T-cell bindin~ activity are also useful in,directin~ cytotoxic T cells a~ainst tar~et cells where the hapten or heterolo~ous polypeptide is capable of bindin~ to a tar~et cell surface receptor.
The precise site at which the fusion is made is variable; particular isolated polypeptide sites are selected in order to optimize the bio!o~ical activity, secretion or bindin~
characteristics of the isolated polypeptide. The optimal site will for a particular application will be det0rmined by routine experimentation. ~:
Substitutional variants are those in which at least one residue in the isolated polypeptide has been removed and a different residue inserted in its place. Such substitutions ~enerally are made in accordance with the followin~ Table 1 when it is desired to finely modulate the characteristics of the isolated polypeptide.
wo 91~1~;12 ,. PCl`tUS9t/02166 -~,3 ~ , o TABL~ 1 Orlainal R~idue Exem~l~ry s-ubgtitutions Ala ~er Arg ly5 Ar~n gln; hi~ ~-~rjp glu ` .
Cyr~ aer; ala Gln ar~n Glu a~p ~
Gly pro : : -Hi~ asn; gln Ile leu; val Leu il~; val Lys arg; gln; ~u Met leu; ile Phe met; leu; tyr Ser thr Thr rJer Trp tyr ~
Tyr trp; phe `
Val ile; l~u Novel amino acid sequences, as well as isosteric analo~s /amino acid or otherwise), as includerJ within the scope of this invention.
Substantial chan~es in function or immunolo~ical identity are made by selectinp substitutions that are less conservative than those in Table 1, i.e., selectin~ residues that differ more si3nificantly in their effect on maintainin~ la) the structure of the polypeptide ~, .
backbone in the area of the substitution, for example as a sheet or helical conformation, lb~
the char~e or hydrophobicity of the molecule at the tar~et site or lcl the bulk of the side chain. Th0 substitutions which in ~eneral are expected to produce the ~reatest chan~es in 30 isolated polypeptide properties will be those in which la) a hydrophilic residue, e.~. seryl or threonyl, is substituted for lor byl a hvdrophobic residue, e.~. Ieucvl, isoleucvl, phenvlalan valyl or alanyl; lbl a cysteine or proline is substituted for lor bVI anV other residue; Ic) a residue havin~ an electropositive side chain, e.~., Iysvl, ar~inyl, or histidvl, is substituted for (or by) an electrone~ative residue, e.~ lutamyl or aspanyl; or Id) a residue havin~ a bulky 35 side chain, e.~., phenylalanine, is substituted for lor by) one not havin~ a side chain, e.
~Iycine.
Some deletions, insenions, and substitutions will not produce radical chan~es in the characteristics of the isola~ed polvpeptide molecule. However, when it is difficult to predict :.
55l2 ~ 7 8 ~ cr/us9l/o2166 the exact effect of the substitution. deletion, or insertion in advance of doin~ so, for example when modifying an immune epitope, one skilled in the art will appreciate that the effect will be evaluated by routine screenin~ assavs. For example, a variant typically is made by site spscific muta~enesis of the isolated polypeptide -encodin~ nucleic acid, expression of the 5 variant nucleic acid in recombinant cell culture and, optionally, purification from the cell culture for example by immunoaffinity adsorption on a polyclonal anti-isolated polypeptide column (in order to adsorb the variant by at least one remainin~ immune epitope). The activity of the cell Iysate or purified isolated polypeptide variant is then screened in a suitable screenin~ assay for the desired characteristic. For example, a chan~e in the immunological 10 character of the isolated polypeptide, such as affinity for T-cell binding, is measured by a competitive-tvpe immunoassay. As more becornes known about the functions in vivo of the isolated polypeptide other assays will become useful in such screenin~. Modifications of ~ -such protein properties as redox or thermal stability, hydrophobicity, susceptibility to proteolytic de~radation, or the tendencv to a~gregate with carriers or into multimers are 15 assayed by methods well known to the artisan.
Another class of isolated polypeptide variants are deletional variants. Deletions are characterized by the removal of one or more amino acid residues from the isolated polypeptide sequence. Typically, deletions are used to affect isolated polypeptide bioloqical activities, however, deletions which preserve the biolo~ical activitV or immune cross-reactivity 20 of the isolated polypeptide are suitable.
Deletions of cysteine or other labile residues also may be desirable, for example in increasin~ the oxidative stability of the isolated polypeptide. Deletion or substitutions of potential proteolysis sites, e.~. Ar~ Ar~, is accomplished by deletin~ one of the basic residues or substitutin~ one by glutaminvl or histidyl residues.
It ~,vill be understood that some variants may exhibit reduced or absent biolo~ical activity. These variants nonetheless are useful as standards in immunoassays for the isolated polypeptide so long as they retain at least one immune epitope of the isolated Polvpeptide.
It is presently believed that the three-dimensional structure of the isolated polypeptides and peptide compositions of the present invention is important to their functioning as 30 described herein. Therefore, all related structural analogs which mimic the active structure of those formed by the isolated polypeptides claimed herein are spscifically incluaed within the scope of thr~ present invention.
Glycosylation variants are included within the scope of the isolatsd polypeptide. They include variants completelY lacking in ~Iycosylation ~un~lycosylated~ and variants havin~ at 35 least one less ~lvcosylated site than ~he native form ~de~lycosylated) as well as variants in which the glycosylation has been chan~ed. Included are deglycosylated and un~lycosylated amino acid sequence variants, deglycosvlated and un~lycosylated isolated polypeptide having the native, unmodified amino acid sequence of the isolated polvpeptide, and other .. . . .. ..
.
. ,.
wo 9~ 512 " PCr/US91/02~6C ~
..
2- :
~Iycosvlation variants. For example, substitu~ional or deletiùnal muta~enesis is empioyed to ~iminate the N- or 0-linked ~Iycosylation sites of the isolated polypeptide, e.~., an asPara~ine residue (not at th~ clip sitel is daleted or substituted for by another basic residue such as Iysine or histidine. Alternatively, flankino residuss makin~ up the ~Iycosylation site are substituted or deleted, even though the aspara~ine residues remain unchan~ed, in order to prevent ~Iycosylation by eliminatin~ the ~Iycosylation reco~nition site.
Unglycosylated isolated pùlypeptide which has the amino acid sequence of the native isolated polypeptide is produced in recombinant prokaryotic cell culture because prokaryotes are incapable of introducin~ ~Iycosylation into polypeptides. .
Glycosylation variants are produced by selectin~ appropriate host cells or by in vitro methods. Yeast, for example, introduce ~Iycosylation which varies si~nificantlY from that of mammalian systems. Similarly, mammalian cells havin~ a different species la.~. hamster, murine, insect, porcine, bovine or ovine) or tissue ori~in (e.~. Iun~, liver, Iymphoid, mesenchymal or epidermall than the source of the isolated polypeptide anti~en are routinely screened for ~he ability to introduce variant ~Iycosylation as characterized for example by ;
elevated levels of mannose or variant ratios of mannose, fucose, sialic acid, and other su~ars typically found in mammalian olycoproteins. In vitro processin~ of the isolated polvpeptide typically is accomplished by enzymatic hydrolysis, e.~. neuraminidase di~estion.Covalent modifications of the isolated polypeptide molecule which do not modify the clip site are included within the scope hereof. Such modifications are introduced by reactin~
tar~eted amino acid residues of the recovered protein with an or~anic derivatizin~ a~ent that ~ -is capable of reactin~ with selected side chains or terminal residues, or by harnessin~
mechanisms of post-translational modification that function in selected recombinant host cqlls. The resultin~ covalent derivatives are useful in pro~rams directed at identifyin~ residues : ;~
important for biolo~ical activity, for immunoassays of isolated polypeptide or for the :
preparation of anti-isolated polypeptide antibodies for immunoaffinity purification of the recombinant isolated polypeptide. For example, complete inactivation of the biolo~ical activity of the protein after reaction with ninhydrin would suo~est that at least one ar~inyl or Iysyl residue is critical for its activity, whereafter the individual rasidues which were modified under the conditions selected are identified by isolation of a peptide fra~ment containin~ the modified amino acid residue. Such modifications are within the ordinary skill in the art and are performed without undue axperimentation.
Derivatization with bifunctional a~ents is useful for preparin~ intermolecular a~re~ates of the isolated polypeptide with polypeptides as well as for cross-linkin~ the isolated poiypeptide to a water insoluble support matrix or surface for use in the assay or affinity purification of its li~ands. In addition, a study of intrachain cross-links will provide direct information on conformational structure. Commonly used cross-linkin~ a~ents include sulfhydryl reagents, 1,1-bisldiazoacetyl)-2-phenylethane, ~lutaraldehyde, N-~, ~ 91/15~12 ~ u 7 ~
hydroxysuccinimide ~st~rs, for exampie sstsrs with 4-azidosalicylic acid, homobifunctional imidoesters includin~ disuccinimidyl esters such as 3,3'-dithiobis Isuccinirnidyl-propionate), and bifunctional maleimides such as bis-N-maleimido-1,B-octane. Derivati~in~ a~ents such as methyl-3~1~p-azido-phenyl)dithio] propioimidate yield photoactivatable intermediates which 5 are capable of formin~ cross-links in th~ presence of li~ht. Alternatively, reactive water insoluble matrices such as cyano~en bromide activated carbohydrates and the systems reactivesubstratesdescribedinU.S.patents3,959,080; 3,969,287;3,691,016;4,195,128;
4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employed for protein imrnobilization and cross-linking.
Polymers 9enerally are covalently linked to the isolated polypeptide herein throu~h a multifunctional crosslinkin~ a~ent which reacts with the polymer and one or more amino acid or su~ar residues of protein. However, it is within the scope of this invention to directly crosslink the polymer by reactin~ a derivatized polymer with ~he isolated polypeptide, or vice versa. Covalent bondin~ to amino ~roups is accomplished by known chemistries based upon 15 cyanuric chloride, carbonyl diimidazole, aldehyde reactive ~roups IPEG alkoxide plus diethyl acetal of bromoacetaidehyde; PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde, succinimidyl active esters, activated dithiocarbonate PEG, 2,4,5-trichlorophenylchloroformate or p-nitrophenylchloroformate activated PEG.
Carboxyl ~roups are derivati~ed by couplinu PEG-amine usinD carbodiimide.
This invention is also directed to polvpeptides of this invention which bV definition or optionally are conformationally stabilized by cyclization. The peptides ordinarily are cyclized by covalently bonding the N and C-terminal domains of one peptide to the correspondin~
domain of another peptide of this invention so as to form cyclooli~omers containin~ two or more iterated peptide sequences, each internal peptide havin~ substantially the same 25 sequence. Further, cyclized peptides ~whether cYclooli~omers or cylomonomersl are crosslinked to form 1 -3 cyclic structures havin~ from 2 to 6 peptides comprised therein. The peptides preferably are not covalently bonded throu~h ~-amino and -carboxyl ~roups Ihead to tail), but rather are cross-linked throu~h the side chains of residues located in the N and C-terminal domains. The linkin~ sites thus ~ensrally will be between the side chains of A, 30 and A10 residues. Substantially identical polypeptides present in the polymerized forms of the peptides hereof are those which e~hibit qualitative isolated polvpeptide activitv, notwithstandin~ the de~ree of amino acid sequence variation amon~ the polypeptides.
Variants which exhibit activity are used as subunits in homo or heteropolymers. In homopolymers th~ peptides are the same. Heteropolymers contain different peptides, each 35 however, chosen from within the parameters described above.
Manv suitable methods per se are known for preparin~ mono- or poly-cvclized peptides as contemplated herein. Lys/Asp cyclization has been accomplished usin~ No-Boc-amino WO 91/15~17 ~ ) PCI/IJS91/0216 acids on solid-phas~ support with Fmoc/OFm side chain protection for LvslAsp; the process is comp~eted by piperidine treatment followed by BoP cyclization.
Glu and Lys side chains also have been crosslinked in preparin~ cyclic or bicyclic peptides: the peptide is synthesized by solid phase chemistry on a p-methylbenzhydrylamine resin. The peptide is cleaved from the resin and depro~ected. The cyclic peptide is formed using diphenylphosphorylazide in dilute dimethylformamide. For an alternative procedure, see Schilleret~ PeptideProteinRes.~25:171-t7711985). SeealsoU.S.Patent4,547,489.
Disulfide crosslinked or cyclized peptides are ~enerated by conventional methods. The method of Pelton et a/. (J. Med. Chem. 29:2370-237~ [1986~) is suitable, except that a ~reater proportion of cyclooligomers are produced by conduc~in~ the reaction in more concentrated solutions than the dilute reaction mixture described by Pelton et ~/. fùr the production of cyclomonomers. The same chemistry is useful for synthesis of dimers lusing A,-A" Pen plus Al-AI~ Cys) or cyclooli~omers or cyclomonomers IPen A1-A,o Cys, or Pen Al-A~o Cys plus Cys A,-A10 Pen). Also useful are thiomethylene brid~es ITetrahedron Letters 25120):2067-2068 i1984~). See also Cody et a/., J. Med. Chem. 28:583 11985).
The desired cyclic or polymeric peptides are purified by ~el filtration followed by reversed-phase high pressure liquids chromato~raphy or other conventional procedures. The peptides are sterile filtered and formulated into conventional pharmacolo~ically acceptable v~hicles .
Certain post-translational derivatizations are the result of the action of recombinant host cells on the e~pressed polypeptide. Glutaminyl and aspara~inyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues.
Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
Other post-translational modifications include hydroxylation of proline and Iysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino ç~roups of iysine, arginine. and histidine side chains tT.E. Creighton, Proteins: Struçture and Molecular ProDerties, W.H. Freeman & Co., San Francisco pp 79-86 119331~, acetvlation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.
DNA encoding the isolated polypeptide is synthesized by in vitro methods or is obtained readily from cDl~iA libraries. The means for synthetic creation of the DNA encodin~ the isolated poiypeptide, either by hand or with an automated apparatus, are generally known to one of ordinary skill in the art, particularly in light of the teachings contained herein. As examples of the current state of the art relating to polynucleotide sYnthesis, one is directed to Maniatis et al., Molecvlar ClomnD--A Lsbora~ory Manual, Cold Spring Harbor Laboratory t1984), and Horvath et al., An Automated DNA Synthesizer Employin~ Deoxynucleoside 3'-Phosphoramidi~es, Methods in Enzymology 154: 313-326, 1987.
wo 91/1~512 ~ ' 8, ~ ~Cr/US91/~2166 Alternasively, to obtain DNA enc~din~ the isola~ed polypeptide, ~ne needs only to conduct hybridization screening with lab~lled DNA encodinQ either the isolated polypeptide or isoiated polypeptide fra~ment lusuaily, ~rsater than about 20, and ordinarily about 50bp) in order to detect clones which contain homolo~ous sequences in the cDNA libraries derived 5 from cells or tissues ot a particular animal, followed by analyzin~ the clones by restriction enzyme analysis and nucleic acid sequencin~ to identify full-len~th clones. If full lenpth clones are not present in the library, then appropriate fragments are recovered from the various clones and li~ated at restriction sites common to the fra~men~s to assemble a full-lenoth clone. DNA encodin~ isolated poiypeptide from various iso~ypes and strains is 10 obtained by probing libraries from hosts of such species with the amino acid ssquences of the isolated polypeptide, or by synthesizin~ the ~enes in vitro.
In ~eneral, prokaryotes are used for clonin~ of DNA sequonces in constructin~ the vectors use~ul in the invention. For exarnple, E. coli K12 strain 294 IATCC No. 31446) is particularly useful. Other microbial strains which may be used include E. co/i ~ and E. coli 15 X1776 (ATCC No. 31537h These examples are illustrative rather than limitin~.
Alternatively, in vitro methods of clonin~, e.~. polymerase chain reaction~ are suitable.
The isolated polypeptides of this invention are expressed directly in recombinant cell culture as an N-terminal methionyl analo~ue, or as a fusion with a polvpeptide heterolo~ous 20 to the hybrid/portion, preferably a si~nal sequence or other polypeptide havin~ a specific cleava~e site at the N-terminus of the hybrid/portion. For example, in constructin~ a prokaryotic secretory expression vector for the isolated polypeptide, the native isolated polypeptide si~nal is employed with hosts that recoanize that si~nal. When the secretory leader is rreco~nized" by the host, the host si~nal peptidase is capable of cleavin~ a fusion 25 of the leader polypeptide fused at its C-terminus to the desired mature isolated polypeptide.
For host prokaryotes that do no~ process the native isolated polypeptide si~nal, the si~nal is substituted by a prokaryotic si~nal selected for example from the ~roup of the alkaline phosphatase, penicillinase, Ipp or heat stable enterotoxin ll leaders. For yeast secretion the native isolated polypeptide si~nal may be substituted by the veast invertase, alpha factor or 30 acid phosphatase leaders. In mammalian cell expression the native isolated polypeptide signal or native HIV env signal is satisfactorv for certain isolated polypeptides, althou~h other mammalian secretorV protein si~nals are suitable, as are viral secretorV leaders, for example the herpes simplex ~D signal.
The isolated polypeptide may be expressed in any host cell, but preferably is 35 synthesized in mammalian hosts. However, host cells from prokaryotes, fun~i, yeast, insects and the like are also are used for expression. Exemplary prokaryotes are ~he strains suitable for clonin~ as well as E. coli W3110 IF-~I prototrophic, ATTC No. 27325~, other ~ :: ~ :': : : : : : : : : ~ : `
WO91/15512 ~ ~, PCl/llS9~/0216 ~ - 1 6-enterobacteriaceae such as Serrati~ marcescans, bacilli and various pseudomonads.
Prr~ferably the host cell should secrete minimal amounts of proteolytic cnzvmes.E~pression hosts typically are transformed with DNA ancodin~ the isolat~d polypeptide which has been li~ated into an expression vector. Such vectors ordinarily carry a replication site (althou~h this is not necessary where chromosomal integration will occur). Expression vectors also includ~ marker sequences which are capable of providing phenotypic selection in transformed cells, as will be discussed further below. For example, f. coli is typically transformed usin~ pBR322, a plasmid ~erived from an E. coli species IBolivar, et a/., Gene 2:
95 119771). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifyin~ transformed cells, whether for purposes of cloninçl or expression. Expression vectors also optimally will contain sequences which are useful for the control of transcription and translation, e.~., promoters and Shine-Dal~arno sequences (for prokaryotes~ or promoters and enhancers If or mammalian cells). The promoters may be, but need not be, inducible; even powerful constitutive promoters such as the CMV promoter for mammalian hosts may produce the isoiated polypeptide without host cell toxicity. While it is conceivable that expression vectors need not contain any expression control, replicative sequences or selection genes, their absence may hamper the identification of transformants and the achievement of high level peptide expression.
Promoters suitable for use with prokaryotic hosts illustratively include the ~-lactamase and lactose promoter systems IChang et al., Narure 275: 615 t19781; and Goeddel et al., Nature 281: 544 119791), alkaline phosphatase, the tryptophan ~trp) promoter system IGoeddel, Nucleic Acids ~es. 8: 4057 l1980) and EP0 Appln. Publ. No. 36,776) and hybrid promoters such as the tac promoter IH. de Boer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 119831). However, other functional bacterial promoters are suitable. Their nucleotide sequences are ~enerally known, thereby enablin~ a skilled worker operably to ligate them to DNA encoding the isolated polypeptide ~Siebenlist et al., Cell 20: 269 119801) using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dal~arno IS.D.~ sequence operably linked to the DNA encodin~ the isolated polypeptide.
In addition to prokarYotes, eukaryotic microbes such as yeast or filamentous fun~i are satisfactory. Saccharomyces cerevislae is the most commonly used eukaryotic microor~anism, althou~h a number of other strains are commonly available, The plasmid YRp7 is a satisfactory expression vector in yeast IStinchcomb, et al., Nature 282: 39 11979);
Kingsman et al, Gene 7: 141 l1979); Tschemper e~ al., Gene 10: 157 (1980)). This plasmid already contains the trp1 ~ene which provides a selection marker for a mutant strain of yeast lackin~ the ability to ~row in tryptophan, for example ATCC no. 44076 or PEP4-1 IJones, Genetics 85: 12119771). The presence of the trp1 lesion as a characteristic of the yeast WO 91/155t2 ~ . 7 ~ cr/us9l/021s6 host cell 0enome then provides an affective environment for detectin~ transformation by ~rowth in the absence of tryptophan.
Suitable promotin~ seqlJ~nces for use with Yeast hosts include the promoters for 3-phospho~lycerate kinase (llitzeman et al., J. ~iol. Chem. 255: 2073 119801) or other glycolytic en2ymes lHess et al., J. Adv. t~nzyme Re~. 7: 149 11968); and Holland, ajochemistry 17: 4900 (1 g78)~, such as enolase, glyceraldehyde-3-phosphate dehydro~enase, hexokinase, pyruvate decarboxylase, phosphofructokinase, ~lucose-~-phosphate isomerase, 3-phospho~lycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho~lucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters havinp tha additional advanta~e of transcription controlled by ~rov~h conditions, are the promoter re~ions for alcohol dehydro~enase 2, isocytochrorne C, acid phosphatase, de~raciative enzymes assor;iated with nitrogen metabolism, metallothionein, ~Iyceraldehyde-3-phosphate dehydro~enase, and enzymes responsible for maltose and ~alactose utilization. Suitable vectors and promoters for USB in yeast expression are further described in R. Hitzeman et a/., European Patent Publication No. 73,657A.
Expression control sequences are known for eucaryotes. Virtually all eukaryotic ~enes have an AT-rich re~ion located approximately 25 to 30 bases upstream ftom the site where transcription is initiated, Another sequence found 70 to 80 bases upstream from the start of transcription of many ~enes is a CXCAAT re~ion where X may be any nucleotide. At the 3' end of most eukaryotic ~enes is an AATAAA sequence which may be the si~nal for addition of the poly A tail to the 3' end of the. codin~ sequence. All of these sequences are inserted into mammalian expression vectors.
Suitable promoters for controlling transcription from vectors in mammalian host cells are readily obtained from various sources, for example, the ~enomes of viruses such as polyoma virus, SV40, adenovirus, MMV ~steroid inducible), retroviruses le-~. the LTR of HIV~, hepatitis-B virus and most preferably cytome~alovirus, or from heterolo~ous mammalian promoters, e.~. the beta actin promoter. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fra~ment which also contains the SV40 viral ori~in of replication. Fiers et ~/., Nature, 273: 113 ~1978). The immediate early promoter of the human cytome~alovirus is conveniently obtained as a Hindlll E restriction fra~ment.
Greenaway, P.J. et a/., Gene 18: 355-360 11982), Transcription of a DNA encodin~ ~he isolated polypeptide by higher eukaryotes isincreased by insertin~ an enhancer sequence into the vector. Enhancers are cis-actin~
elements of DNA, usually about from 10-300bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent havin~ been found 5' (Laimins etal., PNAS78: 993 11981]) and 3' tLusky, M.L., etat., Mot. Cel/t~io. 3:
1108 11983~) to the transcription unit, within an intron IBanerji. J.L. et al., Cet/ 33: 729 WO91/15~12 ", ~ PCr/US~1/02166 l1983)1 as well as within the coding sequence itself (Osborne, T.F., et al., Mot. Cell ~io. 4:
1293 119841). Many enhancer sequences are now known from mammalian ~enes l~lobin, elastase, albumin, a-fetoprot~in and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication ori~in IbP 100-270), the cytome~alovirus early promoter enhancer, the polyoma enhancer on the late side of the replication ori~in, and adanovirus enhancers. ~
Expression vectors used in eukaryotic host cells lyeast, fun0i, insect, plant, animal, - -human or nucleated cells from other multicellular or~anisms) will also contain sequences necessary for the termination of transcription which may affect mP~NA expression. These re~ions are transcribed as poiyadenylated seaments in the untransia~ed portior of the mRNA
encodin~ the hybrid immuno~lobulin. The 3 untranslated re~ions also include transcription termination sites.
Expression vectors may contain a selection ~ene, also termed a selectable marker.
xamples of suitable selectable markers for marnmalian cells are dihydrofolate reductase ~DHFR~, thymidine kinase lTK) or neomycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell is able to survive K placed under selective pressure. There are two widely used distinct cate~ories of selective re~imes. The first Gate~ory ;s based on a cell s metabolism and the use of a mutant cell line which lacks the ability to ~row independent of a supplemented media. Two examples ars CHO DHFR- cells and mouse LTK cells. These cells lack the ability to ~row without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain ~enes necessary for a complete nucleotide synthesis pathway, thev cannot survive unless the missin~ nucleotides are provided in a supplemented media. An alternative to supplementin~
the media is to introduce an intact DHFR or TK ~ene into cells lackin~ the respective ~enes, thus altering their ~rowth requirements. Individual cells which were not transformed with the DHFR or TK ~ene will not be capable of survival in non-supplemented media. In preferred embodiments, herein, CHO cells which are DHFR- are used for recombinan~ expression of the isolated polypeptide.
The second cate~ory of selective re~imes i5 dominant selection which refers to aselaction scheme used in any cell typc and does not require the use af a mutant cell line These schemes typically use a dru~ to arrest ~rowth of a host cell. Those cells which are successfully transformed with a heterolo~ous ~ene express a protein conferring drug resistance and thus survive the selection re~imen. Examples of such dominant selection use the dru~s neomycin ISouthern et al., J. Molec. Appl. Genet. 1: 327 l1982~), mycophenolic acid IMulli~an e~al., Science 209: 1422 l1980)) or hy~romycin lSu~den et al., Mol. Cell. Biol.
5: 410-413 l1985)). The three examples ~iven above employ bacterial ~enes under eukaryotic control to conveY resistance to the appropriate dru~ G418 or neomycin l~eneticin~, x~pt lmycophenolic acid~ or hy~romycin, respectively.
WO 91/15512 r. r~ ~ ~ 4 ~ PC~ S9ltO2166 ~19-~Amplification~ refers tO the increase or replication of an isolated region within a cell s chromosomal DNA. Amplification is achieved usin~ a seiection a~ent, e.o. methotrexate IMTX) which inactivates DHFR. Amplification or the makin~ of successive copies of the DHFR ~ene results in ~reater amounts of DHFR bein~ produced in thrl face of greater amounts 5 of MTX. Amplification pressure is applied notwithstandin~ the presence of endooenous DHFR, by addin~ ever oreater amounts of MTX to the media. Amplification of a desired ~ene can be achieved by cotransfectinS~ a mammalian host cell with a plasmid havin~ a DNA
encodin~ a desired protein and the DHFR or amplification ~en~ permittin~ cointeoration. One ensures that the cell requires more DHFR, which requirement is met by replication of teh t 0 selection ~ene, by selecting only for cells that can ~row in teh presence of ever-~lreater MTX
concentration. So lon~ as the ~ene encoding a desired heterolo~ous protein has cointe~rated with the selection ~ene replication of this ~ene ~ives rise to replication of the ~ene encodin~
the desired protein. The result is that increased copies of the ~ene, i.e. an amplified ~ene, encodin~ the desired heterolo~ous protein express more of the desired protein.
Suitable eukaryotic host cells for expressin~ the isolated polypeptide include monkey kidney CV1 line transformed bv SV40 ~COS-7, ATCC CRL 16511: human embryonic kidney line (293 or 293 cells subcloned for ~rowth in suspension culture, Graham, F,L. e~ al., J. Gen V*ol. 36: 59 (1977j); baby hamster kidney cells ~BHK, ATCC CCL 10~; chinese hamster ovary-cells-DHFR ICHO, Urlaub and Chasin, PNAS ~USAI 77: 4216, 119801~; mouse sertoli cells (TM4, Mather, J.P., Biol. Reprod. 23: 243-251 [1980~); monkey kidney cells (CV1 ATCC
CCL 70i; african green monkey kidney cells (VERO-76, ATCC CRL-1587~; human cervical carcinoma cells (HELA, ATCCCCL 2i; canine kidney cells (MDCK, ATCCCCL 34~; buffalo rat liver cells (BRL 3A, ATCC CRL 1442~; human lùng cells (W138, ATCC CCL 75~; human liver cells (Hep G2, HB 80651; mouse mammary tumor ~MMT 060562, ATCC CCL51); and, TRI
cells ~Mather, J.P. et al., Annals N.Y. Acad. Sci. 383: 44-68 [19821~.
Construction of suitable vectors containin~ the desired coding and control sequences employ standard li~ation techniques. Isolated plasmids or DNA fra3ments are cleaved, tailored, and reli~ated in the form desired to form the plasmids required.
For analysis to confirm correct seqùences in plasmids constructed, the ligation mixtures are used to transform E. co/i K12 strain 294 (ATCC 31446) and successful transformants selccted by ampicillin or tetracvcline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction and/or sequenced by the method of Messin~ et al., Nucleic Acids Res. 9: 309 ~1981) or by the method of Maxam et al., Methods in Enzymolo~y 65: 499 ~1980).
Host cells are transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducin~ promoters, selecting transformants or amplifying the genes encoding the desired sequences. The culture : .' : . . ... . : :. . : ,:,, .:. . : - :: .:
WV ~ 512 ~. r~, ~ PCT/US91/02166 `
2 û-conditions, such as ~emperature, pH and the like, are those previously used with the host cell salected for expression, and will be apparent to the ordinarily skilled artisan.The host c~lls referred ~o in this disclosure encompass cells in in vit,o culture as well as cells which are within a host animal.
~Transformation" means introducin~ DNA into an oroanism so that the DNA is r0plicable, either as an extrachromosomal 01ament or by chromosomal inte~ration. Unless indicated otherwise, the method used herein for transformation of the host cells is the method of Graham, F. and van der Eb, A., Yirolo~y 52: 456-457 It973). However, other methods for introducin~ DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, the preferred method of transfection is calcium treatment using calcium chloride as dèscribed by Cohen, F.N. et al., Proc. Natl. Ac~d. Sci. ~USAI, 69: 2110 (1972).
~Transfection~ refers to the introduction of DNA into a host cell whether or not any codin~ sequences are ultimately expressed. Numerous methods of transfection are known to the ordinarily skilled anisan, for example, CaPO4 and electroporation. Transformation of the host cell is the indicia of successful transfection. ' The nov01 polypeptide of this invention is recovered and purified from recombinant cell cultures by known methods, includin~ ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchan~e chromato~raphy, phosphocellulose chromato~raphy, immunoaffinity chromato~raphy, hydroxyapatite chromato~raphv and lectin chromato~raphv.
See, e.~., the purification methods described in EP 187,041. Moreover, reverse-phase HPLC
and chromato~raphy usin~ ands for the isolated polypeptide are useful for purification. It is presently preferred to utilize ~el permeation chromato~raphy and anion exchan~e ;
25 chromato~raphy, and more preferred to use cation exchan~e and hydrophobic interaction chromato~raphy (HIC) accordin~ to standard protocols.
Optionally, the isolated polypeptide is recovered and purified by passa~e over a column of isolated polypeptide-antibody covalently coupled to aldehyde silica by a standard procedure IRoy et ~I., Journ~l of Chromaro~raphy 303:225-228 (1984~), washin~ of the column with a saline solution, and analyzin~ the eluant by standard methods such as quantitative amino acid analysis. Procedures utilizin~ monoclonal antibodies coupled to ~lvcerol-coated controlled pore qlass are desirable for the practice of this invention. Optionally, low concentrations lapproximatalY 1-5 mM) of calcium ion may be present durin~ purification.
The isolated polypeptide may preferably be purified in the presence of a protease inhibitor ~ `
35 such as PMSF.
The isolated polypeptide is placed into pharmaceutically acceptable, sterile, isotonic `
formulations to~ether with required cofactors, and optionally are administered by standard means well known in the field. The formulation is preferably liquid, and is ordinarily a ' :
wo 91/1~512 ~ 7 ;~ ~ ~PCr/US91/02166 physiolo~ic salt solution containin~ non-phosphate buffer at pH 6.8-7.6, or may be Iyophilized powder.
The isolated polypept;de compositions to be used in therapy will be ~ormula~ed and dosa~es establishsd in a fashion consistent with ~ood medical practice takin~ into account the disorder to be traated, the condition of the individual patient, the site of delivery of the isolated polypeptide, the method of administration and ol~her factors known to practitioners.
The isolated polypeptide is prepared for administration by mixin~ the isolated polypeptide at the d~sired degree of purity with adjuvants or physiolo~ically acceptable carriers i.e. carriers which are nontoxic to recipients at the dosa~es and concentrations employed. Adjuvants and earriers are substances that in themselves shar~ no immune epitopes with the tar~et anti~en, but which stimulate the imrnune response to the tar~et anti~en. Ordinarily, this will entail combinin~ the isolated polypeptide with buffers, low molecular wei~h~ ~less that about 10 residues) polypeptides, proteins, amino acids, carbohydrates includin~ ~lucose or dextrans, chelatin~ agents such as EDTA, and other excipients. Freunds adjuvant (a mineral oil emulsion) oommonly has been used for this purpose, as have a variety of toxic microbial substances such as mycobacterial extracts and cytokines such as tumor necrosis factor and interferon ~amma in U.S. patent 4,963,354.
Althou~h anti~en is desirably administered with an adjuvant, in situations where the initial inoculation is delivered with an adjuvant, boosters with anti~en may not require adjuvant.
Carriers often act as adjuvants, but are generally distin~uished from adiuvants in that carriers comprise water insoluble macromolecular particulate structures which a~re~ate the anti~en, Typical carriers include aluminum hydroxide, latex particles, bentonite and liposomes.
It is envisioned that injections lintramuscular or subcutaneous) will be the primary route for therapeutic administration of the vaccines of this invention, intravenous delivery, or delivery throu~h catheter or other sur~ical tubin~ is also used. Alternative routes include tablets and the like, commercially availa~le nebulizers for liquid formulations, and inhalation of Iyophilized or aerosolized receptors. Liquid formulations may be utilized after reconstitution from powder formulations.
The novel polypeptide may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues includin~ blood. Suitable examples of sustained release carriers inc)ude semipermeable polymer matrices in the form of shaped articles, e.u. suppositories, or microcapsules.
Implantable or microcapsular sustained release matrices include polylactides tU.S. Patent 3,773,919, EP 58,481) copolymers of L-~lutamic acid and ~amma ethyl-L-~lutamate (U.
Sidman etat., ~iopo/ymers~22(1): 547-556, ll985)), poly 12-hydroxyethyl-methacrylate) or ~thylene vinyl acetate IR. Lan~er e~ at., J. ~iomed. Mater. Pes. 15: 1 67-277 (1981) and R.
Lan~er, Chem. Tech. 12: 98-105 l1982)). Liposomes containina the isolated polypeptide are prepared by well-known methods: DE 3,218,121 A; Epstein et al., Proc. ~tatl. Acad. Sci. USA, WO glJ15sl2 ,~ PCr/US91/n2166 82:3688-3692 11985); Hwan~ eral., Proc. Natl. Acad. Sci. US~, 77:4030-4034 i1980): EP
52322A; ~P 36676A; EP 88046A; EP 143949A; EP 14Z541A; Japanese patent apPlication 83-11808; U.S. Patents 4,485,045 and 4,544,545; and UP 102,342A. Ordinarily the liposomes are of the small labout 200-800 &~n~stromsl unilamelar tvpe in which the iipid content is ~reater than about 30 mol. % cholesterol, the seiected proportion bein~ adjusted for the optimal rat~ of the polypeptide leaka~e.
The dose of the isolated polypeptide administered will be dependen~ upon the properties of the isolated polypeptide employed, e.~. its bindino activity and in YiVo plasma half-life, the concentra~ion of the isolated polypeptide in the formulation, the administration route, ~he site and rate of dosa~e, the clinical tolerance of the patient involved, the patholo~ical condition afflictin~ the pa~ient and the like, as is well within the skill of the physician. Generally, doses of from about 0.5 x 10- to 5 x 10' molar of isolated polypeptide per patient per administration are preferred. Different dosa~es are utilized durin~ a series of sequential inoculations: the practitioner may administer an initial inoculation and then boost with relatively smaller doses of isolated polypeptide vaccine.
The isolated polypeptide vaccines of this invention may be administered in a variety of ways and to different classes of recipients. The vaccines are used to vaccinate individuals who may or may not be at risk of exposure to HIV, and additiùnally, the vaccines are desirably administered to seropositive individuals and to individuals who have been previously exposed to HIV (see e.~. Salk, Nature 327:473-476 ~1987); and Salk et al., Science 195:834-847 ~1977)).
The isolated polypeptide may be administered in combination with other anti~ens in a sin~le inoculation "cocktail". The isolated polypeptide vaccines may also be administerad as one of a series of inoculations administered over time. Such a series may include inoculation with the same or different preparations of HIV anti~ens or other vaccines.
The adequacy of the vaccinition parameters chosen, e.~. dose, schedule, adjuvantchoice and the like, is determined by takin~ aliquots of serum from the patient and assayin~
antibody titers durin~ the course of the immunization pro~ram. Alternatively, the presence of T cells may by monitored by conventional methods as described in Example 1 below. In addition, the clinical condition of the patient will be monitored for the desired effect, e.~. anti-infectiva effect. If inadequatc vaccination is achievsd then tha patient can be boosted with funher isolated polypeptlde vacclnations and thc vaccination parameters can be modified in a fashion expected to potentiate the immune response, e.~. increase the amount of anti~en and/or adjuvant, complex the anti~en with a carrier or conju~ate it to an immuno~enic protein, or vary the route of administration.
For use of the isolated polypeptide as a vaccine, it is currently preferred that at least three separate inoculations with isolated polypeptide be administered, with a second inoculation bein~ administered more than two, preferably three to ei~ht, and more preferably ~' W() 91/15~1' PCr/USgl/0216S
8 ~V ~ -S' approximately four weeks fo~lowinD th~ first inoculation. It is preferred that a third inoculation be adrninistered several months later than the second "boost~ inoculation, preferably at least more than five months following the first inoculation, more preferably six months to two years followin~ the first inoculation, and even more preferably ei~ht months 5 to one year followin~ the first inoculation. Pariodic inoculations beyond the third are also desirable to enhance the patient's 'immune memory". See Anderson et ~1., J. Infectio~s Diseases 160(6):960-969 IDec. 1989). Gensrally, infrequent immunizations with isolated polypeptide spaced at relatively lon~ intervals is more preferred than fre~uent immunizations in eliciting maximum antibody responses, and in elicitin~ a protective effect.
The polypeptides of this invention may optionallv be administered alono with other pharmacolo~ic a~ents used to treat AIDS or ARC or other HlV-related dissases and infections, such as AZT, CD4, antibiotics, immunomodulators such as interferon, anti-inflammatory agents, and anti-tumor a~ents.
Antibodies -This invention is also directed to monoclonal antibodies. In accordance with this invention, monoclonal antibodies specifically bindin~ an epitope of an isolated polypeptide or anti~enically active fra~ments thereof are isolated from continuous hybrid cell lines formed by the fusion of anti~en-primed immune Iymphocytes with myeloma cells. The antibodies of the subject invention are obtained throu~h routine screenin~. An assay is usad for 20 screenin~ monoclonal antibodies for their cytotoxic potential as ricin A chain containin~
immunotoxins. The assay involves treatin~ cells with dilutions of the test antibody followed by a Fab fra~ment of a secondary antibody coupled to ricin A chain ~'indirect assay'). The cytotoxicity of tha indirect assay is compared to that of the direct assay where the monoclonal antibody is coupled to ricin A chain. The indirect assay accurately predicts the 25 potency of a ~iven monoclonal antibody as an immunotoxin and is thus useful in screenin~
monoclonal antibodies for U52 as immunotoxins - see also Vitetta et 91., Science238:1098-1104 ~19871, and Weltman et a/., Cancer ~es. 47:5552 ~1987).
Monoclonal antibodies are highly specific, bein~ directed a~ainst a sin~le anti~enic site.
Furthermore, in contrast to conventional antibody IPolYclonal) preparations which typically 30 include different antibodies directed a~ainst different determinants ~epitopes), each monoclonal antibody is directed a~ainst a sin~le determinant on the anti~en. Monoclonal antibodies are useful to improve the selectivity and specificity of dia~nostic and analytical assay methods using anti~en- antibody bindin~. A second advanta~e of monoclonal antibodies is that they are synthesized by the hybridoma culture, uncontaminated by other 35 immuno~lobulins. Monoclonal antibodies may be prepared from supernatants of cultured hybridoma cells or from ascites induced by intra-peritoneal inoculation of hybridoma cells into mice.
WO 91/15~12 ~ ~ PCI'/US91/02166 ~4 The hybridoma techniqu~ described ori~inally by Kohler and M;lstein, Eur. J. Immuno/., 6:511 t1976) has been widelv applied to produce hybrid cell lines that secrete hi~h tevels of monoclonal antibodies a~ainst many specific anti~ens.
In panicular embodiments of this invention, an antibody is obtained by immunizin~ mice such as Balb/c or, preferably C57 BL/6, aoainst an isolated polypeptide and screenin~ for a clonal antibody that, when preincubated with the isolated polypeptide, prevents its bindin~
to isolated polypeptide. Monoclonal antibodies may desirably have differences in affinity, immuno~lobulin class, species of ori~in, or epitope; they may bs antibodias which are expressed in recombinant cell culture or thas are predetermined amino acid sequence variants of known antibodies, includin~ chimeras of antibodies havinq a variable re~ion directed a~ainst an isolated polypeptide, and a human constant re~ion.
The route and schedule of immunization of the host animal or cultured-antibody-producin~ cells therefrom are ~enerally in keepin~ with established and conventional techniques for antibody stimulation and production. Applicants typically have employed mice as the test model althouph it is contemplated that any mammalian subject includin~ human subjects or antibody producin~ cells therefrom can be manipulated accordin~ to the processes of this invention to serve as the basis for production of mammal;an, includin~ human, hybrid cell lines.
After immunization, immune Iymphoid cells are fused with myeloma cells to qenerate a hybrid cell line which can be cultivated and subcultivated indefinitely, to produce lar~e quantities of monoclonal antibodies. For purposes of this invention, the immune Iymphoid cells selected for fusion are Iymphocytes and their normal differentiated pro~eny, taken either from Iymph node tissue or spleen tissue from immunized animals. Applicants prefer to ~.
employ immune spleen cells, since they offer a more concentrated and convenient source of :
antibody producin~ cells with respect to the mouse system. The myeloma cells provide the .
basis for continuous propa~ation of the fused hybrid. Myeloma cells are tumor cells derived from plasma cells~ -It is possible to fuse cells of one species with another. However, it is preferred that the source of immunized antibody producin~ cells and myeloma be from the same species.
The hybrid cell lines can be maintained in culture In vitro in cell culture media. The cell lines of this invention can be selected and/or maintained in a composition comprising the continuous cell line in hypoxanthine-aminopterin thvmidine IHAT) medium. In fact, once the hybridoma cell l;ne is established, it can be maintained on a variety of nutritionally adequate .
media. Moreover, the hybrid cell lines can be stored and preserved in any number of conventional ways, includin~ freezin~ and stora~e under liquid nitro~en. Frozen cell lines can be revived and cultured indefinitely with resumed synthesis and secretion of monoclonal antibody. The secreted antibody is recovered from tissue culture supernatant by conventional methods such as precipitation, lon exchan~e chromato~raphy, affinity chromato~raphy, or WO 91/15512 PCr/US91/02166 -25- . 0~45 the like. The antibodies described herein are also recovered from hybridoma cell cultures by conventional methods for purificasion of I~G or I~M as the case maV be that heretofore have been used to purify these immuno~lobulins from pooled plasma, e.~. ethanol or polyethylene ~Iycol precipitation procedures. The purified antibodies ar0 sterile filtered, and optionally are 5 conju~ated to a detectable marker such as an enzyme or spin label for us~ in dia~nostic assays of isolated polypeptide in test samples.
While the invention covers usin~ mouse monoclonal antibodies, the invention is not so limited; in fact, human antibodies may be used and may prove to be preferable. Such antibodies can be obtained by using human hybridomas ~Cote er ~I., Monocton~l Ant~bodies 10 ~nd C~ncer Therapy, Alan R. Liss, p. 77 /1g85~1. In fact, accordinu to the invention, techniques developed for the production of chimeric antibodies ~Mùrrison et ~/., Proc. N~tl.
Acad. Sci., 81:6851 ~1984~; Neuberger et al., Nature 312:604 11984); Takeda et a/., Narure 314:452 ~1985)) by splicin~ the ~enes from a mouse antibod~ molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate 15 biolo~ical activity ~such as ability to activate human complernent and mediate ADCC) can be used: such antibodies are within the scope of ~his invention.
As another alternative to the cell fusion technique, EBV-immortalized 8 cells are used to produce the monoclonal antibodies of the subject Invention. Other methods for producing monoclonal antibodies such as recombinant DNA, are also contemplated.
20 Immunotoxins This invention is also directed to immunochemical derivatives of the antibodies of this invention such as immunotoxins ~conjugates of the antibody and a cytotoxic moiety). The antibodies are also used to ;nduce Iysis throu~h the naturat complement process, and to interact with antibody dependent cytotoxic cells normally present.
Purified, sterile filtered antibodies are optionally conjugated to a cytotoxin such as ricin for use in AIDS therapy. EPO Publication 0 279 688 published 24 Au~ust 198~ illustrates methods for making and usin~ immunotoxins for the treatment of HIV infection.
Immunotoxins of this invention, capable of specifically bindina regions of HIV env, are used to kill cells that are already infected and are actively ptoducin~ new virus- KillinD iS
30 accomplished by the bindin~ of the immunotoxin to viral coat protein which is expressed on infected cells. The immunotoxin is then internalized and kills the cell. Infected cells that have incorporated viral ~enome into their DNA but are not synthesizing viral protein ~i.e., cells in which the virus is laten~) may not be susceptible to killing by immunotoxin until they begin to synthesize virus. The antibodies of this invention which span the clip site and/or the other 35 antibodies described herein may be used alone or in any combination with for delivering toxins to infected cells. In addition, a toxin-antibodv conjugate can bind to circulating viruses or viral coat protein which will then effect killing of cells that internalize virus or coat protein.
.. , . . ~ ,, . . - . :
WO91/15~12 , ~ PCI'/US91/02166 The subject invention provides a hi~hly selective method of d~stroyin~ HIV infected cells, utilizing the antibodies described herein.
While not ~ishina to be constrained to any particular theory of operation of theinvention, it is believed that ~he expression of the tar~et anti~en on the infected cell surface is transient. The antibodies must be capable of reachin~ the site on the cell surface where the anti~en resides and interactin~ with it. After the antibody complexes with the anti~en, endocytosis takes place carryin~ the toxin into the cell.
The immunotoxins of this invention are particularly helpful in killin~
monocytes/macrophages infected with the HIV virus. In contrast to the transient production of virus from T cells, macrophages produce hi~h !evels of virus for lon~ periods of time.
Current therapy is ineffective in inhibitin~ the production of new viruses in these cells.
Not all monoclonal antibodies specific for an isolated polypeptide make hi~hly cytotoxic immunotoxins, however assays are routinely and commonl~/ used in the field to predict the ability of an antibody to function as part of a immunotoxin. Preferablv the antibodies used cross react with several ~or all) s~rains of HIV.
The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial, funaal, plant or animal ori~in, or an enzymatically active fragment of such a toxin. Enzymatically active toxins and fra~ments thereof used are diphtheria A
chain, nonbindina active fraaments of diphtheria toxin, exotoxin A chain ~from Pseudomonas aeru~mos~), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcln"41eurites fordïï
proteins, dianthin proteins, Phytolaca ameticana proteins ~PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, aelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. In anotherembcdiment, the antibodies are conju~ated to small molecule anticancer dru~s such as cis~
platin or 5FU. Conju~ates of the monoclonal antibody and such cytotoxic moieties are made using a variety of bifunctional protein coupling agents. Examples of such rea~ents are SPDP, IT, bifunctional derivatives of imidoesters such as dimethyl adipimidate HCI, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis ~p-azidobenzovll hexanediamine, bis-diazonium derivatives such as bis- ~p-diazoniumbenzoyl)- ethylenediamine, diisocyanates such as tolylene 2,6-diisocyanate and bis-active fluorine compounds such as 1,5-difluoro- 2,4-dinitrobenzene. The Iysin~ portion of a toxin rnay be joined to the Fab fraament of the antibodies.
Immunotoxins can be made in a variety of ways, as discussed herein. Common~y known crosslinkina reagents can be used to yield stable conju~ates.
Advantaaeously, monoclonai antibodies specifically bindin~ the domain of the protein which is exposed on the infected cell surface, are coniuaated to ricin A chain. Most advantageously the ricin A chain is deglycosylated and produced through recombinant means.
WO 91/1~512 -27- ,~ ~ 7 ~ ~ ~C~/U~91/02166 An advanta~eous method of makin~ the ticin immunotoxin is described in Vitetta et ~1., Science 238:1098 11987).
When used to kill infected human cells in vitro for dia~nostic purposes, the conju~ates will typically be added to the cell culture medium a~ a concentration of at least about 10 nM.
5 The formulation and mode of administration for in vitro use are not critical. Aqueous formulations that are cornpatible with the culture or peRusion rnedium will norrnally be used.
Cytotoxicity may be read by conventional techniques.
Cytotoxic radiopharmaceuticals for treatin~ infected cells rnay be made by conju~atin~
radioactive isotopes le.g. 1, Y, Pr~ to the antibodies. Advanta~eously alpha particle-emittin~
1 û iso~opes are used. The term 'cytotoxic moir~ty" as used herein is intended to include such isotopes.
In a preferred embodiment, ricin A chain is de~lycosylated or produced without oli~osaccharides, to decrease its clearance by irrelevant clearance mechanisms (e.~., the liver). In another embodiment, whole ricin (A chain plus B chain) is conjugated to antibody 15 if the ~alactose bindin~ property of B-chain can be blocked ~blocked ricin~l.In a further embodiment toxin-conju~ates are made with Fab or Flab'12 fra~ments.Because of their relatively small size these fralaments can better penetrate tissue to reach infacted cells.
In another embodiment, fuso~enic liposomes are filled with a cytotoxic dru~ and the 20 liposomes are coated with antibodies specifically bindinp HIV env.
Antibodv Decendent Cellular Cvtotoxicitv The present invention also involves a method based on the use of antibodies which are lal directed a~ainst an isolated polypeptide, and (b) belon~ to a subclass or isotype that is capable of mediatin~ the Iysis of HIV virus infected cells to which the antibody molecule 25 binds. More specifically, these antibodies should belon~ to a subclass or isotype that, upon complexin~ with cell surface proteins, activates serum complement and/or mediates antibody dependent cellular cytotoxicity ~ADCCI by activating effector cells such as natural killer cells or macropha~es.
The present invention is also directed to the use of these antibodies, in their na~ive 30 form, for AIDS therapy. For example, IgG2a and IgG3 mouse antibodies which bind HlV associated cell suRace anti~ens can be used in viuo for AIDS therapy. In fact, since HIV
env is present on infected monocytes and T-lymphocytes, the antibodies disclosed herein and their therapeutic use have ~eneral applicability.
Biolo~ical activity of antibodies is known to be determined, to a lar~e extent, by the 35 Fc re~ion of the antibody molecule ~Uananue and Benacerraf, Textbook of lmmunoloDy, 2nd Edition, Williams & Wilkins, p. 21 8 119841). This includes their ability to activate complement and to mediate antibody-dependent cellular CytotoxiCitY ~ADCCI as effected by leukocytes.
Antibodies of different classes and subclasses differ in this respect, and, accordin~ to the WO 91/15~12 c ~ ') PCI/US91/02166 present invention, antibodies of those classes havin~ the desired biolo~ical activity are selected. For example, mouse immunoQlobulins of the I~G3 and I~G2a class are caPable of activatin~ serum complement upon bindin~ to the tar~et cells which express the co~nate anti~en.
In ~en0rall antibodies of the I~G2a and I~G3 subclass and occasionally I~G1 can mediate ADC~, and antibodies of the I~G3, ioG~a, and I~M subclasses bind and activate serum complement. Complement activation ~enerally requires the bindin~ of at least two I~G
molecules in close proximity on the tar~et cell. However, the bindin~ of only one 10M
molecule activates serum complement.
O The ability ~f any particular antibody to mediate Iysis of the tar~et cell by complement activation andlor ADCC can be assayed. Ths cells of interest are ~rown and labeled in vitro;
the antibody is added to the cell culture in combination with either serum complement or immune cells which may be activated by the an~i~en antibody complexes. Cytolysis of the tar~et cells is detected by the release of label from the Iysed cells. In fact, antibodies can be screened usin~ the patient's own serum as a source of complement and/or immune cells. The antibody that is capable of activatin~ complement or mediatin~ ADCC in the in vitfo test can then be used therapeutically in that panicular patient.
Antibodies of virtually any ori~in can be used for this purpose provided they bind an isolated polypeptide epitope ~ can activate complement or mediate ADCC. Monoclonal antibodies offer the advanta~e of a continuous, ample supply.
TheraDeutic and Other Uses of the Antibodies When used In vivo for therapy, the antibodies of the subject invention are administered to the patient in therapeutically effec~ive amounts li.e. amounts that restore T cell counts).
They will normally be administered parenterally. The dose and dosa~e re~imen will depend upon the de~ree of the infection, the characteristics of the particular irnmunotoxin lwhen used~, e.g., its therapeutic index, the patient, and the patient's history. Advama~eously the immunotoxin is administered continuously over a period of 1-2 weeks, intravenously to treat cells in the vasculature and subcutaneously and intraperitoneally to treat re~ional Iymph nodes. Optionally, the administration is made durin0 the course of adjunct therapy such as combined cycles of turnor necrosis factor and interferon or other immunomodulatory a0ent.
For parenteral administration the antibodies will be formulated in a unit dosageinjectable form ~solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic, and non-therapeutic.
Examples of such vehicles are water, saline, Rin~er's solution, dextrose solution, and 5%
human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicitY and chemical stabilitY, e.~., buffers and WO 91/15512 PCr/US91/02166 h V 7 ~
preservatives. The antibodies will tvpicalh~ be formula~ed in such vehicles at concentrations of about 1 m~/ml to 10 m~/ml.
Use of I~M antibodies is not currently preferred, since the anti~en is hi~hly specific for the target cells and rarely occurs on normal cells. I~G molecules by bein~ smaller may be more able than I~M molecules to localize to infacted cells.
There is evidence that cornplement activation in vivo leads to a variety of biolo~ical effects, includin~ the induction of an inflammatory response and the activation of macropha~es lUananue and Benecerraf, Texrbook of Immunoloay, 2nd Edition, Williams &
Wilkins, p. 218 ll 984~h The increased vasodilation accompanyin~ inflammation may increase the abilitv of various anti-AlDS a~ents to localize in infected cells. Therefore, anti~en-antibody cornbinations of the ~ype specified by this invention can be used therapeutically in many ways. Addilionally, purified anti~ens IHakomori, Ann. Rev. Immunol 2:103 11~84)) or anti-idiotypic antibodies INeporn et a/., Proc. Natl. Acad. Scl: 81:2864 (1985); Koprowski et al., Proc; Natl. Acad. Sci. 81:216 11984)) relatin~ ~o such anti~ens could be used to induce an active immune response in human patients. Such a tesponse includes the formation of antibodies capable of activatin~ human complement and mediatin~
ADCC and by such mechanisms cause infacted cell destruction.
The antibodies of the subject invention ate also useful in the dia~nosis of HIV in test samples. They are employed as one axis of a sandwich assay for an isolated polypeptide of HIV env, toqether with a polyclonal or monoclonal antibody directed at another stericallv-free epitope of HIV env. For use in some embodiments of sandwich assays the anti-isolated polypeptide antibody is bound to an insolubilizin~ support or is labelled with a detectable moiety followin~ conventional procedures used with other monoclonal antibodies. In another embodiment a labelled antibody, e.p. Iabelled Goat anti-murine I~G, capable of bindin~ the anti-isolated polypeptide antibody is employed to detect the isolated pol~peptide or ~IIV env bindin~ usin~ procedures previously known pef se.
The antibody compositions used in therapy are formulated and dosa~es established in a fashion consistent with ~ood rnedical practice takin~ into account the disorder to be treated, the condition of the individual patient, the site of deliverv of the composition, the method of administration and other factors known to practitioners. The antibody compositions are prepared for administration accordin~ to the description of preparation of polvPeptides for administration, Infra.
In order ~o facilitate understandin~ of the followin~ examples certain frequently occurrin~ methods and/or terms will be described.
~ Plasmids" are desi~nated by a lower case p preceded and/or followed bV capital letters and/or numbers. The startin~ plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord ,.
: : . . . : . : -WO 91/1~12 ~ .Q, ~ PCTIUS91/02166 with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
In particular, it is preferred that these plasmids have some or all of the follov~in~
characteristics~ possess a minimal number of host-organism sequences; (2) be stable in the desired host; (31 be capable of being present in a high copy numbet in the desired host;
(4) possess a re~ulatabls promoter; and (5) have at least one DNA sequence codin~ for a selectable trait present on a portion of the plasmid separate from that where the novel DNA
sequence will be inserted. Alteration of plasmids to meet the above criteria are easily performed by those of ordinary skill in the art in light of the available literature and the teachin~s herein. It is to be understood that additional clonin0 vectors maV now exist or will be discovered which have the above-identified properties and are therefore suitable for use in the present invention and these vectors are also contemplated as bein~ within the scope of this invention.
~Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, tvpically 1 ~ of plasmid or DNA fra~ment is used with about 2 units of enzyme in about 20 ~JI of buffer solution. For the purpose of isolatin~ DNA fra~ments for plasmid construction, typically 5 to 50 119 of DNA are di~ested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular r~striction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37C are ordinarily used, but may vary in accordance with the supplier's instructions. After di~estion the reaction is electrophoresed directly on a polyacrylamide ~el to isolate the desired fra~ment.
Size separation of the cleaved fra~ments is performed using 8 percent polyacrylamide ~el described by Goeddel, D. et a/., Nucleic Acids Res. 8: 4057 11980).
~PCR" lpolymerase chain reaction~ refers to a technique whereby a piece of DNA is amplified. Oli~onucleotide primers which correspond to the 3' and 5' ends Isense or antisense strand-check) of the se~ment of the DNA to be amplified are hybridized under appropriate conditions and the enzyme Taq polymerase, or eqùivalent enzyme, is used to synthesize copies of the DNA located between the primers.
"Dephosphorylation~ refers to the removal of the terminal 5' phosphates by treatment with bacterial alkaline phosphatase ~BAP). This procedure prevents the two restriction cleaved ends of a DNA fra~ment from "circularizin~ or forming a closed loop that would impede insertion of another DNA fra~ment at the restriction site. Procedures and reagents for dephosphorylation are conventional. Maniatis, T. et al., Molecular Clonino pp. 133-134 11982). Reactions usin~ BAP are carried out in 50mM Tris at 68C to suppress the activity ":: . ' . ' ', ~ , ; : ':' : , , :, :" ' : : , WO 91/15512 PCI`/U~91/ûZ166 f~ V J ~ 5 of any exonucleases which are present in the anzyme pteparations. Reactions are run for 1 hour. Followin~ the reaction the DNA fra~ment is ~el purified.
~ OIi~onucleotides~ refers to either a sin~le stranded polydeoxynucleotide or two complementarv polydeoxynucleotide strands which may be chemically synthesized. Such 5 synthetic oli~onucleotides have no 5' phosphate and thus will not li~ate to another oli~onucleotide without addin~ a phosphate with an ATP in the presence of a kinase. A
synthetic oli0onucleotide will li~ate to a fragrnent that has not been dephosphorylated.
aLi~ation~ refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T. et a/.., Id., p. 1461. Unless otherw;se provided, 10 li~ation is accomplished using known buffers and conditions with 10 units of T4 DNA li~ase ~"ligase~) per 0.5 ,u~ of approximately equimolar arnounts af the DNA fra~ments to be li~ated.
~ Filling~ or ~blunting~ refers to the procedures bV which the single stranded end in the cohesive terminus of a restriction enzyme-cleav~d nucleic acid is converted to a double strand. This eliminates the cohesive terminus and forms a blunt end. This process is a 15 vPrsatile tool for convertin~ a restriction cut end that may be cohesive with the ends created bV only one or a few other restriction enzymes into a terminus compatible with any blunt-cutting restriction endonuciease or other filled cohesive terminus. Typically, bluntin~ is accornplished by incubating 2-15 JJ~ of the tar~et DNA in 1 OmM M~CI~, 1 mM dithiothreitol, 50mM NaCI, 10mM Tris IpH 7.5) buffer at about 37C in the presence of 8 units of the 20 Klenow fra~ment of DNA polymerase I and 250 ~M of each of the four deoxynucleoside triphosphates. The incubation generally is terminated after 30 min. phenol and chloroform extraction and ethanol precipitation.
It is understood that the application of the teachings of the present invention to a specific problem or situation will be within the capabilities of one havin~ ordinary skill in the 25 art in light of the teachings contained herein. Examples of the products of the present invention and representative processes for their isolation, use, and manufacture appear below, but should not be construed to limit the invention.
EXAMPLE
We have been able to produce large amounts of two different r~pl20 fusion proteins 30 in a mammalian cell system (Laskv et a/., 1986~. This has allowed us to elucidate all nine of the disulfide bonds, the positions of the ~Iycosylation sites that are utilized and the type of oli~osaccharide moiety present at each site in r~p120 from the 1119 isolate of HIV-1 produced in CHO cells.
This sxample describes the structural characterization of the recombinant envelope 35 glycoprotein Ir~pl20) of human immunodeficienc~/ virus type 1 produced by expression in Chinese hamster ovary cells. Enzymatic cleavage of rgp120 and reversed-phase high performance liquid chromato~raphy were used to confirm the primary structure of the protein, to assign intrachain disulfide bonds and to characterize potential sites for N-glycosylation '~:
WO 91/15~12 ~ 'J PCI/US91/0216fi J
~ - 3 2 -All of the tryptic peptides identified were consistent with the primary structure predicted from the cDNA sequenc~ Tryptic mappin~ studies combined with treatment of isolat~d peptides with S. ~ureus V8 protease or with peptide: N-~lycosidase F (PNGase F) followed by endoproteinase Asp-N permitted the assi~nment of all nine intrachain disulfide bonds of r~p120. The 24 potential sites for N-~lycosylation were characterized by determinin~ the susceptibilities of the attached carbohydrate structures to PNGase F and to endo-,B-N-acetyl~lucosaminidase H. Tryptic mappin~ of enzymaticaily de~lycosYlated r~pl20 was used in conjunction with Edman de~radation and fast atom bombardment-mass spectrometry of individually treated peptides to determine which of these sites are ~Iycosylated and what types of structures are present. The results indicate that all 24 sites of ~p120 are utilized, includin~ 13 that contain complex-type oli~osaccharides as the predominant structures, and 11 ~hat contain primarily hi~h mannose-type andlor hybrid-type oli~osaccharide structures~
For convenience, complete biblio~raphic references are ~iven at the end of this Example.
EXPERIMENTAL PROCEDURES
The abbreviations used throu~hou~ this example are: AAA, amino acid analysis; AIDS, acquired immunodsficiency syndrome; amu, atomic mass unit; CHO, Chinese hamster ovary;
Drr, dithiothreitol; endo H, endo-,8-N-acetyl~lucosaminidase H; FAB-MS, fast atom bombardment-mass spectrometry; ~D1, herpes simplex type 1 Dlycoprotein D; ~p, ~Iycoprotein; HIV, human immunodeficiency virus; HPLC, hi~h performance liquid chromato~raphy; IAA, iodoacetic acid; PNGase F, peptide: N-~lycosidase F; PTH, phenylthiohydantoin; RCM, reduced and S carboxymethylated; r~p, recombinant ~Iycoprotein;
SIV, simian immunodeficiency virus; TFA, trifluoroacetic acid; TPCK, 2 5 L- 1 p-tosylamido-2-phenylethyl chloromethyl ketone~
Materials-- Recombinant ~pl 20 proteins were produced in CHO cells and purified by immunoaffinity chromato~raphy as previously described (Lasky et al., 1986). DTT, iAA, and 2-acetamido-1-,6-(L-aspartamido~-1,2-dideoxy-D-glucose ~GIcNAc-Asnl were obtained from Si~ma Chemical Company. HPLC/Spectro Grade trifluoroacetic acid tPierce), Acetonitrile UV
~American B&JI, and Milli Q" water ~Millipore) were used for reversed-phase HPLC. The enzymes used were TPCK trypsin from Worthin~ton 8iomedical Corp., endoproteinase Asp N
~"sequencin~ ~rade") obtained from Boehrin~er Mannheim t;mbH, S. aureus V8 protease from ICN ImmunoBiolo~icals, and PNGase F ~N-Glycanasen) and endo H from Genzyme.
Reduction and S-Carboxymethylation-- Recombinant ~pl 20 ~2.0 mg of CL44 ISEQ. ID NO.
121) was dialyzed a~ainst 0.36 M Tris buffer, pH 8.6, containin~ 8 M urea and 3 mM EDTA.
DTT was added to a concentration of 10 mM and the sample was incubated for 4 hours at ambient temperature. The sample was then treated with 25 mM IAA in the dark for 30 .
WO 91/15~12 PCT/l S91/02166 -33- h ~ 8 ~
minutes at ambient tsmperature. The reaction was ~uenched with excess Dl~, the sample was dialyzed auainst 0.1 M ammonium bicarbonate, and ~hen Iyophilked.
Treatment of RCM toP120 with PNGase F-- RCM r~pl20 10.5 m~) was reconstituted in 0.1 ml of 0.25 M sodium phosphate, pH 8.6, containin~ 10 mM EDTA and 0.02% NaN, to a5 concentration of 5 mg per mL. Tryptic peptides w0re reconstituted to the same molar concentration in 0.05 mM sodium phosphate, pH 7.0, con~ainin~ 0.02% NaN~. PNGase F
was arlded to the sample in the ratio of 12.5 units per mg of protein ~nd the sample was incubated overni~ht at 37C. RCM r~pl 20 treated with PNGase F was dialy~ed a~ainst 0.1 M ammonium bicarbonate.
10 Treatment of RCM r~p 120 with Endo ~/- RCM rgp120 (0.5 m~ was reconstituted in 0.1 ml of 0.05 M sodium phosphate, pH 6.0, containin~ 0.02% NaN3. Endo H (2 unitslml) was added to the sample in the ratio of 0.1 unit per m~ of protein anrJ the sample was incubated overni~ht at 37 C. RCM r~pl 20 treat0d with endo H was dialyzed a~ainst O. t M ammoniurn bicarbonate.
15 Treatment with TPCK-Trypsm- Samples of untreated, PNGase F-treated and endo H-treated RCM r~pl20 (0.5 m~ aliquots of CL44 ISEQ. ID N0. 121) in 0.1 M ammonium bicarbonate were treated at ambient temperatur~ with TPCK-trypsin by the addition of aliquots of enzyme ~enzyme to substrate rstio of 1:100 w/w~ at 0 and 6 hours of incuba~ion. The dioestion was stopped after 24 hours by freezing the samples. For disulfide determinations, a sample ot 20 r~pl 20 (0.5 m~ of 9AA [SEû. ID N0. 11 l~ was treated with TPCK-trypsin usin~ the same conditions.
Treatmenf of Trypt;c Peprides with PNG~se F Followed by Endoprotemase Asp-N - Paptides (ran~in~ ~rom 0.5 nmol to 3.7 nmol) purifi~d by reversed-phase HPLC of a 9AA tryptic di~est were reconstituted in 0.05 M sodium phosphate, pH 7.0, containin~ 0.02% NaNJ 10.05 ml).
25 PNGase F (5 units in 0.06 ml of 0.05 M sodium phosphate, pH 7.0, containin~ 0.02% NaN3) was added and the samples were incubated for 20 hours at 37C. Endoproteinase Asp-N (2 micro~raml was then added and the samples were incubated for 20 hours at 37C.
Treatment of Tryptic Peptides with S. aureus V8 Pfotease-- Peptides 13.0 nmol) purifir~d by 30 reversed-phase HPLC of a 9AA tryptic di~est were reconstituted in 0.05 M sodium phosphate, pH 7.0, containin~ 0.02% NaN3 10.04 ml). V8 protease 15 micro~ram) was added at 0 and 7 hours and the sample was incubatr~d for 24 hours at 37C.
Treatment of CL44 Peptides with Endo H Fo/lowed by PNGase F-- Peptides (tYpicallY 3 nmol) purified by reversed-phase HPLC were reconstituted in 0.05 M sodium phosphats, pH 6.0, 35 containin~ 0.02% NaN3 (0.1 ml). Endo H 10.05 unit in 0.025 ml of 0.05 M sodium phosphate, pH 6.0, containin~ 0.02% NaN3) was added and the sample was incubated for 20 hours at 37C. PNGase F 16.25 units~ and 0.5 M sodium phosphate, pH 10.3, containin~
~'` ~': ' :.' ' .
WO 91/15512 ~ ~ P~/US~1/02166 `~, 3 0.02 M EDTA and 0.02% NaN, 10.125 ml) were then added and the sample was incubated for 20 hours at 37C.
P~eversed-phase 11PLC-- Tryptic diDests were fractionated by reversed-phase HPLC on a 5 micron Vydac C18 endcapped column ~4.6 mm x 250 mm). After equilibration with 0.1%
5 aqueous TFA, the elution of tryptic peptides was carried out at 1 ml per minute with a linear ~radien~ from O to 45% acetonitrile containing 0.08% TFA in 90 minutes. The system used was a Waters gradient liquid chromato~raph consistin~ of two 6000A pumps, a 720 controller, and a WISP 71 OB injector, and a Perkin-Elmer LC75 sin~le wavelength UV detector set at 214 nm. ;
Peptides subjected to further manipulations were fractionated by reversed-phase HPLC
on a Vydac C18 column 12. 1 mm x 250 mm) equilibrated in 0. 1% aqueous TFA at a flow rate of 0.2 ml per minuts and a temperature of 40C. These peptides were eluted with a linear ~radient from O to 60% acetonitrile Icontaining 0.08% TFA) in 60 minutes. The system used was a Hewlett-Packard tO9OM liquid chromato~raph.
15 Peptjde Identific~tion-- Peptides collected from reversed-phase HPLC were identified by AAA
and/or N-terminal sequence analysis. Samples for AAA were treated with constant boilin~
IICI a~ 110C in vacuo for either 24 or 72 hours, dependin~ upon extent of elycosylation.
The extended hvdrolysis degrades ~lucosamine, which would otherwise interfere with quantitation of lle and Leu. Analysis was performed on a Beckman Model 6300 amino acid 20 analyzer with ninhydrin detection.
N-terminal sequence analysis was performed on an Applied ~iosystems Model 477At120A. The acetonitrile concentration in the equilibration buffer of the PTH analysis system was decreased from 10 to 9% to resolve the PTH derivative of GlcNAc-Asn from Drr.
25 fA~-MS-- FAB mass spectra were acquired on a JEOL HX110HF/HX110HF tandem massspectrometer operated in a normal two-sector rnode. FAB-MS was performed with 6 keV
xenon atoms 110 mA emission currentl. Data were acquired over a mass ranae of 380-4000 amu.
R E~LTS
Lasky et al. 11986) expressed ~pt 20 in CHO cells as a fusion proteln usin~ the signal peptide of the herpes simplex ~D1. Two such fusion proteins were used in this study. The recombinant ~Iycoprotein used in most of this study lCL44 lSEQ. ID NO. 121) was expressed as a 498-amino-acid fusion protein containing the first 27 residues of gD1 fused to residues 31-501 of gp120 ILasky et a/., 19861. This construction lacks the first cysteine residue of 35 mature ~p120. Disulfide assignment~ were carried out on another recombinant fusion protein 19AA ISEQ. ID NO. 111~ which conta;ns the first 9 residues of gD1 fused to residues 4-501 of gp12û. This restores the first cysteine residue, Cys 24. Carboxy-terminal analysis of CL44 ISEQ. ID NO. 1 2l usin~ carboxypeptidase digestions ind;cated that ~lutamic acid residue WO 91/15~12 PCI/US91/02166 -35~ .~
479 is the carboxy t~rminus of the fully processed rnolecule sect0ted by CH0 cells Idata not shown). The amino acid saquences of these two constructions are ~iven in Fi~ure 1.
RCM CL44 Tryptic M~p- Reversed-phase HPLC tryptic mappino was used to confirm the primary structure of the molecule, to assign intrachain disulfide bonds and to characterize potential sites for N-~lycosylation. In a~periments not intended to ~ive information about disulfides, the protein was RCM prior to di~estion with trYpsin. This treatment unfolds the protein and disrupts disulfide bonds, thereby resultin~ in smaller tryptic fra~ments than would be obtained with the native mol0cule.
Thc reversed-phass HPLC trvptic map of RCM CL44 is shown in Fi~urs 2. Tryptic ~ p 10 peptides were separated by reversed-phase HPLC usin~ an acetonitrile/water system with TFA as the ionic modifi2r. As will be discussed below, much of the pe~k heterogeneity derives from the extremely hi~h laPProximatelY 50% of total mass) carbohydrate content of the molecule. Peaks were collected and subjected to AAA for identification ITable 1). In some cases, N-terminal sequence analysis was used for confirmation Ithese peaks are 15 indicated in Table l? The peaks not assi~ned a label in Fi~ure 2 were not identified.
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WO 91/1~511 PCr/U~i~1/02166 -37~
All of th~ peptidea identified were consis~ent with the primary structu~e predicted from the cDNA sequence. Of tha 38 predict0d peptides with 3 or more amino acids, 36 were identified in the tryptic map of RCM CL44. In addition, 4 predicted peptides consisting of 2 amino acids each were alsc identified IH3, H4, T23, and T35). The tripeptide composed of residues 139-141 IVQK) was not identified in the map and was not given a label in Fi~ure 2.
The only other peptide not identified was T13 (CNNK). Aspara~ine residue 200 of peptide T13 is a potential glycosylation site and the peptide lacks hydrophobic amino acids.
Therefore, this ~Iycopeptide is likely ~o be extremely hydrophilic and poorly resolved from the salt fracticn on the reversed-phase column.
Tryptic cleava~e did not occur between peptides T5 and T6 and between peptides T8 and T9. These are designat~d in Figure 2 as two T-numbers separated by a comma ~T5,6 and T8,9). The absence of cleavage was confirmed by N-terminal sequence analysis of the peptides, In both of these cases, the asparagine residue to the C-terminal side of the cleava~e site is a potential N-~lycosylation site and it is likely that the carbohydrate moiety interferes with the action of tr~/psin. Incomplete tryptic cleavage was also observed between peptides H4 and T2' and between peptides T23 and T24 IH4,T2' and T23,241.
Several peptides arisin~ from non-tryptic cleavages were observed in the tryptic map of RCM CL44. Two of the predicted tryptic peptides were further cleaved by ~chymotrypsin-like~ cleava~es. Peptide T12 was completely cleaved af~er tyrosine residue 187 and phenylalanine residue 193 to yield peptides T12a, T12b, and T12c. Peptide T4 was partially hydrolyzed after leucine residue 95 to yield peptides T4a and T4b. Intact peptide T4 was also present.
One of the tryptic peptides, T22 ~QAHCNISRl ISEQ. ID NO.14l eluted at two different positions 132.4 minutes and 34.1 minutesl in the RCM CL44 tryptic map. Deglycosylation studies ~discussed below) with PNGase F and endo H indicated that the different retention times of the $wo forms of peptide T22 are not due to carbohydrate differQnces. It is possible that this retention time heterogeneity results from partial conversion of the N-terminal glutamine residue to pyroglutamic acid ISanger and Thompson, 19531.
Disulfide Ass/6~nments ~ p12~- Mature gp120 CQntains 18 cysteine residues lshaded in Figure 1) and therefore could contain 9 intrachain disulfide bonds. The CL44 ~SEQ. ID NO.
12] construction lacks ~ys-24, the first cysteine residue of gp120 ILasky ~t a/., 1986);
therefore, a different construction 19AA îSEQ. ID NO. t 11l, in which the first cysteine residue was restored, was expressed and purified to approximatelY the same degree as CL44 (L.
Riddle, T. Gregory and D. Dowbenko, unpublished data). Ellman's reagent (Ellman, 1959) was used to demonstrate the absence of free sulfhydryl groups in 9AA ISEQ. ID NO. 111 Idata not shown). Therefore, disulfide assignments were determined for the 9AA
construction.
WO 91/15 i l 2 ~ PCI-/US91/02166 Tryptic rnappin~ studies performed without S-carboxymethyiation of cysteine residues allowed panial assi~nment of disulfides. The tryptic map of 9AA is shown in Fi~ure 3. Peaks were identified by N-tsrminal sequence analysis ITable ll). These identitications allowed unequivocal assi~nment of three ot the nine disulfide bonds: between Cys-101 and Cys-127 ~Peak A, Table ll~; between Cys~266 and Cys-301 (Peak B, Table ll); and between Cys-24 and Cys-44 ~Peak E, Table ll).
Peptides containin~ the remainin~ cysteine residues were also identified (Table ll).
Peptide T28 contains three cysteine residues and coelutes with peptide T31, which contains one cysteine residue (Peak D, Table ll). Peptide T11 contains two cysteine residues and coelutes with peptides T3 and T4, each of which contains a sin~le cysteine residue ~Peak F, Table ll). Similarly, peptide T14 contains two cysteine residues and coelutes with peptides T12 and T13, each of which has a sin~le cysteine residue (Peaks C and E, Table ll). In each of these cases more than one disulfide bond was present in the ~roup of tryptic peptides, thereby preventin~ unambi~uous assi~nment. These tryptic peptides were further manipulated as described below to introduce selective cleava~e between cysteine residues located on a sin~le peptide.
wo 91/~5;12 39 PCr/VS91/02t66 . able 11. Identification of Cyste;ne-containing Peptides ir~ ge~ 5 T/yptic Ma~ gAA.
Cys-containing peaks trom the ~ryptic map ot 9AA were identified by N-~erminal sequence analysis. Cysteines in boxes joined by a solid line represent disulfide bond assignments. Cysteines in boxes joined by dotted lines represent dis~ulfide bonds that could not be assigned unarnbiguously in this experiment. Partial cleavages are indicated by a parenthesis. Cysteines are labelled by an amino acid number and peptides are labelled with T-numbers corresponding to the nomenclature used in Figure 1.
Peak Cys-Containing Peptides _ _ _ _ _ 101 (~5,6) A [~TDLKNDTNTNSSSGR
(GEIK)NE~3SFNISTSIR
127 ~ra _ - ~
266 (T16) B TIIVQLNQSVEIN[~3TRPNNNTR
QAH E~¦NISR
301 (T22) (T12a.~) 188 C VSFEPIPIHYl~APAGF E~NNK
/ ~ ~ ~, ' (T14a) l ~ ~
TFNGTGP[~TNVSTVQ~}THGIR
_ _ .
D QSSGGDPEIVTHSFN~GGEFFY~lNSTQLFNSTWFNSTWSTE--GSNNTEGSDTITLP R ~ _ ~ (T31) 388 E~SSNITGLLLTR
_ :
24 ~Tt) E EATTTLF~3ASDAK
AYDTEVHNVWATHA ~3VPTDPNPaEVVLVNVTENFNMWK
ITl2) 188 VSFEPIPIHY~APAGFAILK 198 (T13) / ~ _ ~ _ TFhGTGP~ITNVSTVQ~3THGlRPVVsTQLLLNGsLAErrVVlR
-- . :
NDMVEQMHEDllSLWDaSLKP~VK ~ (T4) LDllplDNDTTsyTLTs~3NTsvlTQA~3pK -.
WO 91/15~12 ~ 3~ ~ PCI/U!~9t/02166 Each of the pepsides has a potential N-linked ~IycosylaTion site located between the cysteine residues. The peptides were treated with PNGase F, which removes asparaoine-linked carbohydrate while conver~in~ the attachment aspara~ine residue to aspartic acid (Tarentino e~ a/., 1985~. The resultin~ aspartic acid residue serves as a point 5 for selective cleava~e of the peptides Witt1 endoprot~inase Asp-N ~Drapeau, 1980). The peptides were separated by reversed-phase HPLC and identified by N-terminal sequence analysis.
The HPLC chromato~ram obtained after treatment of peptides T12, T13, and T14 (Peak C, Fi~ure 3) with PNGase F follow~d by endoproteinase Asp-N is ~iven in Fi~ura 4a, and 10 the sequences of rel0vant peptides are given in Table lll. The results indicate that r~pl 20 has disulfide bonds be~ween Cys-198 and Cys-209 and between Cys-188 and Cys 217 ITable Ill). Treatment of peptides T3, T4, and T11 IPeak F, Fi~ure 3) with PNGase F followed by endoproteinase Asp-N allowed the recovery of fra~ments that demonstrated the presence of disulfide bonds between Cys-89 and Cys-175 and between Cys-96 and Cys-166 IFi~ure 4b 15 and Table lll) : : ~ :
WO 91~ 12 ~ V 7 8 ~P~/US~I/02166 Table lll. Assignment of Disu!fides from Peptides isolated in Figure 4.
. The tryptic peptides that could not be assigned unambiguously in Table li were further rnanipulated as described in Figure 4. Peaks were identified by N-terminal sequence analysis.
Peak Sequence :
.... . . .~
198 ~
1 QN ~ :
, . .
DGTG PlJT ~ :
~:
2EPIPIHY~}APAGF :~
DVSTVQ1~3THG( I R) . 3 DQSLKP~lVK ;:
DTSVITQA [~1P K
.
4 LTPL~VSLK
DDrrSYTLTS
IVTHSFN~GGE
I~SSNITGLLLTR
6FFY~NSTQLFNSTWFNSTWSTE
TITLPL~IR ~:
'' WO 91/1~512 ~ PCT/US91/02166 The last two disulfida bonds wsre assioned by treatinD peptides T28 and T31 IPeak D, Fi~ure 31 with V8 protease to cleave to tha carboxy side of the ~lutamic acid and aspartic acid residuss IDrapeau et ~/., 1972) locatad between the cystsine residues of T28. The chromato~ram obtained after V8 protease dipestior~ of T28 and T31 is ~iven in Fi~ure 4c and 5 the sequences of the relevant peptides are oiven in Table lll. The results demonstrated the presence of disulfide bonds between Cys-348 and Cys-415 and between Cys-355 and Cys-388.
Thus, th0 combined results of the tlyptic mapping analysis and the further selective deDradations permitted the assi~nment of all nine intrachain disulfid3 bonds of r~pl20.
10 Parallel experiments performed on CL44 ~SEQ. ID N0. 121 produced similar results for the 8 disulfide bonds remainin~ in that construction (not shown). The disulfide bond assi~nments of rgp120 are summarized in Fi~ure 6.
Glycosy/ation Sites of ~p t2~- Mature ~pl 20 contains 24 potential sites for N-~lycosylation, as recoanized by the sequence: Asn-Xaa-SerlThr) IKornfeld and Kornfeld, 1985~. These sites 15 are indicated by a dot above the correspondin~ aspara~ine residue in Fi~ure 1A. tn the present study, tryptic mappin~ of enzymatically de~lycosylated CL44 [SEQ. ID N0. 121 was used in conjunction with Edman de~radation and FAB-MS of individually treated peptides to determine which of the 24 potential N-~lycosylation sites are Dlycosylated and which contain Iess fully proccssed li.e. hi~h mannose-type or hybrid-type) oli~osaccharides.
The two enzymes used for de~lycosylation were PNGase F and endo H. PNGase F
releases all types of N-linked oli~osaccharide structures by cleava~e of the ~-aspartylplucosylamine linka~e ITarentino et ol., 1985). Endo H releases only hi~h mannose-type and hybrid-type oli~osaccharide structures by cleavinp between the two core N-acetyl~lucosamine residues ITai et ~/., 1977). De~lycosylation of a peptide can be 25 monitored by the increase in retention time of the peak correspondin~ to the Glycopeptide in the reversed-phase elution profile. Thus, it was possible to determine which peptides were 31ycosylated by treatment with PNGase F and, on the basis of susceptibility tn cndo H, to distin~uish those with attached high mannose~type andlor hybrid-type oli~osaccharides as the predominant structures.
The 24 potential ~Iycosylation sites of CL44 ~SEQ. ID N0. 121 are contained in 14 tr~ptic clycopeptides. Thineen of thesr~ olycopeptides were identified in the tryptic map of RCM CL44 lFiaure 2~. As mentioned above, T13 ICNNK) lSEQ. ID N0. 15~ was not identified. The tryptic maps of PNGase F-treated RCM CL44 and endo H-treated RCM CL44 are compared with the RCM CL44 tryptic map in Fi~ure 5. The peaks correspondinp to 35 ~Iycopeptides are labelled in each of thc three tryptic maps.
As would be expected for a heavily ~Iycosylated mo!ecule, treatment of RCM CL44 with PNGase F ~Fi~ure 5b~ simplified ~he tryptic map si~nificantly. Typically, the peaks correspondin~ to potential ~Iycopeptides in the RCM CL44 tryptic map IFi~ure 5a) were broad WO 91/15~1' PCI/US91/02166 -43- 7 ~
and often appeared as muitiplets. De~iycosylation resulted in sharp, sin~l~ peaks tor each pep~ide, indicatin~ that the g!ycopeptide psak multiplicity and broadness was due to carbohydrate hetero~eneity. ~ .
All of the 13 potential plvcopeptides that had been identified in the tryptic map of RCM
CL44 were shifted to later retention times in the tryptic map of PNGase F-treated material.
This dsrnonstrates that at least 13 of th0 24 potential sites are ~Iycosylated. Peptide T28 was not recover0d after de~lycosylation. This peptide contains a lar0e number of non-polar i~
amino acids and, after removal of the hydrophilic carbohydrate moieties, may bind irreversibly ~;
to the HPLC column. As described above, peptide T22 elutes at 2 positions in tha RCM GL44 tryptic map presumably as a result of conversion of the N-terminal ~lutamine to pyroglutamic acid. The retention times of both of the T22 peaks were altered in the de~lycosylated material produced by treatment with both PNGase F and endo H, confirmin~ that the difference between these forms of peptide T22 in the RCM CL44 tryptic map was not due to carbohydrate hetero~eneity. .
The ~ryptic map of endo H-treated RCM CL44 (Fi~ure 5c~ indicated that 6 of the 13 tryptic ~Iycopeptides were endo H-susceptible ~p~ptides T14, Tl 6, T22, T24, T28, and T31~.
In addition, a small amount of peptide T15 showed endo H susceptibility. For each of these ~Iycopeptides, the elution time of the endo H-treated glycopeptide was eariier than that of the correspondin~ PNGase F-treated ~Iycopeptide. This is due to the hydrophilic N-ace~yl~lucosamine residue that remains attachcd to the asparagine residue followin~ endo H treatment. Peptide Tl 6 was not identified in the tryptic map of endo H-treated RCM CL44.
This peptide contains 3 potential glycosylation sites and was poorly recovered under any circumstances.
Conclusions as to the type of ~Iycosylation present on each of the tryptic :
~Iycopeptides based on susceptibility to PNGase F and endo H are summarized in Table IV.
S0ven of the 13 ~Iycopeptides identified in the tryptic map of RCM CL44 contain only a sin~le ~Iycosylation site and thus could be characterized unambi~uously with re~ard to enzyms susceptibili~y. Peptides T2' lAsn-58), T26 ~Asn-3261, and T32 ~Asn-433) were de~lycosylated only by PNGase F and, therefore, contain attached complr~x-type oli~osaccharide structures. Peptides T22 ~Asn-302), T24 (Asn-309), and T31 ~Asn-418) were susceptible to both PNGase F and endo H and, therefore, ~arry high mannose-type and/or hybrid-typc oli~osaccharide strtJctures. Peptide T15 is only partially susceptible to endo H; therefore, Asn-246 carries primarily complex-type oli~osaccharides but must also have some attached hi~h mannose-type and/or hybrid-type oli~osaccharide structures.
WO 91~15512 ~ PCI/US91/0~166 Table IV. Assignmen~ ~I Glycosyla~ion Type ~o RCM CL44 Tryp~ic Peptides by Susceplibility to PNGase F and Endo H.
Suscep~ibili~y to PNGase F or endo H was determined by an increase in ~he retention ~ime ol a peptide in the tryptic map o~ FICM CL44 . PNGase F reieases all types ot N-linked oligosaccharide s~ructures, whereas endo H releases only high mannose and hybridoligosaccharide structures.
Tryp~ic GlycosylaVon Si~es SuscepHble Susceptible Glycosyblion Pep~ide a (Asn Residue #) To PNGase F To Endo H Type _ T2 5~ Yes No Complex T6 106,111 Yes No Complex b -T9 126,130 Yes No Complex b T11 156,167 Yes No Complex b T14 204,211,232 Yes Yes High Mannose, Hybrid, and/or Complex c T15 246 Yes Trace Complex (Trace High Mannose and/or Hybrid) T16 259,265,271Yes Yes High Mannose, Hybrid, and/or ComplexC
T22 302 Yes Yes High Mannose and/or Hybrid T24 309 Yes Yes High Mannose and/or Hybrid T26 326 Yes No Complex T28 356,362,367,376 Yes Yes High Mannose, Hybrid, and/or ComplexC
T31 418 Yes Yes High Mannose and/or Hybrid T32 433 Yes No Complex a T13 not tound.
b Eilher or both siles glycosylaled.
c Endo H susceplible glycosylalion at one or more sitels).
WO 91/1~12 . ~ 5~ Pcr/us9l/0~l~6 Peptides T6, T9, and Tl 1 each c~ntain 2 p~t~ntial ~Iycosylation sites. Each peptide was de~lycosylated by PNGase F but not by endo H indicatin~ the presence of mostly complex-type oli~osa~-charide struc~ures. In order to determine wh~th~r one or both of the potential olycosylation sites in each peptide were actually glycosylated, tha PNGase F-treated 5 olycopeptides were subjected to either FAB-MS or Edman dearadation. Treatment with PNGase F converts the attachment aspara~ine residue to aspartic acid during de~lycosylation tTarentino et ol., 1985), This conversion can be detected by FAB-MS as an increase of 1 amu in the mass of the peptide for aach site de~lycosylated tCarr and Roberts, 1986) or by Edman degradation by the appaarance of th~ PTH derivative of aspartic acid at the 10 appropriate cycles. FAB-MS of de~lYcosylated peptide T5,6 rsvealed an ion correspondin~
to the peptide mass plus 2 amu ~[MH]~ observed: m/z 1772.6; calculated: m/z 1772.7).
FAB-MS of deglycssylated pep~ide T9 gavs similar results (IMH1- observed: m/z 1301.8;
calculated: m/z 1301.5). Edman de~radation was performed instead of FAB-MS on de~lycosyla~ed peptide T11 because of its hi~h molecular wei~ht t~2000 amu). Aspartic 15 acid was observed in cycles 8 Iderived from Asn-156~ and 19 (derived from Asn-l 67). These combined results indicate the presence of complex-type oligosaccharide structures attached to Asn residues 106, 1 1 1, 126, 130, 156, and 167.
The remainin~ 3 glycopeptides identified in the tryptic map of RCM CL44 contained multiple potential glycosylation sites and were endo H-susceptible. Peptides T14, T16, and 20 T28 account for a total of 10 potential glycosylation sites. Characterization of each ~Iycosylation site was achieved by Edman degradation of HPLC-purified peptides that had been subjected to treatment with endo H followed by PNGase F.
When endo H releases the high mannose-type and hybrid-type oli~osaccharide structures, it leaves an N-acetylglucosamine residue attached to the aspara~ine residue of the 25 peptide ~Tarentino et ~/., 1974~. PNGase F will not remove this N-acetyl~lucosamine residue, but will release the remainin~ N-linked oli~osaccharide structures by cleavage of the ,B-aspar~ylglucosvlamine bond, resulting in conversion of the attachment asparagine residu0 to aspartic acid lChu, 1986). Therefore, treatment with Endo H followed by PNGase F will yield aspara~ine at an unglycosylated site, GlcNAc-Asn at a ~Iycosylation site that contained 30 primarily hi~h mannose-type and/or hybrid-type oli~osaccharide structures, and aspartic acid at a ~Iycosylation site that carried primarily complex-type oli~osaccharide structures. Paxton et ~/. It 987~ have shown that it is possible to detect the PTH derivative of GlcNAc-Asn after Edman de~radation. Using this approach, it was possible to characterize the remainder of the ~Iycosylation sites of CL44 ISEQ. ID N0. 12~. For example, treatment of ~Iycopeptide T16 35 which contains 3 potential N-glycosylation sites, with endo H followed by PNGase F resulted in the appearance of the PTH derivative of GlcNAc-Asn at cycles 7 and 13 and theappearance of PTH-Asp at cycle 19 during Edman degradation. Thus, ~Iycopeptide T16 carries primarily high mannose-type and/or hybrid-type oligosaccharides at Asn-259 and wo 91/15S12 . ~, ` PCI/US91/02166 Asn-265 and complex-type olioosaccharides at Asn-271. The rosults of shese experiments are sumrr,arized in Table V and indicate that CL44 ISEQ. ID N0. 12] contains complex-type oli~osaccharide structwes at Asn residuas 271, 367, and 376 and high mannoss-tYPe and/or hybrid-type oligosaccharide structures at Asn residues 204, 211, 232, 259, 265, 356, and 5 362.
~ ' ' ~ ' ' I . ' . ' ' , ' ' ' ' ' ' ~ ' , ' . , : " ' ' , ' ' `,`: ' ~ ' '.' ' ' ~ ' ' ' ' ~ . .
WO 91/1~;512 PCr/US91/02166 Table V. Assignment of Glycosylation Type ~o RCM CL44 Tryptic Glycopeptides Containing Multiple Potential Glycosylation Sites.
Characterization of multiple potential glycosylation sites on RCM
CL44 tryptic glycopeptides was achieved by Edman degradation of HPLC purified peptides subjected to treatment wi~h endo H followed by PNGase F. Edman degradation of deglycosylated peptides shows either an Asn residue at an unglycosylated site, a GlcNAc-Asn at a glycosylation site to which had been attached high mannose or hybrid oligosaccharide structures, or an Asp residue at a glycosylation site which had carried complex type oligosacc~aride structures.
Tryptic Asn ~esidue Peptide Residue # Observed Gtycosylation Type . ~ ..... . _ .. .. ....
T14 204 GlcNAc-Asn High Mannose and/or Hybrid 211 GlcNAc-Asn High M~nnose and/or Hybrid 232 GlcNAc-Asn High Mannose and/or Hybrid T16 259 GlcNAc-Asn High Mannose and/or Hybrid 265 GlcNAc-Asn High Mannose and/or Hybrid 271 Asp Complex T28 356 GlcNAc-Asn High Mannose and/or Hybrid 362 GlcNAc-Asn High Mannose and/or Hybrid 367 Asp Complex 376 Asp Complex .. , . : . , ~. . .. . - ., , , ~ , , . . .. . . . . , ,., , . , ~ .; . ... .... . .
wo 91/15~12 ~ ) PCI/US91/02166 -~8-Peptide T13, which con~ains the remainin~ ~Iycosylation site, was not identified in any of the tryptic maps presented in this paper. However, FAB-MS data obtained from the void peak of a tryptic map of RCM CL44 treated with endo H followed by PNGase F revealed an ion correspondin~ to MH~ for that peptide containin~ an attached N-acetyl~lucosamine residue lobserved: m/z 740.1; calculated: m/z 740.4). The prescnce of peptide T13 in the void peak was further confirmed by AAA. Therefore, we conclude that Asn-200 is ~Iycosylated and carries primarily hi~h mannose-type and/or hybrid-type oli~osaccharide structures.
The data presented here demonstrate that all 24 potential ~Iycosylation sites of ~p120 10 are utilized, that 13 sites contain primarily complex-type oli~osaccharide struc~ures while 11 sites contain primarily hi~h mannose-type and/or hybrid-type oli~osaccharide structures. The type of glvcosylation at aach site is summarized in Figure 6.
DISCU~SION
We have determined the disulfide bondin~ pattern and the attachment positions of15 oli~osaccharide moieties of r~p120 from the 11i8 isolate of HIV-1. A schematic representation of this information is presented in Fi~ure ô [SEQ. ID N0. 10]. The r~p120 molerules from which the structural data were obtained possess the functional properties attributed to ~p120 produced by HIV-1 virions includin~ hiqh-affinity CD4 bindinq ~Lasky et ~1., 1987), and HIV-1 neutralizin~ anti~enicity tLasky et a/., 1986). We therefore conclude that the CH0-expressed 20 gp120 is properly folded and that the disulfide-bonded domains reported here for the recombinant molecules are representative of those occurrin~ in gp120 produced by HIV-1 virions.
functiona/Aspectsofop120Structute--Theqp120moleculecomprisesfivedisulfide-bondedloop structures. The first and fourth are simple loops formed by sinqle disulfide bonds while 25 the second, third and fifth are more complex arrays of loops formed by nested disulfide bonds. The fourth disulfide-bonded domain kesidues 266-301) has been shown to contain siqnificant type-sp0cific neutralizin~q epitopes lMatsushita et ~/., 1988; Rusche et a/., 1988;
Goudsmit et ~/., 1988; Javaherian et ~/., 1989~ and the fifth disulfide-bonded domain Iresidues 348-415) has been shown to be important for CD4 bindin~ ILaskY et a/., 1987;
30 Kowalski et a/., 1987). No direct functional correlates have been described for the other three disulfide bonded domains. The amino acid sequence of ~p120 varies to a larqe extent between different viral isolates but the majority of the variability is localized in hypervariable re~ions which punctuate the otherwise relatively conserved sequences ~Willey et al., 1986;
Modrow et a/. 1987~. Modrow et ~ 1987) have identified five hypervariable re~ions which 35 are characterized by sequence variation, insertions and deletions. Four of these hypervariable reaions correspond to well delineated loops as indicated in Fi~ure 6. With the excep~ion o~
the third hypervariable loop ~disulfide-bonded domain IV~ the functional si~nificance of these re~ions is unknown.
WO 91/1~512 PC~/US91/02166 The positions ot the :ystein~ residues and, presumably, the disulfids bondino pattern in op120 are hi~h~y conserved b~twe~n isolat~s. Amon~ HIV-1 isolates, the only ~xception to this c~nservation is the Z3 isolate ~Will~y ~t ~J., 1986) which has an additional pair of cysteine residues in the fourth hypervariable domain ~residues 363-384i. These residues most likely forrn a tanth disulfide bond in the ~p120 from this isolate. The presence of this extra bond in such a hypervariable region probably has no more effect on the structure and function of the molecule than the other sequence variations that occur in that re~ion.
As shown in Fi~. 7 in HIV-2 lSEQ. ID NO. 13], and similarly in SIV ~da~a not shown) the positions of the cystaine r~sidues in disulfide-bonded dorr~ains 1, Il, IV and V are conserved IHuman ~etroviruses and AIDS ~1989). G. Myeres, A. P~abson, S. Josephs, T.
Smith, J. ~erzofsky and F. Won~-Stahl, Editors. U.S. Government Printin~ Offica, Los Alamos National Laboratory, Los Alamos, New Mexico, LA-UR, 89-743). In domain lll there are two additional pairs of cysteine residues ~three in SIV isolate MM142) which are presumed to be disulfide bonded within a fin~er-like domain lll structure analo~ous to that illustrated in Figure 6. Another major differenc0 between HIY-1, IIIV-2 and SIV is that hypervariabls reDion V2 is reduced to five amino acids in HIV-2 and SIV. Ths functional si~nificance of the differences between HIV-1, HIV-2 and SIV is unknown at this time.
One of the most important functions of gp120 is its ability to bind to CD4 and thereby mediate the attachment of virions to susceptible cells IKlatzman et al., 1984; Dal~10ish et ~1., 1984). The CD4-bindin~ function has been locilized by mutagenesis and sttustural studies ILasky et ~1., 1987; Kowalski et al., 1987~ to the r0~ion between residues 320 and 450, which includes the fifth disulfide-bonded domain. Lasky et a/. (1987~ showed that deletion of residues 396 to 407 and muta~enesis of Ala-402 to Asp abolished CD4 binding. They also mapped the epitope of a monoclonal antibody that bloclcs ~pl20-CD4 bindin~ to residues 392-402. Kowalski et ~/. 11987~ identified three re~ions as bein~ involved with CD4 bindin~
Insertions between residues 333-334, 388-390 and 442-443 abolished CD4 bindin~. In addition, a deletion of residues 441-479 abolished CD4 bindin~ while deletion of residues 362-369 within the fourth hypervariable re~ion had no effect on bindin~. Cordonnier et ~1 11989) have shown that muta~enesis of Trp-397 to Tyr or Phe dacreases CD4 binding and chan~es to Ser, Gly, Val or Ar~ abolish bindin~. Nygren et ~ 1988) have teported that a protflolytic fra~ment of ~p120 from residue 322 to near the C-terminus retains the ability to bind to CD4. The results of these studies indicate that the CD4 bindin~ capacity of ~pl 20 is localized to the re~ion between residues 320 and 450 and more specifically to the residues around 333-334, 442-443 and the sequence between 388 and 407.
In the course of efforts to map the epitope of monoclonal antibody 5C2-E5 which blocks ~pl 20-CD4 bindina, Lasky et a/. (19871 treated r~pl 20 (CL44 lSEQ. ID NO 1 2l~ with acetic acid to cleave the protein at aspartic acid residues (In~ram, 1963~ and isolated the peptide fra~ment 383-426 from a column of immobili~ed anti-~p120 monoclonal antibody WO ~/15~12 ~ ') P~/US~ 166 ` ~
,. 50-5C2 E5. Dior~stion of reduc~d rDpl20 yielded the sarre fra~m~nt. ConssquentlY~ il was concludad that a disulfide bond existed between Cys residues 388 and 415. In the analysis reported here we hava failed to find this disulfide bond and, instead, have consistentlY found the disulfide bonds betwe0n Cys 355 and Cys 388, and between Cys 348 and Cys 415 as 5 summarized in Fi~ure 6. We beliflve that the true disulfide bond assi~nment is as indicated in Figure 6 and that the acetic acid dioestion produced some disulfide bond rearran~ement (Ryle and San~er, 1955) in the earlier work.
The 01igosacchat;des of oP12~ Approximately 50% of the apparent molecular mass of ~pl 20 is carbohydrate. The struçtures of the oli~osacchari~e moieties released by 10 hydrazinolysis of CL44 ISEQ. ID N0.
121 r~pl 20 have been exhaustively analyzed (Mizuochi et a/., 1 988a; Mizuochi et ~/., 1 988b).
These authors found that 33% of the N linked oli~osaccharides were of the high mannose type, 4% were of the hybrid type, and 63% were of the complex type. Of the complex olioosacchatides 90% were fucosylated and 94% were sialylated. The complex 15 structures were approximately 4% monoantennary, 61% biantennary, 19% triantennary and 16% tetraantennary. ND 0-linked oligosaccharides were found. Geyer er a/. 11988) have analyzed the oli~osaccharides of ~pl20 from the lll~ isolate of HlV-1 infected human cells.
They found that high mannose type oli~osaccharides accounted for approximately 50% of the carbohydrate structures. The remainin~ structures were fucosylated, partially sialylated 20 bi, tri, and tetraantennary complex type oli~osaccharides. No novel carbohydrate structures, or moieties that would be expected to act as heterophile anti~ens in man, have been isolated from ~pl20 from either source.
We have shown here that all 24 glycosylation sites are utilized, and that 13 of the 24 :
sites contain complex type oli~osaccharides as the predominant structures while 11 contain 25 primarily hybrid and/or hi~h mannose structures. The demonstration of endo H susceptible structures at 11 of the 24 sites is consistent with the earlier results of Mizuochi et al.
~1988a,1988b~ who determined that nearly 40% of the total oli~osaccharide structures released from r~pl 20 were hybrid and/or hi~h mannose type oli~osaccharides.
The 24 potential N linked ~Iycosylation sites in the ~pl 20 sequence are conserved to 30 a lar~e extent between different viral isolates IWilley et a/., 1986; Modrow et a/., 1987~.
Based on the ~pl20 sequence comparisons in these references, 13 of the sites on ~pl20 from the ill9 isolate of HIV 1 are absolutely conserved; these include 8 of the 11 sites that carry predominantly hybrid type and/or hi~h mannose type oli~osaccharides. Thus, the less fully processed li.e. Endo H susceptible) oli~osaccharides of ~p120 are found preferentially 35 at the most conserved ~Iycosylation sites. The remainin~ sites ~8 complex and 3 hybrid/hi~h mannose~ are relatively conserved, even thou~h many of them occur in the hypervariable re~ions. The positions of these sites may shift or be deleted, but there is always one or more new sitels) within 5 to 10 residues of the reference 1119 site. Studies by Willey et a/. 11988) ` ' :
WO 91/15~12 PCrlUS91/02166 -51 h ~ S
d~monstrated that muta~n~sis of Asn-232 to Gln decreased the inf~c~ivity of virions containing th~ mutant ~pl 20 molr~cules without aff~r,tin~ CD4 bindin~ or syncytium formation. At this tim~, no particular functional si~nificanc~ can be attributed to the type of oli~osaccharide structur~ at any of the sitr~s.
The rol~ of th~ carbohydrate moi~ties on ~p120 in CD4 bindin~ has been investi~ated by several authors ILifson et a/., 1986; Matthews e~ a/., 1987; Fenouillet et a/., 1989).
Those that smployed enzymatic dr~lycosylation in the presence of deter~ents ILifson et ~I., 1986; Matthews er ~/., 1987) have conclud~d that the carboh~/drates ar~ not directly involved with the bindin~, but that th~y are required to maintain the conformation o~ ~p120 10 necessary for bindin~. In contrast, Fenouillet et ~/. 11989) enzymatically de~lycosylated ~pl 20 without deter~ent and demonstrated that the CD4 bindin~ affinity was preserved. It ~herefore appears that the carbohydrate moie~ies of ~p120 are not required for its bindin~ to CD4 but that the conformational stabiiity of ~p120 to deter~ents is lost after de~lycosylation.
The r~pl20 used for these determinasions is functionally and structurally equivalent 15 to gp120 produc~d by HIV-1 infected cells.
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.. ' "' ''' ' . . : ' :
WO 91/tS512 ~ PCr/US91/02166 ~ SEQUENCE LISTING
(1) sENERAL INFORMATION:
~i) APPLICANT: G~n~nt~ch, Inc.
(ii) TITLE OF INVENTION: HIV Envelope Polypeptide~
(iii) NUM~ER OF SEQUENCES: 15 ~iv) CORRESPONDENC~ ADDRESS:
~A) ADDRESSEE: Genentech, Inc.
(B) STREET: 460 Polnt San Bruno Blvd : :
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(A) TELEPHONE: 415/266-2614 (B) TELEFAX: 415/952-9881 ~ `
(C) TELEX: 910/371-7168 ~`
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS: ~ .
(A) LENGTH: 18 amino acid~
(B) TYPE: amino acid ~D) TOPOLOGY: lLnear txi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Cys Val Lys Leu Thr Pro Leu Cy8 Cya Asn Thr Ser Val Ile Thr Gln Ala Cy~
WO 91/15512 PCr/US91/02166 -55~ 3 4 ~
t2) INFORMATION POR SEQ ID NO:2:
(i) SEQUENCE CHARAC~ERIS~ICS:
(A) LENGTH: 40 amLno ~cid~
( B ) TYPE: ~mino acid (D) TOPOLOCY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 Pro Ile Hi~ Tyr Cy~ Pro Al~ Gly Phe Ala Ile Leu Ly~ Cy~
A~n Asn Lyu Thr Ph~ A~n Cly Thr Gly Pro Cy8 Thr A~n Val 5er -~
20 25 30 ~:
Thr Val Gln Cy8 Thr His Gly Ile Arg Pro ~:
- 35 40 .- :
:
(2) INFO~MATION FOR SEQ ID NO:3: ,.
`
(i) SEQUENCE CHARACTERISTICS~
~A) LENGTH: 12 amino acids ~) TYPE: amino acid : : :
~D) TOPOLOGY: linear :
~5 ~xi) SEQUENCE DESCRIP~ION: SEQ ID NO:3: ~:
Cy~ Asn Asn Ly~ Thr Phe A~n Gly Thr Gly Pro CYD
30~
~2) INFORMATION FOR SEQ ID NO:4:
.
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 ~mino ~cids ~B) TYPE: amino acid ~D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
40 Cys Ala Pro Ala Gly Phe Ala Ile Leu Ly~ Cy8 Cy5 Thr A~n Val Ser Thr Val Gln Cy~
~2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS: ~ .
(A) LENaTH: 12 amino acida ~:
50 . ~ S ) TYPE: amino acid :~
(D) TOPOLOGY: linear (xl) SEQU~NCE DESCRIPTION: SEQ ID NO:5: ~
55Pro Ile His Tyr Cys Cy~ Thr Hia Gly Ile Arg Pro ~:
1 5 . 10 12 ~.
:
, .:
WO 91/15~12~ PCI/U591/02166 ~
2) INFOF~ATION FOR SEQ ID NO:6:
(i) SEQUFNC~ C~A~ACTERISTICS:
(A) LENGTH: 58 amino acid~
~B) TYPE: ~mlno acid (D) TOPOLOCY: line~r (xi) SEQUENCE DESCRIPTSON: SEQ ID NO:6: ~i Gly Gly ABP Pro Glu Ile Val Thr H$~ Ser Phe A~n Cys Gly Gly Glu Ph~ Ph~ Tyr Cy~ Asn Ser Leu Pro Cy~ Arg Ile Ly~ Gln Phe 20 25 30 ~.`
Ile Aqn Met Trp Gln Glu Yal Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser G-y Gln Ile Arg Cy~ Ser Ser A~n Ile Thr aly 58 .
(2) INFORMATION FOR SEQ ID NO:7:
~i) SEQUENCE C~ARACTERISTICS:
~A) LENGT~: 36 amino acids tB) TYPE: amino acid ~D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Cy~ Gly Gly Glu Phe Phe Tyr Cy~ Cy~ Arg Ile Ly~ Gln Phe Ile 1 5 10 15 : :
A~n Met Trp Cln Glu Val Gly Lyn Ala ~et Tyr Ala Pro Pro Ile : .
Ser Gly Gln Ile Arg Cy~
(2) INFORMATION FOR SEQ ID NO.8: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 ~mino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:B:
Cys Ala Ser Asp Ala Ly~ Ala Tyr Asp Thr Glu Val Hi~ A3n Val Trp Ala T~r Hl~ Ala Cy~
~2) INFORMATION FOR SEQ ID NO:9:
.-.
W O 91/1~512 PCT/USg1/02166 (1) SEQUENCE CHAR~cTERISTICS~
(A) LBNGTH: 32 amino aeid~
(~) TYPE: ~Imino acid ~:~
tD~ TOPOLOGY: 1i~e~r (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Ly~ Ala Tyr Asp Thr ,~ -Glu Val Hi~ A~n Val Trp Ala Thr Hi~ Ala Cy~ Val Pro Thr Asp Pro A~n - .
(2) INFORMATI0N FOR SEQ ID N0:10:
( i ) SEQUENCE CHZ~CTERISTICS: ;-20(A) LENGTB: 479 amino ac~ d~
(B) TYPE: amino alcid (D) TOPOLOGY: lin~ar (Xi) SEQUENCE DESCRIP2ION: SEQ ID NO:10:
Thr Glu Lyl3 Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp 5 10 15 : .
Lyl3 Glu Ala Thr Thr Thr Leu Phe Cya Ala Ser Aap Ala Ly~ Ala :.
3020 25 30 ~, Tyr A~p Thr Clu Val Hi~ A~n Val Trp Ala Thr Hi~ Ala Cy~ Val 35 40 45 ~ -35Pro Thr A~p Pro Asn Pro Gln Glu Val Val Leu Val Asn Val Thr Glu A~n Phe A~n Met Trp Lys Asn A~p Met VB1 Glu Glrl Met Hi~
. 65 70 75 Glu A~p Ile Ile Ser Leu Trp A~p Gln Ser Leu Ly~ Pro Cy5 Val 80 85 90 ' Ly~ Leu Thr Pro Leu Cy~ Val Ser Leu Ly~ Cy~ Thr A~p Leu LyfJ
A~n A~p Thr A~n Thr A~n Ser Ser Ser Gly Arg Met Ile Me'c Glu 50Ly~ Gly Glu Ile Ly~ Asn Cy3 Ser Phe A~n Ile Ser Thr Ser Ile ~:
Arg Gly Ly~ Val Gln Lys Glu Tyr Ala Phe Phe Tyr Ly~ Leu Asp Ile Ile Pro Ile A-p A-n Asp Thr Thr Ser Tyr Thr Leu Thr Ser ~, .
WO ~5~12,. '~ ~', PCI/US9t/0~166 Cy~ A~n Thr Ser Val Ile Thr Gln Ala Cy~ Pro Ly~ Val Ser Phe G}u Pro Ile Pro Ile Hi~ Tyr Cy~ Ala Pro Ala Gly Phe Ala Ile Leu Ly~ CYB Aan A~n LyE~ Thr Phe A~n Gly ~hr Gly Pro Cyl3 Thr Asn Val Ser Thr Val Cln Cy~ Thr Hi~ Cly Il~ Arg Pro Val Val 15Ser Thr Gln LBU Leu Leu A~n Gly Ser Leu Ala Glu Glu Glu Val 230 ~35 240 Val Ile ~rg Ser Ala AE~n Phe Thr A~3p A~n Ala Lyn Thr Ile Ile Val Gln Leu Asn Gln Ser Val Glu Ile A~n Cys Thr Arg Pxo A~n A~n Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Ly~ Ile Gly A~n Met Arg Gln Ala Hi~
30Cy~ Asn Ile Ser Arg Ala Ly~ Trp Asn Asn Thr L~u Lys Gln Ile Asp Ser Lya Leu Arg Glu Gln Phe Gly Asn Asn Ly~ Thr Ile Ile Phe LYB Gln Ser Ser Gly Gly A~p Pro Glu Ile Val Thr Hi~ Ser Phe Asn Cy~ Gly Gly Glu Phe Phe Tyr Cy~ Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Phe A~n Ser Thr Trp ser Thr Glu Gly Ser 45Asn Asn Thr Glu Gly Ser Asp Thr Ile Thr Leu Pro Cy~ Arg Ile LYB Gln Phe Ile Asn Met Trp Gln Glu Val Gly Ly~ Ala Het Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cy~ Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arq A~p Gly Gly A~n A~n A~n Asn Clu Ser ~, .: , ,"" "" ~
WO 91/15;12 PCI/US!Jl/02166 -5~ 7 ~
Glu Il~3 Phe Arg Pro Gly Gly Gly Asp M~t Arg A~p A3n Trp Arg Ser Clu Leu Tyr Ly~ Tyr Ly0 Val Val Ly~ Ile Glu Pro Leu Gly ~;
Val Ala Pro Thr Ly~ Ala Ly~ Arg Arg Val Val Cln Arg Glu ( 2 ) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE C~ACTERISl`ICS: ~, (A) LENGTH: 9 amino Acid ~ B ) TYPE: amino acid (D) TOPOLOGY: linear ~
(xi) SEQVENCE DESCRIPTION: SEQ ID NO:11: ; ;
Ly~ Tyr Ala Leu Ala A~p Ala Ser Leu ~ . .
1 5 9 ~ .
(2) INFORMP~TION FOR SEQ ID NO:12 ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 nmino ~Icid~
( B ) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ly~ Tyr Ala Leu Ala A~p Ala Ser Leu Ly~ Met Ala Asp Pro A~n Arg Phe Arg Cly Lys A~p L~u Pro Vai Leu A~p Gln 27 ~
(2) INFORMATION FOR SEQ ID NO:13: ~ ;
( i ) SEQUENCE CHMACTERISTICS:
(A) LENGTH: 481 amino ac~ds ( B ) TYPE: amino acid ( D ) TOPOLWY: l inear :
(xi) SEQVENCE DESCRIPTION: SEQ ID NO:13:
Thr Gln Tyr Val Thr Val Phe Tyr Gly Val Pro Thr T~p Lys Asn Ala Thr Ile Pro Leu Phe Cys Ala Thr Arg Asn Arg Asp Thr Trp Gly Thr Ile Gln Cy~ Leu Pro Asp Asn Asp Asp Tyr Gln Glu Ile 55 Thr Leu Asn Val Thr Glu Ala Phe Asp Ala Trp Asn Asn Thr Val ':
,~
WO ')1/1551~ P~/US91/V2166 Thr Glu Cln Ala Ile Glu Asp Val Trp Hi~ Leu Phe Glu Thr Ser I1Q Ly~ Pro Cy~ Val Ly~ Leu Thr Pro Leu Cy8 Val Ala M~t L~
Cys Ser Ser Thr Glu sQr Ser Thr Cly Asn A~n Thr Thr Ser Ly~
10Ser Thr Ser Thr Thr Thr Thr Thr Pro Thr A3p Gln Glu Gln Glu 110 llS 120 Ile Ser Clu A~p Thr Pro Cy~ Ala Arg Ala A3p A~n Cy~ 5er Gly Leu Gly Glu Glu Glu Thr Ile Asn Cy~ Cln Phe Asn 2let Thr G,ly Leu Glu Arg Asp Lys LYR Lys Gln Tyr Asn Glu Thr Trp Tyr S6!r Ly~ A~p Val Val Cy~ Glu $hr A~n Asn Ser Thr Asn Gln Thr Gln 170 175 lB0 25Cys Tyr Mat Asn His Cy~ AEln Thr Ser Val Ile Thr Glu Ser Cy~
Asp Lyn Hi~ Tyr Trp Asp Ala Ile Arg Phe Arg Tyr Cy~ Ala Pro Pro Gly Tyr Ala Leu Leu Arg Cy8 Asn Asp Thr Asn Tyr Ser Gly Phe Ala Pro Asn CYB S~r Lys Val Val Ala Ser Thr Cys Thr Arg Met Met Glu Thr Gln Thr Ser Thr Trp Phe Gly Phe Asn Gly Thr 40Arg Ala Glu Asn Arg Thr Tyr Ile Tyr Trp Hi3 Gly Arg Asp Asn Arg Thr Ile Ile Ser Leu Asn Lys Tyr Tyr A~n Leu Ser Leu Hi~
Cys Lyu Arg Pro Gly A~n Ly~ Ile V~ll Ly~ Gln Ile Met Leu Met Ser Gly His Val Phe His Ser Hil3 Gln Pro Ile Asn Lys Arg Pro Arg Gln Ala Trp CYB Trp Phe Lys Gly Lys Trp Lys Asp Ala ~let 55Gln Glu Val Lys Glu Thr Leu Ala LYB His Pro Arg Tyr Arg Gly W ~ ~ItlS~12 PCTtUS91/~21~6 -61~ 5 Thr A~n A Jp Th~ Arg Asn Il~ ser Phe Aln Ala Pro Gly Ly~ Gly Ser A~p Pro GIu Val Ala Tyr Met Trp Thr A~n Cy~ Arg Gly Glu Ph~ Leu Tyr Cy~ A~n Met Thr Trp Ph~ Leu Asn Trp Ile Glu Aan 3~0 385 390 10Ly~ Thr ~ia Arg A~n Tyr Ala Pro Cyn ~l~ Ile Ly~ Gln Ile Ile Asn Shr Trp Hi~ Ly~ Val Gly ~rg A~n VA1 Tyr L~u Pro Pro Arg Glu Gly Glu Leu Ser Cy~ Asn Ser ~hr Val Thr Ser Ile Ile Ala A~n Ile Asp Trp Gln Aan A~n A~n Gln Thr Asn Ile Thr Phe Ser Ala Glu Val Ala Glu Leu Tyr Arg Leu Glu Leu Gly Asp Tyr Ly~
4~5 460 465 25 L~u Val Glu Ile Thr Pro Ile Gly Phe Ala Pro Thr Ly~ Glu LYB
Arg (2) INFORMATION FOR SEQ ID NO~14:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear txi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Gln Ala HiD Cy A~n Ile Ser Arg l 5 B
~2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids ~) TYPE: amino acid ~D) TOPOLOGY: linear .,.
~0(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Cy~ Asn Asn Ly~
l 4 58 `
......... . . . . . . . . .. . . .
. . . ~ . : . : , , ~
Many work~rs in the fi~ld hav~ prspared mutagenic and fraçlment variants of ~p120.
Sae, e.~.: Matsushita er al., _'. Vitolo~y 62:~107-2114 (19881; Rusche et al., Proc. N~rl.
Ac~d. Sci. USA 85:3198-3202 11988); Goudsmitet~/.,AlDS2:157-164 11988);.Javaherian et ~I., Proc. N~tl. Ac~d. Sci. USA 86:6768~6772 ~19891; Lasky et al., Cel/ 50:975-985 (1987); Kowalski et al., Science 237:1351-1355 (19871; Willey et at., Proc. Natl. Ac~d. Sci. .
USA 83;5038-5042 (19861; Modrow et ~/., J. Virolooy 61:570-578 (1987).
The disulfide bondin~ pattern within gpl 20 and the positions of actual oli~osaccharide moieties on the molscule would be useful inforrnation for directin~ muta0enesis and fragmentation studies aimed at defining the func~ional domains of ~p120 and sites for :
potential pharmacolo~ical interruption of its functions (e.~., type-cammon neutralizin~
epi~opes). This information has been difficult to obtain due to the small amounts of ~pl 20 available from natural sources, the complexity of the disulfide bondin~ and oli~osaccharide structures in Qp 120, and uncertainty re~arding the functionality or structural relevance (Moore e~ al., in press) of r~pl 20 produced in non-mammalian systems. ~ -The invent~rs herein have surprisin~ly discovered that certain regions of native gp120 exist in specif;c three-dimensional conformation, ~,vhich conformation is conserved over isotype and strain. `It is an object of this invention to provide novel polypeptides which are useful as diagnostic tools for assayin~ biolo~ical samples for evidence of HIV infection.
It is a further object of this invention to provide novel polypeptides which are usable for vaccines, and for pharmacolo~ic interruption of the course of HIV infection.It is a further object of this invention to provide methods for preparin~ such polypeptides, and antibodies directed to such polypeptides.
Other objects, features, and characteristics of the present in~/ention will become ~ -apparent upon consideration of the following description and the appended claims.
Summarv of the Invention The objects of this invention are accomplished by the preparation and administration of compositions compr;sin~ isolated cyclized polypeptides which are suitable foradministration to a human or non-human patient having or at risk of having HIV infection.
These cyclized polypeptides are selected from the following:
a) C V K L T P L C C N T S V I T a A C ISEC~. ID NO~ 11 and containin~ less than about 28 amino acid residues;
bl P I H Y C A P A G F A I L K C N N K T F N G T G P C T N V S T V Q C T H G
I R P lSEO. ID NO. 21 and containin~ less than about 45 amino acid residues;
c~ C N N K T F N G T G P C lSEO. ID NO. 3] and containin~ less than about 22 amino acid residues:
dl CAPAGFAlLKCCTNVSTVQClSEO.lDNO.41andcontainingless than about 30 amino acid residues;
~,:
WO 9t/1531~ PCI/IJS91/0216 el P I H Y C C T H G I R P ISEQ. ID NO. 51 and containin~ less than about 22 amino acid residues;
f) G G D P E I V T H S F N C G G E F F Y C N S L P C R I K Cl F I N M W Q E V G
K A M Y A P P I S G Q I R C S S N I T G ISEQ. ID. NO. 61 and containin~ less than about 65 amino ac;d residues;
~) C G G E F F Y C C R I K Q F I N M W Q E V G K A M Y A P P I S G Q I R C
ISEQ. ID NO. 71 and containino less than about 45 amino acid residues;
h) CASDAKAYDTEVHNVWATHAClSEQ.lDNO.81andcontainin~
less than about 30 amino acid residues; and i) T T T L F C A S D A K A Y D T E V H N V W A T H A C V P T D P N [SEQ. lt) ;
NO. 91 and containing less than about 50 amino acid residues.
Addiionall~, this invention is also directed to compositions comprising an isolated ~ :
polypeptide havin~ an antigenic determinant or determinants immunolo~ically cross-reactive with a determinant of the HIV env polypeptide of strain HTLV-IIIB havin~ an amino acid 15 sequence selected from the ~roup consistin~ of a) residues 1-80;
b) residues 8-180; . :~
c) residues 165-260;
dl residues 160-260;
e) residues 260-310; and f) residues 320-479.
This invention is particularly directed to vaccines comprising the compositions of this invention. The compositions of this invention, including variant analo~ues thereof, are also useful in dia~nostic assays for HIV neutralizing antibody in patient samples.
Monoclonal antibodies directed to the isolated polypeptides of this invention are provided, characteri7ed by their affinity for li~and, epitope binding, and ability to a) block CD4/~p120 bindin~, bl neutralize HIV virions, c~ reduce reverse transcriptase activity in vitro, and d) inhibit syncytia formation. .
These antibodies are useful as dia~nostics for the presence of HIV infection in a patient or patient sample, and for affinity purification of HIV env. Thesn antibodies are also useful in passively immunizin~ patients infected with I IIV. In certain embodiments, antibodies are provided which are conju~ated to a cytotoxin, a water-insoluble matrix, or to a detectable marker~
Antibodies directed to HIV env epitopes have been described in the literature; however, it should be noted that, due to the variety and confusion amon~ authors currently as to numbarina systems for HIV env sequences, not all antibodies described in the literature as directed to certain re~ions will actually the same residue numbers as defined herein ~see e.g. ~ :
Matsushitaeta/.,J. Vlfol 62:2107~2114(1988);EPOApplicalionNo.EP339504; Rusche "~
~:
W~ 91/15~12 PCI`/US91/02166 5 ~,78~4~ :~
er a/.. Proc. N~t~. Acdd. Sci. USA, 85:3198-3202 ~19881; Looney et al., Science 241:357- ~ :
359 (19881;
Brief DescriDtion of the Drawin~s FIGURE 1 provides the amino acid sequences of la) the mature envelope ~Iycoprotein (~p1201 from the IIIB isolate of HIV-1 lSEQ. ID N0. 101, and ~b) the N-terminal sequence portion of the recombinant fusion glycoproteins (9AA [SEQ. ID N0. 1 1 ] or CL44 ISEQ. ID N0.
121) from the herpes simplex gD1. Fusion sites between the ~D1 and ~p120 se~ments in the 9AA and CL44 constructions are marked with (-I and (~-~, respectively. The letter T refers ~
to observed tryptic cleava~a of the ~p120 se~ment, and ~he peptides are ordered sequentially ~:
starting at the N-terminus of the molecule. Lower case letters following the T number indicate other unexpected proteolytic cleava~es. The letter H refers to the observed tryptic cleava~e of the herpes simplex ~D1 protein portion of CL44. Peptide T2' contains the fusion site in CL44. The cysteine residues of gp120 are shaded, and potential N-olycosylation sites are indicated with a dot above the correspondin~ asparagine residue.
~5 FIGURE 2 shows a reversed-phase HPLC tryptic map of RCM CL44. This chromato~ram was ~enerated with 7.5 nmol of trvpsin-di~ested RCM CL44. Chromato~raphy conditions were as described in Experimental Procedures. Peaks were collected and identified by AAA and in some cases confirmed by N-terminal sequence analysis ~Table 1). Identified peaks are labelled accordin~ to the nomenclature ~iven in Fi~ure 1. Peptides containino poten~ial tryptic sites that were not hydrolyzed ara desi~nated by two T numbers separat0d by a comma.
FIGURE 3 shows a reversed-phase HPLC tryptic map of 9AA. This chromatogram was ~enerated with 6.8 nmol of sample. Chromato~raphy conditions were as described in the Example herein. Peaks containin~ cysteine residues were identified bv N-terminal sequence ; -analysis. These identifications are summarized in Table ll.
FIGURE 4 shows the results of further manipulations of tryptic peptides from the map of 9AA to isolate individual disulfides. The chromatograms are details of microbore 30 reversed-phase HPLC separations of peptides rèsultin~ from: ~a) treatment of peptides T12, T13, and T14 (Peak C, Fi~ure 3~ with PNGase F follûwed by endoproteinase Asp-N, ~b1 treatment of peptides T3, T4, and T11 ~F'eak F, Fi~ure 3) with PNGase F followed by endoproteinase Asp-N, and Ic) treatment of peptides T28 and T31 (Peak D, Figure 31 with S. aureus V8 protease. Chromato~raphy conditions were as described in the Example herein.
35 Peak identifications were determined by N-terminal sequence analysis and are ~iven in Table 111. :
FIGURE 5 shows reverse-phase HPLC tryptic maps of endo~lycosidase treated RCM
CL44. The chromato3rams are tryptic maps of: ~a) untreated RCM CL44, (b~ PNGase : - . .. . . ..
: . : , ~, , .. , .. ,~ , ... . ..
W(~91/15:~12 . l~ J ' PCT/US9~/02166 F-treated RCM CL~4, and lc~ endo H-treated RCM CL44. Each tryptir, map was ~enerated with 7.5 nmol of sample. Chromato~raphy conditions were as described in Experimental Procedures. Peaks were collected and identified by AAA Idata not shown). Glycopeptide peaks are labelled accordin~ to the nomenclature in Fi~ure 1.
FIGURE 6 is a schematic representation of gp 120 of the Illa isolate of HIV-1 showin~
disulfides and ~Iycosylation sites, with the amino acids represented in single-letter code lSEQ.
ID N0. 101. Roman numerals label the five disulfide bonded domains. The five hypervariable re~ions of Modrow e~ a/., J. Virol. 61 :570-S78 11987~ are enclosed in boxes and labelled V1- :
V5. Glycosylation sites containin~ hi~h mannose-type and/or hybrid-type oli~osaccharide t 0 structures are indicated by a branchin~-Y symbol, and glycosylation sites containing complex-type oli~osaccharide structures are indicated by a V-shaped symbol.
FIGURE 7 shows a schematic representation of the HIV env 31ycoprotein ~pl 20 of HIV-2, showing disulfides and potential glycosylation sites ~SEQ. ID N0. 131. Glycosylation sites are indicated by a shaded box around a N residue. i~oman numerals label five disulfide-bonded domains.
Detailed DescriDtion of the Invention .
HIV env is de~ined herein as the envelope polypeptide of Human Immunodeficiency Virus as described above, together with its amino acid sequence variants and derivatives produced by covalent modification of HIV env or its variants in vitro, as discussed herein~
As used herein, the term "HIV env" encompasses all forms of Dpl20 and/or 160, e.~.
including fra~ments, fusions of op160/120 or their fragments with other peptides, and variantly ~Iycosylatad or un~lycosylated HIV env. The HIV env of this invention is recovered free of active virus.
HIV env and its variants are conventionally prepared in recombinant cell culture. For example, see EP publication No. 187041. Henceforth, gp120 prepared in recombinant cell culture is referred to as r~pl 20. Recombinant s~nthesis is preferred for reasons of safety and economy, but it is known to prepare peptides by chemical synthesis and to purify HIV env from viral culture; such env preparations are included within the definition of HIV env herein.
Genes encoding HIV env are obtained from the genomic cDNA of an HIV strain or from available subDenomic clones containinD the Dene encodinD HIV env.
This invention Is dirflcted to isolated polypeptides. Certain of thflse isolatedpolypeptides are defined as cyclized polypeptides comprisin~ a particular amino acid sequence, and certain isolated polypeptides are described by reference to specific amino acid residue numbers. The amino acid numbering reflects the mature HIV-1 ~pl20 amino acid sequence as shown by Fi~. 6. and Fiq. lA ISEQ. ID N0. 10], not counting any signal sequence or other upstream re~ions, and is used throu~hout this description to conveniently connote the intended residues. however it is understood that this invention is not limited to .
~' ~
those specific residue numbers. For gp120 sequences which includa the native HIV-IIIB N-tsrminal si~nal sequence, numberin~ may differ. The same nuclsotide and amino acid residue numbers may not be applicable in other strains where upstream deletions or insertions chan~e the len~th of the viral ~enome and HIV env, but the re~ion encodin~ this portion of ~p120 5 is readily identified by reference to the teachin~s herein. Also, variant si~nal sequences (such as those resultin~ from a fusion with a fra~mented or heterolooous si~nal sequence as discussed below may lead to a sli~ntly different numberin~, however the precise amino acid sequences are discerned for all embodiments by reference to Fi~. 6 and/or Fi~ 1A iSEQ. ID
N0. 101.
1~ Included within the scope of the isolated polypeptides of this invention, as those terms are used herein are polypeptides havin~ spacified amino acid sequences, de~lycosylated or unglycosylated derivatives, homolo~ous amino acid sequence variants, and homolo~ous in vitro-~enerated variants and derivatives, and which variants are capable of exhibiting a biological activity in common with the HlV env of Fi~. 6 or Fig. 7.
Isolated polypeptide biological activity is defined as either 11 immunolo~ical cross-reactivitV with at least one isolated polypeptide, or 2) the possession of at least one adhesive or effector function qualitatively in common with the isolated polypeptide. Examples of the qualitative biolo~ical activities of an isolated polypeptide include the ability to bind to the viral receptor CD4 or known monoclonal antibodies, and the ability of ~pl 20 ta interact with ~p41 20 to induce fusion of the viral and host cell membranes.
Immunolo~ically cross-reactive as used herein means that the candidate polypeptide is capable of competitively inhibitin~ the qualitative biolo~ical activity of an isolated polypeptide havin~ this activity with polycional antisera raised a~ainst the known active analo~ue. Such antisera are prepared in conventional fashion by injectinU qoats or rabbits, 25 for example, subcutaneously with the known ac~ive analo~ue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds.
The ordinarily skilled worker may use the disulfide bondin~ pattern within qp120 and the positions of actual oli~osaccharide moieties on the molecule as described herein for directing mutagenesis and fragmentation variants of the claimed isolatnd polypeptides. It i5 30 intended that the variants of this invention include isolated polypeptides in which one or more residues have been substituted, deletions of one or more residues, and insartions of one or more amlno acid residues.
This inventlon also contemplates amino acid sequence variants of the isolated polypeptides. Amino acid sequence variants are prepared with various objectives in mind, 35 includin~ increasin~ the affinity of the isolated polypeptide for a liaand or antibody, facilitatin~
the stability, purification and preparation of the isolated polypeptide, modifyin~ its plasma half life, improvin~ therapeutic efficacy, and lessening the severity or occurrence of side effects durin~ therapeutic use of the isolated polypeptide. In the discussion below, amino acid . ` , ;.
WO ~ 512 PCl/US91/021~ -sequenc~,~nants of the isolated polypep~ide are provided, exemplary of the variants that may be selected.
Amino acid sequence variants of isolated polypeptide fall into one or more of three classes: Insertional, substitutional, or deletional variants. These variants ordinarily are 5 prepared by site-specific muta~enesis of nucleotides in the DNA encodin~ the isolated polypeptide, by which DNA encodin~ the variant is obtained, and thereafter expressin~ the DNA in recombinant cell culture. However, fra~ments havin~ up to about 100-150 amino acid residues are prspared conveniently bV in vitro synthesis. The followin~ discussion applies to any isolated polypeptide to the exten~ it is applicabie to its structure or function.
The amino acid sequence variants of the isolated polypeptide are predetermined variants not found in nature or naturally occurrin~ alleles. The isolated polypeptide variants typically exhibit the same qualitative biolo~ical--for example, antibody binding--activity as the naturally occurrin~ isola~ed polypeptide or isolated polypeptide analogue. However, isolated polypeptide variants and derivatives that are not capable of bindin~ to antibodies are useful 15 nonetheless (a) as a rea~ent in dia~nostic assays for isolated polypeptide or antibodies to the isolated polypeptide, ~b) when insolubilized in accord with known methods, as a~ents for purifyin~ anti-isola~ed polypeptide antibodies from antisera or hybridoma culture supernatants, and ~c) as immunogens for raisin~ antibodies to isolated polypeptide or as immunoassay kit components llabelled, as a competitive rea~ent for the native isolated 20 polvpeptide or unlabelled as a standard for isolated polypeptide assay) so long as at least one isolated polypeptide epitope remains active.
While the site for introducin~ an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize theperformance of a mutation at a given site, random or saturation mutagenesis ~where all 20 25 possible residues are inserted) is conducted at the tar~et codon and the expressed isolated polypeptide variant is screened for the optimal combination of desired activities. Such screenin~ is within the ordinary skill in the art.
Amino acid insertions usually will be on the order of about from 1 to 10 arnino acid residues; substitutions are typically introduced for sin~le residues; and deletions will ran~e .
30 about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. It will be amply apparent from the following discussion that substitutions, deletions, insertions or any combination thereof are introduced or combined to arrive at a final construct. Insertional amino acid sequence variants of the isolated polypeptide are those in which one or more amino acid residues i~
35 extraneous to the isolated polypeptide are introduced into a predetermined site in the tar~et isolated polypeptide and which displace the preexistinp residues.
Commonly, insertional variants are fusions of heterologous proteins or polypeptides to the amino or carboxyl terminus of the isolated polypeptide. Such variants are referred to as : . . .. , .; , .:
WO 91/15~12 ~ 8 ~ 4 ~D~/US91/02166 fusions of the isolated poivpeptide and a polyp~ptide containin~ a sequance which is other than that which is normally found in the isolated polypeptida at the inserted position. Several ~roups of fusions are contemplated herein.
The novel isolated polypeptides of this invention are useful in dia~nostics or in purification of the antibodies or li~ands by known immunoaffinity techniques.
Desirable fusions of the isolated polypeptide, which may or may not also be immunolo~ically active, include fusions of the mature isolated polypeptide sequence with a si~nal se~uence heterolooous to a native isolated polypeptide as mentioned above. Si~nal sequence fusions ars employed in order to more expeditiously direct the secretion of the isolated polypep~ide. The heterolo~ous siynal replaces tha native isolated polypep1ide signal, and wh0n the resultin~ fusion is reco~nized, i.e. processed and cleaved by the host cell, the isolated polypeptide is secreted. Si~nals are selected based on the intEnded host cell, and ~
may include bacterial yeast, mammalian and viral sequences. The native HIV env signal or the herpes gD ~Iycoprotein si~nal is suitable for use in mammalian expression systems.
C-terminal or N-terminal fusions of the isolated polypeptide or isolated polypeptide fra~ment with an immuno~enic haptan or heterologous polypeptide are useful as vaccine components for the immunization of patients against HIV infection. Fusions of the hapten or heterologous polypeptide with isolated polypeptide or its active fragments which retain T-cell bindin~ activity are also useful in,directin~ cytotoxic T cells a~ainst tar~et cells where the hapten or heterolo~ous polypeptide is capable of bindin~ to a tar~et cell surface receptor.
The precise site at which the fusion is made is variable; particular isolated polypeptide sites are selected in order to optimize the bio!o~ical activity, secretion or bindin~
characteristics of the isolated polypeptide. The optimal site will for a particular application will be det0rmined by routine experimentation. ~:
Substitutional variants are those in which at least one residue in the isolated polypeptide has been removed and a different residue inserted in its place. Such substitutions ~enerally are made in accordance with the followin~ Table 1 when it is desired to finely modulate the characteristics of the isolated polypeptide.
wo 91~1~;12 ,. PCl`tUS9t/02166 -~,3 ~ , o TABL~ 1 Orlainal R~idue Exem~l~ry s-ubgtitutions Ala ~er Arg ly5 Ar~n gln; hi~ ~-~rjp glu ` .
Cyr~ aer; ala Gln ar~n Glu a~p ~
Gly pro : : -Hi~ asn; gln Ile leu; val Leu il~; val Lys arg; gln; ~u Met leu; ile Phe met; leu; tyr Ser thr Thr rJer Trp tyr ~
Tyr trp; phe `
Val ile; l~u Novel amino acid sequences, as well as isosteric analo~s /amino acid or otherwise), as includerJ within the scope of this invention.
Substantial chan~es in function or immunolo~ical identity are made by selectinp substitutions that are less conservative than those in Table 1, i.e., selectin~ residues that differ more si3nificantly in their effect on maintainin~ la) the structure of the polypeptide ~, .
backbone in the area of the substitution, for example as a sheet or helical conformation, lb~
the char~e or hydrophobicity of the molecule at the tar~et site or lcl the bulk of the side chain. Th0 substitutions which in ~eneral are expected to produce the ~reatest chan~es in 30 isolated polypeptide properties will be those in which la) a hydrophilic residue, e.~. seryl or threonyl, is substituted for lor byl a hvdrophobic residue, e.~. Ieucvl, isoleucvl, phenvlalan valyl or alanyl; lbl a cysteine or proline is substituted for lor bVI anV other residue; Ic) a residue havin~ an electropositive side chain, e.~., Iysvl, ar~inyl, or histidvl, is substituted for (or by) an electrone~ative residue, e.~ lutamyl or aspanyl; or Id) a residue havin~ a bulky 35 side chain, e.~., phenylalanine, is substituted for lor by) one not havin~ a side chain, e.
~Iycine.
Some deletions, insenions, and substitutions will not produce radical chan~es in the characteristics of the isola~ed polvpeptide molecule. However, when it is difficult to predict :.
55l2 ~ 7 8 ~ cr/us9l/o2166 the exact effect of the substitution. deletion, or insertion in advance of doin~ so, for example when modifying an immune epitope, one skilled in the art will appreciate that the effect will be evaluated by routine screenin~ assavs. For example, a variant typically is made by site spscific muta~enesis of the isolated polypeptide -encodin~ nucleic acid, expression of the 5 variant nucleic acid in recombinant cell culture and, optionally, purification from the cell culture for example by immunoaffinity adsorption on a polyclonal anti-isolated polypeptide column (in order to adsorb the variant by at least one remainin~ immune epitope). The activity of the cell Iysate or purified isolated polypeptide variant is then screened in a suitable screenin~ assay for the desired characteristic. For example, a chan~e in the immunological 10 character of the isolated polypeptide, such as affinity for T-cell binding, is measured by a competitive-tvpe immunoassay. As more becornes known about the functions in vivo of the isolated polypeptide other assays will become useful in such screenin~. Modifications of ~ -such protein properties as redox or thermal stability, hydrophobicity, susceptibility to proteolytic de~radation, or the tendencv to a~gregate with carriers or into multimers are 15 assayed by methods well known to the artisan.
Another class of isolated polypeptide variants are deletional variants. Deletions are characterized by the removal of one or more amino acid residues from the isolated polypeptide sequence. Typically, deletions are used to affect isolated polypeptide bioloqical activities, however, deletions which preserve the biolo~ical activitV or immune cross-reactivity 20 of the isolated polypeptide are suitable.
Deletions of cysteine or other labile residues also may be desirable, for example in increasin~ the oxidative stability of the isolated polypeptide. Deletion or substitutions of potential proteolysis sites, e.~. Ar~ Ar~, is accomplished by deletin~ one of the basic residues or substitutin~ one by glutaminvl or histidyl residues.
It ~,vill be understood that some variants may exhibit reduced or absent biolo~ical activity. These variants nonetheless are useful as standards in immunoassays for the isolated polypeptide so long as they retain at least one immune epitope of the isolated Polvpeptide.
It is presently believed that the three-dimensional structure of the isolated polypeptides and peptide compositions of the present invention is important to their functioning as 30 described herein. Therefore, all related structural analogs which mimic the active structure of those formed by the isolated polypeptides claimed herein are spscifically incluaed within the scope of thr~ present invention.
Glycosylation variants are included within the scope of the isolatsd polypeptide. They include variants completelY lacking in ~Iycosylation ~un~lycosylated~ and variants havin~ at 35 least one less ~lvcosylated site than ~he native form ~de~lycosylated) as well as variants in which the glycosylation has been chan~ed. Included are deglycosylated and un~lycosylated amino acid sequence variants, deglycosvlated and un~lycosylated isolated polypeptide having the native, unmodified amino acid sequence of the isolated polvpeptide, and other .. . . .. ..
.
. ,.
wo 9~ 512 " PCr/US91/02~6C ~
..
2- :
~Iycosvlation variants. For example, substitu~ional or deletiùnal muta~enesis is empioyed to ~iminate the N- or 0-linked ~Iycosylation sites of the isolated polypeptide, e.~., an asPara~ine residue (not at th~ clip sitel is daleted or substituted for by another basic residue such as Iysine or histidine. Alternatively, flankino residuss makin~ up the ~Iycosylation site are substituted or deleted, even though the aspara~ine residues remain unchan~ed, in order to prevent ~Iycosylation by eliminatin~ the ~Iycosylation reco~nition site.
Unglycosylated isolated pùlypeptide which has the amino acid sequence of the native isolated polypeptide is produced in recombinant prokaryotic cell culture because prokaryotes are incapable of introducin~ ~Iycosylation into polypeptides. .
Glycosylation variants are produced by selectin~ appropriate host cells or by in vitro methods. Yeast, for example, introduce ~Iycosylation which varies si~nificantlY from that of mammalian systems. Similarly, mammalian cells havin~ a different species la.~. hamster, murine, insect, porcine, bovine or ovine) or tissue ori~in (e.~. Iun~, liver, Iymphoid, mesenchymal or epidermall than the source of the isolated polypeptide anti~en are routinely screened for ~he ability to introduce variant ~Iycosylation as characterized for example by ;
elevated levels of mannose or variant ratios of mannose, fucose, sialic acid, and other su~ars typically found in mammalian olycoproteins. In vitro processin~ of the isolated polvpeptide typically is accomplished by enzymatic hydrolysis, e.~. neuraminidase di~estion.Covalent modifications of the isolated polypeptide molecule which do not modify the clip site are included within the scope hereof. Such modifications are introduced by reactin~
tar~eted amino acid residues of the recovered protein with an or~anic derivatizin~ a~ent that ~ -is capable of reactin~ with selected side chains or terminal residues, or by harnessin~
mechanisms of post-translational modification that function in selected recombinant host cqlls. The resultin~ covalent derivatives are useful in pro~rams directed at identifyin~ residues : ;~
important for biolo~ical activity, for immunoassays of isolated polypeptide or for the :
preparation of anti-isolated polypeptide antibodies for immunoaffinity purification of the recombinant isolated polypeptide. For example, complete inactivation of the biolo~ical activity of the protein after reaction with ninhydrin would suo~est that at least one ar~inyl or Iysyl residue is critical for its activity, whereafter the individual rasidues which were modified under the conditions selected are identified by isolation of a peptide fra~ment containin~ the modified amino acid residue. Such modifications are within the ordinary skill in the art and are performed without undue axperimentation.
Derivatization with bifunctional a~ents is useful for preparin~ intermolecular a~re~ates of the isolated polypeptide with polypeptides as well as for cross-linkin~ the isolated poiypeptide to a water insoluble support matrix or surface for use in the assay or affinity purification of its li~ands. In addition, a study of intrachain cross-links will provide direct information on conformational structure. Commonly used cross-linkin~ a~ents include sulfhydryl reagents, 1,1-bisldiazoacetyl)-2-phenylethane, ~lutaraldehyde, N-~, ~ 91/15~12 ~ u 7 ~
hydroxysuccinimide ~st~rs, for exampie sstsrs with 4-azidosalicylic acid, homobifunctional imidoesters includin~ disuccinimidyl esters such as 3,3'-dithiobis Isuccinirnidyl-propionate), and bifunctional maleimides such as bis-N-maleimido-1,B-octane. Derivati~in~ a~ents such as methyl-3~1~p-azido-phenyl)dithio] propioimidate yield photoactivatable intermediates which 5 are capable of formin~ cross-links in th~ presence of li~ht. Alternatively, reactive water insoluble matrices such as cyano~en bromide activated carbohydrates and the systems reactivesubstratesdescribedinU.S.patents3,959,080; 3,969,287;3,691,016;4,195,128;
4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employed for protein imrnobilization and cross-linking.
Polymers 9enerally are covalently linked to the isolated polypeptide herein throu~h a multifunctional crosslinkin~ a~ent which reacts with the polymer and one or more amino acid or su~ar residues of protein. However, it is within the scope of this invention to directly crosslink the polymer by reactin~ a derivatized polymer with ~he isolated polypeptide, or vice versa. Covalent bondin~ to amino ~roups is accomplished by known chemistries based upon 15 cyanuric chloride, carbonyl diimidazole, aldehyde reactive ~roups IPEG alkoxide plus diethyl acetal of bromoacetaidehyde; PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde, succinimidyl active esters, activated dithiocarbonate PEG, 2,4,5-trichlorophenylchloroformate or p-nitrophenylchloroformate activated PEG.
Carboxyl ~roups are derivati~ed by couplinu PEG-amine usinD carbodiimide.
This invention is also directed to polvpeptides of this invention which bV definition or optionally are conformationally stabilized by cyclization. The peptides ordinarily are cyclized by covalently bonding the N and C-terminal domains of one peptide to the correspondin~
domain of another peptide of this invention so as to form cyclooli~omers containin~ two or more iterated peptide sequences, each internal peptide havin~ substantially the same 25 sequence. Further, cyclized peptides ~whether cYclooli~omers or cylomonomersl are crosslinked to form 1 -3 cyclic structures havin~ from 2 to 6 peptides comprised therein. The peptides preferably are not covalently bonded throu~h ~-amino and -carboxyl ~roups Ihead to tail), but rather are cross-linked throu~h the side chains of residues located in the N and C-terminal domains. The linkin~ sites thus ~ensrally will be between the side chains of A, 30 and A10 residues. Substantially identical polypeptides present in the polymerized forms of the peptides hereof are those which e~hibit qualitative isolated polvpeptide activitv, notwithstandin~ the de~ree of amino acid sequence variation amon~ the polypeptides.
Variants which exhibit activity are used as subunits in homo or heteropolymers. In homopolymers th~ peptides are the same. Heteropolymers contain different peptides, each 35 however, chosen from within the parameters described above.
Manv suitable methods per se are known for preparin~ mono- or poly-cvclized peptides as contemplated herein. Lys/Asp cyclization has been accomplished usin~ No-Boc-amino WO 91/15~17 ~ ) PCI/IJS91/0216 acids on solid-phas~ support with Fmoc/OFm side chain protection for LvslAsp; the process is comp~eted by piperidine treatment followed by BoP cyclization.
Glu and Lys side chains also have been crosslinked in preparin~ cyclic or bicyclic peptides: the peptide is synthesized by solid phase chemistry on a p-methylbenzhydrylamine resin. The peptide is cleaved from the resin and depro~ected. The cyclic peptide is formed using diphenylphosphorylazide in dilute dimethylformamide. For an alternative procedure, see Schilleret~ PeptideProteinRes.~25:171-t7711985). SeealsoU.S.Patent4,547,489.
Disulfide crosslinked or cyclized peptides are ~enerated by conventional methods. The method of Pelton et a/. (J. Med. Chem. 29:2370-237~ [1986~) is suitable, except that a ~reater proportion of cyclooligomers are produced by conduc~in~ the reaction in more concentrated solutions than the dilute reaction mixture described by Pelton et ~/. fùr the production of cyclomonomers. The same chemistry is useful for synthesis of dimers lusing A,-A" Pen plus Al-AI~ Cys) or cyclooli~omers or cyclomonomers IPen A1-A,o Cys, or Pen Al-A~o Cys plus Cys A,-A10 Pen). Also useful are thiomethylene brid~es ITetrahedron Letters 25120):2067-2068 i1984~). See also Cody et a/., J. Med. Chem. 28:583 11985).
The desired cyclic or polymeric peptides are purified by ~el filtration followed by reversed-phase high pressure liquids chromato~raphy or other conventional procedures. The peptides are sterile filtered and formulated into conventional pharmacolo~ically acceptable v~hicles .
Certain post-translational derivatizations are the result of the action of recombinant host cells on the e~pressed polypeptide. Glutaminyl and aspara~inyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues.
Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
Other post-translational modifications include hydroxylation of proline and Iysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino ç~roups of iysine, arginine. and histidine side chains tT.E. Creighton, Proteins: Struçture and Molecular ProDerties, W.H. Freeman & Co., San Francisco pp 79-86 119331~, acetvlation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.
DNA encoding the isolated polypeptide is synthesized by in vitro methods or is obtained readily from cDl~iA libraries. The means for synthetic creation of the DNA encodin~ the isolated poiypeptide, either by hand or with an automated apparatus, are generally known to one of ordinary skill in the art, particularly in light of the teachings contained herein. As examples of the current state of the art relating to polynucleotide sYnthesis, one is directed to Maniatis et al., Molecvlar ClomnD--A Lsbora~ory Manual, Cold Spring Harbor Laboratory t1984), and Horvath et al., An Automated DNA Synthesizer Employin~ Deoxynucleoside 3'-Phosphoramidi~es, Methods in Enzymology 154: 313-326, 1987.
wo 91/1~512 ~ ' 8, ~ ~Cr/US91/~2166 Alternasively, to obtain DNA enc~din~ the isola~ed polypeptide, ~ne needs only to conduct hybridization screening with lab~lled DNA encodinQ either the isolated polypeptide or isoiated polypeptide fra~ment lusuaily, ~rsater than about 20, and ordinarily about 50bp) in order to detect clones which contain homolo~ous sequences in the cDNA libraries derived 5 from cells or tissues ot a particular animal, followed by analyzin~ the clones by restriction enzyme analysis and nucleic acid sequencin~ to identify full-len~th clones. If full lenpth clones are not present in the library, then appropriate fragments are recovered from the various clones and li~ated at restriction sites common to the fra~men~s to assemble a full-lenoth clone. DNA encodin~ isolated poiypeptide from various iso~ypes and strains is 10 obtained by probing libraries from hosts of such species with the amino acid ssquences of the isolated polypeptide, or by synthesizin~ the ~enes in vitro.
In ~eneral, prokaryotes are used for clonin~ of DNA sequonces in constructin~ the vectors use~ul in the invention. For exarnple, E. coli K12 strain 294 IATCC No. 31446) is particularly useful. Other microbial strains which may be used include E. co/i ~ and E. coli 15 X1776 (ATCC No. 31537h These examples are illustrative rather than limitin~.
Alternatively, in vitro methods of clonin~, e.~. polymerase chain reaction~ are suitable.
The isolated polypeptides of this invention are expressed directly in recombinant cell culture as an N-terminal methionyl analo~ue, or as a fusion with a polvpeptide heterolo~ous 20 to the hybrid/portion, preferably a si~nal sequence or other polypeptide havin~ a specific cleava~e site at the N-terminus of the hybrid/portion. For example, in constructin~ a prokaryotic secretory expression vector for the isolated polypeptide, the native isolated polypeptide si~nal is employed with hosts that recoanize that si~nal. When the secretory leader is rreco~nized" by the host, the host si~nal peptidase is capable of cleavin~ a fusion 25 of the leader polypeptide fused at its C-terminus to the desired mature isolated polypeptide.
For host prokaryotes that do no~ process the native isolated polypeptide si~nal, the si~nal is substituted by a prokaryotic si~nal selected for example from the ~roup of the alkaline phosphatase, penicillinase, Ipp or heat stable enterotoxin ll leaders. For yeast secretion the native isolated polypeptide si~nal may be substituted by the veast invertase, alpha factor or 30 acid phosphatase leaders. In mammalian cell expression the native isolated polypeptide signal or native HIV env signal is satisfactorv for certain isolated polypeptides, althou~h other mammalian secretorV protein si~nals are suitable, as are viral secretorV leaders, for example the herpes simplex ~D signal.
The isolated polypeptide may be expressed in any host cell, but preferably is 35 synthesized in mammalian hosts. However, host cells from prokaryotes, fun~i, yeast, insects and the like are also are used for expression. Exemplary prokaryotes are ~he strains suitable for clonin~ as well as E. coli W3110 IF-~I prototrophic, ATTC No. 27325~, other ~ :: ~ :': : : : : : : : : ~ : `
WO91/15512 ~ ~, PCl/llS9~/0216 ~ - 1 6-enterobacteriaceae such as Serrati~ marcescans, bacilli and various pseudomonads.
Prr~ferably the host cell should secrete minimal amounts of proteolytic cnzvmes.E~pression hosts typically are transformed with DNA ancodin~ the isolat~d polypeptide which has been li~ated into an expression vector. Such vectors ordinarily carry a replication site (althou~h this is not necessary where chromosomal integration will occur). Expression vectors also includ~ marker sequences which are capable of providing phenotypic selection in transformed cells, as will be discussed further below. For example, f. coli is typically transformed usin~ pBR322, a plasmid ~erived from an E. coli species IBolivar, et a/., Gene 2:
95 119771). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifyin~ transformed cells, whether for purposes of cloninçl or expression. Expression vectors also optimally will contain sequences which are useful for the control of transcription and translation, e.~., promoters and Shine-Dal~arno sequences (for prokaryotes~ or promoters and enhancers If or mammalian cells). The promoters may be, but need not be, inducible; even powerful constitutive promoters such as the CMV promoter for mammalian hosts may produce the isoiated polypeptide without host cell toxicity. While it is conceivable that expression vectors need not contain any expression control, replicative sequences or selection genes, their absence may hamper the identification of transformants and the achievement of high level peptide expression.
Promoters suitable for use with prokaryotic hosts illustratively include the ~-lactamase and lactose promoter systems IChang et al., Narure 275: 615 t19781; and Goeddel et al., Nature 281: 544 119791), alkaline phosphatase, the tryptophan ~trp) promoter system IGoeddel, Nucleic Acids ~es. 8: 4057 l1980) and EP0 Appln. Publ. No. 36,776) and hybrid promoters such as the tac promoter IH. de Boer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 119831). However, other functional bacterial promoters are suitable. Their nucleotide sequences are ~enerally known, thereby enablin~ a skilled worker operably to ligate them to DNA encoding the isolated polypeptide ~Siebenlist et al., Cell 20: 269 119801) using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dal~arno IS.D.~ sequence operably linked to the DNA encodin~ the isolated polypeptide.
In addition to prokarYotes, eukaryotic microbes such as yeast or filamentous fun~i are satisfactory. Saccharomyces cerevislae is the most commonly used eukaryotic microor~anism, althou~h a number of other strains are commonly available, The plasmid YRp7 is a satisfactory expression vector in yeast IStinchcomb, et al., Nature 282: 39 11979);
Kingsman et al, Gene 7: 141 l1979); Tschemper e~ al., Gene 10: 157 (1980)). This plasmid already contains the trp1 ~ene which provides a selection marker for a mutant strain of yeast lackin~ the ability to ~row in tryptophan, for example ATCC no. 44076 or PEP4-1 IJones, Genetics 85: 12119771). The presence of the trp1 lesion as a characteristic of the yeast WO 91/155t2 ~ . 7 ~ cr/us9l/021s6 host cell 0enome then provides an affective environment for detectin~ transformation by ~rowth in the absence of tryptophan.
Suitable promotin~ seqlJ~nces for use with Yeast hosts include the promoters for 3-phospho~lycerate kinase (llitzeman et al., J. ~iol. Chem. 255: 2073 119801) or other glycolytic en2ymes lHess et al., J. Adv. t~nzyme Re~. 7: 149 11968); and Holland, ajochemistry 17: 4900 (1 g78)~, such as enolase, glyceraldehyde-3-phosphate dehydro~enase, hexokinase, pyruvate decarboxylase, phosphofructokinase, ~lucose-~-phosphate isomerase, 3-phospho~lycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho~lucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters havinp tha additional advanta~e of transcription controlled by ~rov~h conditions, are the promoter re~ions for alcohol dehydro~enase 2, isocytochrorne C, acid phosphatase, de~raciative enzymes assor;iated with nitrogen metabolism, metallothionein, ~Iyceraldehyde-3-phosphate dehydro~enase, and enzymes responsible for maltose and ~alactose utilization. Suitable vectors and promoters for USB in yeast expression are further described in R. Hitzeman et a/., European Patent Publication No. 73,657A.
Expression control sequences are known for eucaryotes. Virtually all eukaryotic ~enes have an AT-rich re~ion located approximately 25 to 30 bases upstream ftom the site where transcription is initiated, Another sequence found 70 to 80 bases upstream from the start of transcription of many ~enes is a CXCAAT re~ion where X may be any nucleotide. At the 3' end of most eukaryotic ~enes is an AATAAA sequence which may be the si~nal for addition of the poly A tail to the 3' end of the. codin~ sequence. All of these sequences are inserted into mammalian expression vectors.
Suitable promoters for controlling transcription from vectors in mammalian host cells are readily obtained from various sources, for example, the ~enomes of viruses such as polyoma virus, SV40, adenovirus, MMV ~steroid inducible), retroviruses le-~. the LTR of HIV~, hepatitis-B virus and most preferably cytome~alovirus, or from heterolo~ous mammalian promoters, e.~. the beta actin promoter. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fra~ment which also contains the SV40 viral ori~in of replication. Fiers et ~/., Nature, 273: 113 ~1978). The immediate early promoter of the human cytome~alovirus is conveniently obtained as a Hindlll E restriction fra~ment.
Greenaway, P.J. et a/., Gene 18: 355-360 11982), Transcription of a DNA encodin~ ~he isolated polypeptide by higher eukaryotes isincreased by insertin~ an enhancer sequence into the vector. Enhancers are cis-actin~
elements of DNA, usually about from 10-300bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent havin~ been found 5' (Laimins etal., PNAS78: 993 11981]) and 3' tLusky, M.L., etat., Mot. Cel/t~io. 3:
1108 11983~) to the transcription unit, within an intron IBanerji. J.L. et al., Cet/ 33: 729 WO91/15~12 ", ~ PCr/US~1/02166 l1983)1 as well as within the coding sequence itself (Osborne, T.F., et al., Mot. Cell ~io. 4:
1293 119841). Many enhancer sequences are now known from mammalian ~enes l~lobin, elastase, albumin, a-fetoprot~in and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication ori~in IbP 100-270), the cytome~alovirus early promoter enhancer, the polyoma enhancer on the late side of the replication ori~in, and adanovirus enhancers. ~
Expression vectors used in eukaryotic host cells lyeast, fun0i, insect, plant, animal, - -human or nucleated cells from other multicellular or~anisms) will also contain sequences necessary for the termination of transcription which may affect mP~NA expression. These re~ions are transcribed as poiyadenylated seaments in the untransia~ed portior of the mRNA
encodin~ the hybrid immuno~lobulin. The 3 untranslated re~ions also include transcription termination sites.
Expression vectors may contain a selection ~ene, also termed a selectable marker.
xamples of suitable selectable markers for marnmalian cells are dihydrofolate reductase ~DHFR~, thymidine kinase lTK) or neomycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell is able to survive K placed under selective pressure. There are two widely used distinct cate~ories of selective re~imes. The first Gate~ory ;s based on a cell s metabolism and the use of a mutant cell line which lacks the ability to ~row independent of a supplemented media. Two examples ars CHO DHFR- cells and mouse LTK cells. These cells lack the ability to ~row without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain ~enes necessary for a complete nucleotide synthesis pathway, thev cannot survive unless the missin~ nucleotides are provided in a supplemented media. An alternative to supplementin~
the media is to introduce an intact DHFR or TK ~ene into cells lackin~ the respective ~enes, thus altering their ~rowth requirements. Individual cells which were not transformed with the DHFR or TK ~ene will not be capable of survival in non-supplemented media. In preferred embodiments, herein, CHO cells which are DHFR- are used for recombinan~ expression of the isolated polypeptide.
The second cate~ory of selective re~imes i5 dominant selection which refers to aselaction scheme used in any cell typc and does not require the use af a mutant cell line These schemes typically use a dru~ to arrest ~rowth of a host cell. Those cells which are successfully transformed with a heterolo~ous ~ene express a protein conferring drug resistance and thus survive the selection re~imen. Examples of such dominant selection use the dru~s neomycin ISouthern et al., J. Molec. Appl. Genet. 1: 327 l1982~), mycophenolic acid IMulli~an e~al., Science 209: 1422 l1980)) or hy~romycin lSu~den et al., Mol. Cell. Biol.
5: 410-413 l1985)). The three examples ~iven above employ bacterial ~enes under eukaryotic control to conveY resistance to the appropriate dru~ G418 or neomycin l~eneticin~, x~pt lmycophenolic acid~ or hy~romycin, respectively.
WO 91/15512 r. r~ ~ ~ 4 ~ PC~ S9ltO2166 ~19-~Amplification~ refers tO the increase or replication of an isolated region within a cell s chromosomal DNA. Amplification is achieved usin~ a seiection a~ent, e.o. methotrexate IMTX) which inactivates DHFR. Amplification or the makin~ of successive copies of the DHFR ~ene results in ~reater amounts of DHFR bein~ produced in thrl face of greater amounts 5 of MTX. Amplification pressure is applied notwithstandin~ the presence of endooenous DHFR, by addin~ ever oreater amounts of MTX to the media. Amplification of a desired ~ene can be achieved by cotransfectinS~ a mammalian host cell with a plasmid havin~ a DNA
encodin~ a desired protein and the DHFR or amplification ~en~ permittin~ cointeoration. One ensures that the cell requires more DHFR, which requirement is met by replication of teh t 0 selection ~ene, by selecting only for cells that can ~row in teh presence of ever-~lreater MTX
concentration. So lon~ as the ~ene encoding a desired heterolo~ous protein has cointe~rated with the selection ~ene replication of this ~ene ~ives rise to replication of the ~ene encodin~
the desired protein. The result is that increased copies of the ~ene, i.e. an amplified ~ene, encodin~ the desired heterolo~ous protein express more of the desired protein.
Suitable eukaryotic host cells for expressin~ the isolated polypeptide include monkey kidney CV1 line transformed bv SV40 ~COS-7, ATCC CRL 16511: human embryonic kidney line (293 or 293 cells subcloned for ~rowth in suspension culture, Graham, F,L. e~ al., J. Gen V*ol. 36: 59 (1977j); baby hamster kidney cells ~BHK, ATCC CCL 10~; chinese hamster ovary-cells-DHFR ICHO, Urlaub and Chasin, PNAS ~USAI 77: 4216, 119801~; mouse sertoli cells (TM4, Mather, J.P., Biol. Reprod. 23: 243-251 [1980~); monkey kidney cells (CV1 ATCC
CCL 70i; african green monkey kidney cells (VERO-76, ATCC CRL-1587~; human cervical carcinoma cells (HELA, ATCCCCL 2i; canine kidney cells (MDCK, ATCCCCL 34~; buffalo rat liver cells (BRL 3A, ATCC CRL 1442~; human lùng cells (W138, ATCC CCL 75~; human liver cells (Hep G2, HB 80651; mouse mammary tumor ~MMT 060562, ATCC CCL51); and, TRI
cells ~Mather, J.P. et al., Annals N.Y. Acad. Sci. 383: 44-68 [19821~.
Construction of suitable vectors containin~ the desired coding and control sequences employ standard li~ation techniques. Isolated plasmids or DNA fra3ments are cleaved, tailored, and reli~ated in the form desired to form the plasmids required.
For analysis to confirm correct seqùences in plasmids constructed, the ligation mixtures are used to transform E. co/i K12 strain 294 (ATCC 31446) and successful transformants selccted by ampicillin or tetracvcline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction and/or sequenced by the method of Messin~ et al., Nucleic Acids Res. 9: 309 ~1981) or by the method of Maxam et al., Methods in Enzymolo~y 65: 499 ~1980).
Host cells are transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducin~ promoters, selecting transformants or amplifying the genes encoding the desired sequences. The culture : .' : . . ... . : :. . : ,:,, .:. . : - :: .:
WV ~ 512 ~. r~, ~ PCT/US91/02166 `
2 û-conditions, such as ~emperature, pH and the like, are those previously used with the host cell salected for expression, and will be apparent to the ordinarily skilled artisan.The host c~lls referred ~o in this disclosure encompass cells in in vit,o culture as well as cells which are within a host animal.
~Transformation" means introducin~ DNA into an oroanism so that the DNA is r0plicable, either as an extrachromosomal 01ament or by chromosomal inte~ration. Unless indicated otherwise, the method used herein for transformation of the host cells is the method of Graham, F. and van der Eb, A., Yirolo~y 52: 456-457 It973). However, other methods for introducin~ DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, the preferred method of transfection is calcium treatment using calcium chloride as dèscribed by Cohen, F.N. et al., Proc. Natl. Ac~d. Sci. ~USAI, 69: 2110 (1972).
~Transfection~ refers to the introduction of DNA into a host cell whether or not any codin~ sequences are ultimately expressed. Numerous methods of transfection are known to the ordinarily skilled anisan, for example, CaPO4 and electroporation. Transformation of the host cell is the indicia of successful transfection. ' The nov01 polypeptide of this invention is recovered and purified from recombinant cell cultures by known methods, includin~ ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchan~e chromato~raphy, phosphocellulose chromato~raphy, immunoaffinity chromato~raphy, hydroxyapatite chromato~raphv and lectin chromato~raphv.
See, e.~., the purification methods described in EP 187,041. Moreover, reverse-phase HPLC
and chromato~raphy usin~ ands for the isolated polypeptide are useful for purification. It is presently preferred to utilize ~el permeation chromato~raphy and anion exchan~e ;
25 chromato~raphy, and more preferred to use cation exchan~e and hydrophobic interaction chromato~raphy (HIC) accordin~ to standard protocols.
Optionally, the isolated polypeptide is recovered and purified by passa~e over a column of isolated polypeptide-antibody covalently coupled to aldehyde silica by a standard procedure IRoy et ~I., Journ~l of Chromaro~raphy 303:225-228 (1984~), washin~ of the column with a saline solution, and analyzin~ the eluant by standard methods such as quantitative amino acid analysis. Procedures utilizin~ monoclonal antibodies coupled to ~lvcerol-coated controlled pore qlass are desirable for the practice of this invention. Optionally, low concentrations lapproximatalY 1-5 mM) of calcium ion may be present durin~ purification.
The isolated polypeptide may preferably be purified in the presence of a protease inhibitor ~ `
35 such as PMSF.
The isolated polypeptide is placed into pharmaceutically acceptable, sterile, isotonic `
formulations to~ether with required cofactors, and optionally are administered by standard means well known in the field. The formulation is preferably liquid, and is ordinarily a ' :
wo 91/1~512 ~ 7 ;~ ~ ~PCr/US91/02166 physiolo~ic salt solution containin~ non-phosphate buffer at pH 6.8-7.6, or may be Iyophilized powder.
The isolated polypept;de compositions to be used in therapy will be ~ormula~ed and dosa~es establishsd in a fashion consistent with ~ood medical practice takin~ into account the disorder to be traated, the condition of the individual patient, the site of delivery of the isolated polypeptide, the method of administration and ol~her factors known to practitioners.
The isolated polypeptide is prepared for administration by mixin~ the isolated polypeptide at the d~sired degree of purity with adjuvants or physiolo~ically acceptable carriers i.e. carriers which are nontoxic to recipients at the dosa~es and concentrations employed. Adjuvants and earriers are substances that in themselves shar~ no immune epitopes with the tar~et anti~en, but which stimulate the imrnune response to the tar~et anti~en. Ordinarily, this will entail combinin~ the isolated polypeptide with buffers, low molecular wei~h~ ~less that about 10 residues) polypeptides, proteins, amino acids, carbohydrates includin~ ~lucose or dextrans, chelatin~ agents such as EDTA, and other excipients. Freunds adjuvant (a mineral oil emulsion) oommonly has been used for this purpose, as have a variety of toxic microbial substances such as mycobacterial extracts and cytokines such as tumor necrosis factor and interferon ~amma in U.S. patent 4,963,354.
Althou~h anti~en is desirably administered with an adjuvant, in situations where the initial inoculation is delivered with an adjuvant, boosters with anti~en may not require adjuvant.
Carriers often act as adjuvants, but are generally distin~uished from adiuvants in that carriers comprise water insoluble macromolecular particulate structures which a~re~ate the anti~en, Typical carriers include aluminum hydroxide, latex particles, bentonite and liposomes.
It is envisioned that injections lintramuscular or subcutaneous) will be the primary route for therapeutic administration of the vaccines of this invention, intravenous delivery, or delivery throu~h catheter or other sur~ical tubin~ is also used. Alternative routes include tablets and the like, commercially availa~le nebulizers for liquid formulations, and inhalation of Iyophilized or aerosolized receptors. Liquid formulations may be utilized after reconstitution from powder formulations.
The novel polypeptide may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues includin~ blood. Suitable examples of sustained release carriers inc)ude semipermeable polymer matrices in the form of shaped articles, e.u. suppositories, or microcapsules.
Implantable or microcapsular sustained release matrices include polylactides tU.S. Patent 3,773,919, EP 58,481) copolymers of L-~lutamic acid and ~amma ethyl-L-~lutamate (U.
Sidman etat., ~iopo/ymers~22(1): 547-556, ll985)), poly 12-hydroxyethyl-methacrylate) or ~thylene vinyl acetate IR. Lan~er e~ at., J. ~iomed. Mater. Pes. 15: 1 67-277 (1981) and R.
Lan~er, Chem. Tech. 12: 98-105 l1982)). Liposomes containina the isolated polypeptide are prepared by well-known methods: DE 3,218,121 A; Epstein et al., Proc. ~tatl. Acad. Sci. USA, WO glJ15sl2 ,~ PCr/US91/n2166 82:3688-3692 11985); Hwan~ eral., Proc. Natl. Acad. Sci. US~, 77:4030-4034 i1980): EP
52322A; ~P 36676A; EP 88046A; EP 143949A; EP 14Z541A; Japanese patent apPlication 83-11808; U.S. Patents 4,485,045 and 4,544,545; and UP 102,342A. Ordinarily the liposomes are of the small labout 200-800 &~n~stromsl unilamelar tvpe in which the iipid content is ~reater than about 30 mol. % cholesterol, the seiected proportion bein~ adjusted for the optimal rat~ of the polypeptide leaka~e.
The dose of the isolated polypeptide administered will be dependen~ upon the properties of the isolated polypeptide employed, e.~. its bindino activity and in YiVo plasma half-life, the concentra~ion of the isolated polypeptide in the formulation, the administration route, ~he site and rate of dosa~e, the clinical tolerance of the patient involved, the patholo~ical condition afflictin~ the pa~ient and the like, as is well within the skill of the physician. Generally, doses of from about 0.5 x 10- to 5 x 10' molar of isolated polypeptide per patient per administration are preferred. Different dosa~es are utilized durin~ a series of sequential inoculations: the practitioner may administer an initial inoculation and then boost with relatively smaller doses of isolated polypeptide vaccine.
The isolated polypeptide vaccines of this invention may be administered in a variety of ways and to different classes of recipients. The vaccines are used to vaccinate individuals who may or may not be at risk of exposure to HIV, and additiùnally, the vaccines are desirably administered to seropositive individuals and to individuals who have been previously exposed to HIV (see e.~. Salk, Nature 327:473-476 ~1987); and Salk et al., Science 195:834-847 ~1977)).
The isolated polypeptide may be administered in combination with other anti~ens in a sin~le inoculation "cocktail". The isolated polypeptide vaccines may also be administerad as one of a series of inoculations administered over time. Such a series may include inoculation with the same or different preparations of HIV anti~ens or other vaccines.
The adequacy of the vaccinition parameters chosen, e.~. dose, schedule, adjuvantchoice and the like, is determined by takin~ aliquots of serum from the patient and assayin~
antibody titers durin~ the course of the immunization pro~ram. Alternatively, the presence of T cells may by monitored by conventional methods as described in Example 1 below. In addition, the clinical condition of the patient will be monitored for the desired effect, e.~. anti-infectiva effect. If inadequatc vaccination is achievsd then tha patient can be boosted with funher isolated polypeptlde vacclnations and thc vaccination parameters can be modified in a fashion expected to potentiate the immune response, e.~. increase the amount of anti~en and/or adjuvant, complex the anti~en with a carrier or conju~ate it to an immuno~enic protein, or vary the route of administration.
For use of the isolated polypeptide as a vaccine, it is currently preferred that at least three separate inoculations with isolated polypeptide be administered, with a second inoculation bein~ administered more than two, preferably three to ei~ht, and more preferably ~' W() 91/15~1' PCr/USgl/0216S
8 ~V ~ -S' approximately four weeks fo~lowinD th~ first inoculation. It is preferred that a third inoculation be adrninistered several months later than the second "boost~ inoculation, preferably at least more than five months following the first inoculation, more preferably six months to two years followin~ the first inoculation, and even more preferably ei~ht months 5 to one year followin~ the first inoculation. Pariodic inoculations beyond the third are also desirable to enhance the patient's 'immune memory". See Anderson et ~1., J. Infectio~s Diseases 160(6):960-969 IDec. 1989). Gensrally, infrequent immunizations with isolated polypeptide spaced at relatively lon~ intervals is more preferred than fre~uent immunizations in eliciting maximum antibody responses, and in elicitin~ a protective effect.
The polypeptides of this invention may optionallv be administered alono with other pharmacolo~ic a~ents used to treat AIDS or ARC or other HlV-related dissases and infections, such as AZT, CD4, antibiotics, immunomodulators such as interferon, anti-inflammatory agents, and anti-tumor a~ents.
Antibodies -This invention is also directed to monoclonal antibodies. In accordance with this invention, monoclonal antibodies specifically bindin~ an epitope of an isolated polypeptide or anti~enically active fra~ments thereof are isolated from continuous hybrid cell lines formed by the fusion of anti~en-primed immune Iymphocytes with myeloma cells. The antibodies of the subject invention are obtained throu~h routine screenin~. An assay is usad for 20 screenin~ monoclonal antibodies for their cytotoxic potential as ricin A chain containin~
immunotoxins. The assay involves treatin~ cells with dilutions of the test antibody followed by a Fab fra~ment of a secondary antibody coupled to ricin A chain ~'indirect assay'). The cytotoxicity of tha indirect assay is compared to that of the direct assay where the monoclonal antibody is coupled to ricin A chain. The indirect assay accurately predicts the 25 potency of a ~iven monoclonal antibody as an immunotoxin and is thus useful in screenin~
monoclonal antibodies for U52 as immunotoxins - see also Vitetta et 91., Science238:1098-1104 ~19871, and Weltman et a/., Cancer ~es. 47:5552 ~1987).
Monoclonal antibodies are highly specific, bein~ directed a~ainst a sin~le anti~enic site.
Furthermore, in contrast to conventional antibody IPolYclonal) preparations which typically 30 include different antibodies directed a~ainst different determinants ~epitopes), each monoclonal antibody is directed a~ainst a sin~le determinant on the anti~en. Monoclonal antibodies are useful to improve the selectivity and specificity of dia~nostic and analytical assay methods using anti~en- antibody bindin~. A second advanta~e of monoclonal antibodies is that they are synthesized by the hybridoma culture, uncontaminated by other 35 immuno~lobulins. Monoclonal antibodies may be prepared from supernatants of cultured hybridoma cells or from ascites induced by intra-peritoneal inoculation of hybridoma cells into mice.
WO 91/15~12 ~ ~ PCI'/US91/02166 ~4 The hybridoma techniqu~ described ori~inally by Kohler and M;lstein, Eur. J. Immuno/., 6:511 t1976) has been widelv applied to produce hybrid cell lines that secrete hi~h tevels of monoclonal antibodies a~ainst many specific anti~ens.
In panicular embodiments of this invention, an antibody is obtained by immunizin~ mice such as Balb/c or, preferably C57 BL/6, aoainst an isolated polypeptide and screenin~ for a clonal antibody that, when preincubated with the isolated polypeptide, prevents its bindin~
to isolated polypeptide. Monoclonal antibodies may desirably have differences in affinity, immuno~lobulin class, species of ori~in, or epitope; they may bs antibodias which are expressed in recombinant cell culture or thas are predetermined amino acid sequence variants of known antibodies, includin~ chimeras of antibodies havinq a variable re~ion directed a~ainst an isolated polypeptide, and a human constant re~ion.
The route and schedule of immunization of the host animal or cultured-antibody-producin~ cells therefrom are ~enerally in keepin~ with established and conventional techniques for antibody stimulation and production. Applicants typically have employed mice as the test model althouph it is contemplated that any mammalian subject includin~ human subjects or antibody producin~ cells therefrom can be manipulated accordin~ to the processes of this invention to serve as the basis for production of mammal;an, includin~ human, hybrid cell lines.
After immunization, immune Iymphoid cells are fused with myeloma cells to qenerate a hybrid cell line which can be cultivated and subcultivated indefinitely, to produce lar~e quantities of monoclonal antibodies. For purposes of this invention, the immune Iymphoid cells selected for fusion are Iymphocytes and their normal differentiated pro~eny, taken either from Iymph node tissue or spleen tissue from immunized animals. Applicants prefer to ~.
employ immune spleen cells, since they offer a more concentrated and convenient source of :
antibody producin~ cells with respect to the mouse system. The myeloma cells provide the .
basis for continuous propa~ation of the fused hybrid. Myeloma cells are tumor cells derived from plasma cells~ -It is possible to fuse cells of one species with another. However, it is preferred that the source of immunized antibody producin~ cells and myeloma be from the same species.
The hybrid cell lines can be maintained in culture In vitro in cell culture media. The cell lines of this invention can be selected and/or maintained in a composition comprising the continuous cell line in hypoxanthine-aminopterin thvmidine IHAT) medium. In fact, once the hybridoma cell l;ne is established, it can be maintained on a variety of nutritionally adequate .
media. Moreover, the hybrid cell lines can be stored and preserved in any number of conventional ways, includin~ freezin~ and stora~e under liquid nitro~en. Frozen cell lines can be revived and cultured indefinitely with resumed synthesis and secretion of monoclonal antibody. The secreted antibody is recovered from tissue culture supernatant by conventional methods such as precipitation, lon exchan~e chromato~raphy, affinity chromato~raphy, or WO 91/15512 PCr/US91/02166 -25- . 0~45 the like. The antibodies described herein are also recovered from hybridoma cell cultures by conventional methods for purificasion of I~G or I~M as the case maV be that heretofore have been used to purify these immuno~lobulins from pooled plasma, e.~. ethanol or polyethylene ~Iycol precipitation procedures. The purified antibodies ar0 sterile filtered, and optionally are 5 conju~ated to a detectable marker such as an enzyme or spin label for us~ in dia~nostic assays of isolated polypeptide in test samples.
While the invention covers usin~ mouse monoclonal antibodies, the invention is not so limited; in fact, human antibodies may be used and may prove to be preferable. Such antibodies can be obtained by using human hybridomas ~Cote er ~I., Monocton~l Ant~bodies 10 ~nd C~ncer Therapy, Alan R. Liss, p. 77 /1g85~1. In fact, accordinu to the invention, techniques developed for the production of chimeric antibodies ~Mùrrison et ~/., Proc. N~tl.
Acad. Sci., 81:6851 ~1984~; Neuberger et al., Nature 312:604 11984); Takeda et a/., Narure 314:452 ~1985)) by splicin~ the ~enes from a mouse antibod~ molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate 15 biolo~ical activity ~such as ability to activate human complernent and mediate ADCC) can be used: such antibodies are within the scope of ~his invention.
As another alternative to the cell fusion technique, EBV-immortalized 8 cells are used to produce the monoclonal antibodies of the subject Invention. Other methods for producing monoclonal antibodies such as recombinant DNA, are also contemplated.
20 Immunotoxins This invention is also directed to immunochemical derivatives of the antibodies of this invention such as immunotoxins ~conjugates of the antibody and a cytotoxic moiety). The antibodies are also used to ;nduce Iysis throu~h the naturat complement process, and to interact with antibody dependent cytotoxic cells normally present.
Purified, sterile filtered antibodies are optionally conjugated to a cytotoxin such as ricin for use in AIDS therapy. EPO Publication 0 279 688 published 24 Au~ust 198~ illustrates methods for making and usin~ immunotoxins for the treatment of HIV infection.
Immunotoxins of this invention, capable of specifically bindina regions of HIV env, are used to kill cells that are already infected and are actively ptoducin~ new virus- KillinD iS
30 accomplished by the bindin~ of the immunotoxin to viral coat protein which is expressed on infected cells. The immunotoxin is then internalized and kills the cell. Infected cells that have incorporated viral ~enome into their DNA but are not synthesizing viral protein ~i.e., cells in which the virus is laten~) may not be susceptible to killing by immunotoxin until they begin to synthesize virus. The antibodies of this invention which span the clip site and/or the other 35 antibodies described herein may be used alone or in any combination with for delivering toxins to infected cells. In addition, a toxin-antibodv conjugate can bind to circulating viruses or viral coat protein which will then effect killing of cells that internalize virus or coat protein.
.. , . . ~ ,, . . - . :
WO91/15~12 , ~ PCI'/US91/02166 The subject invention provides a hi~hly selective method of d~stroyin~ HIV infected cells, utilizing the antibodies described herein.
While not ~ishina to be constrained to any particular theory of operation of theinvention, it is believed that ~he expression of the tar~et anti~en on the infected cell surface is transient. The antibodies must be capable of reachin~ the site on the cell surface where the anti~en resides and interactin~ with it. After the antibody complexes with the anti~en, endocytosis takes place carryin~ the toxin into the cell.
The immunotoxins of this invention are particularly helpful in killin~
monocytes/macrophages infected with the HIV virus. In contrast to the transient production of virus from T cells, macrophages produce hi~h !evels of virus for lon~ periods of time.
Current therapy is ineffective in inhibitin~ the production of new viruses in these cells.
Not all monoclonal antibodies specific for an isolated polypeptide make hi~hly cytotoxic immunotoxins, however assays are routinely and commonl~/ used in the field to predict the ability of an antibody to function as part of a immunotoxin. Preferablv the antibodies used cross react with several ~or all) s~rains of HIV.
The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial, funaal, plant or animal ori~in, or an enzymatically active fragment of such a toxin. Enzymatically active toxins and fra~ments thereof used are diphtheria A
chain, nonbindina active fraaments of diphtheria toxin, exotoxin A chain ~from Pseudomonas aeru~mos~), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcln"41eurites fordïï
proteins, dianthin proteins, Phytolaca ameticana proteins ~PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, aelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. In anotherembcdiment, the antibodies are conju~ated to small molecule anticancer dru~s such as cis~
platin or 5FU. Conju~ates of the monoclonal antibody and such cytotoxic moieties are made using a variety of bifunctional protein coupling agents. Examples of such rea~ents are SPDP, IT, bifunctional derivatives of imidoesters such as dimethyl adipimidate HCI, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis ~p-azidobenzovll hexanediamine, bis-diazonium derivatives such as bis- ~p-diazoniumbenzoyl)- ethylenediamine, diisocyanates such as tolylene 2,6-diisocyanate and bis-active fluorine compounds such as 1,5-difluoro- 2,4-dinitrobenzene. The Iysin~ portion of a toxin rnay be joined to the Fab fraament of the antibodies.
Immunotoxins can be made in a variety of ways, as discussed herein. Common~y known crosslinkina reagents can be used to yield stable conju~ates.
Advantaaeously, monoclonai antibodies specifically bindin~ the domain of the protein which is exposed on the infected cell surface, are coniuaated to ricin A chain. Most advantageously the ricin A chain is deglycosylated and produced through recombinant means.
WO 91/1~512 -27- ,~ ~ 7 ~ ~ ~C~/U~91/02166 An advanta~eous method of makin~ the ticin immunotoxin is described in Vitetta et ~1., Science 238:1098 11987).
When used to kill infected human cells in vitro for dia~nostic purposes, the conju~ates will typically be added to the cell culture medium a~ a concentration of at least about 10 nM.
5 The formulation and mode of administration for in vitro use are not critical. Aqueous formulations that are cornpatible with the culture or peRusion rnedium will norrnally be used.
Cytotoxicity may be read by conventional techniques.
Cytotoxic radiopharmaceuticals for treatin~ infected cells rnay be made by conju~atin~
radioactive isotopes le.g. 1, Y, Pr~ to the antibodies. Advanta~eously alpha particle-emittin~
1 û iso~opes are used. The term 'cytotoxic moir~ty" as used herein is intended to include such isotopes.
In a preferred embodiment, ricin A chain is de~lycosylated or produced without oli~osaccharides, to decrease its clearance by irrelevant clearance mechanisms (e.~., the liver). In another embodiment, whole ricin (A chain plus B chain) is conjugated to antibody 15 if the ~alactose bindin~ property of B-chain can be blocked ~blocked ricin~l.In a further embodiment toxin-conju~ates are made with Fab or Flab'12 fra~ments.Because of their relatively small size these fralaments can better penetrate tissue to reach infacted cells.
In another embodiment, fuso~enic liposomes are filled with a cytotoxic dru~ and the 20 liposomes are coated with antibodies specifically bindinp HIV env.
Antibodv Decendent Cellular Cvtotoxicitv The present invention also involves a method based on the use of antibodies which are lal directed a~ainst an isolated polypeptide, and (b) belon~ to a subclass or isotype that is capable of mediatin~ the Iysis of HIV virus infected cells to which the antibody molecule 25 binds. More specifically, these antibodies should belon~ to a subclass or isotype that, upon complexin~ with cell surface proteins, activates serum complement and/or mediates antibody dependent cellular cytotoxicity ~ADCCI by activating effector cells such as natural killer cells or macropha~es.
The present invention is also directed to the use of these antibodies, in their na~ive 30 form, for AIDS therapy. For example, IgG2a and IgG3 mouse antibodies which bind HlV associated cell suRace anti~ens can be used in viuo for AIDS therapy. In fact, since HIV
env is present on infected monocytes and T-lymphocytes, the antibodies disclosed herein and their therapeutic use have ~eneral applicability.
Biolo~ical activity of antibodies is known to be determined, to a lar~e extent, by the 35 Fc re~ion of the antibody molecule ~Uananue and Benacerraf, Textbook of lmmunoloDy, 2nd Edition, Williams & Wilkins, p. 21 8 119841). This includes their ability to activate complement and to mediate antibody-dependent cellular CytotoxiCitY ~ADCCI as effected by leukocytes.
Antibodies of different classes and subclasses differ in this respect, and, accordin~ to the WO 91/15~12 c ~ ') PCI/US91/02166 present invention, antibodies of those classes havin~ the desired biolo~ical activity are selected. For example, mouse immunoQlobulins of the I~G3 and I~G2a class are caPable of activatin~ serum complement upon bindin~ to the tar~et cells which express the co~nate anti~en.
In ~en0rall antibodies of the I~G2a and I~G3 subclass and occasionally I~G1 can mediate ADC~, and antibodies of the I~G3, ioG~a, and I~M subclasses bind and activate serum complement. Complement activation ~enerally requires the bindin~ of at least two I~G
molecules in close proximity on the tar~et cell. However, the bindin~ of only one 10M
molecule activates serum complement.
O The ability ~f any particular antibody to mediate Iysis of the tar~et cell by complement activation andlor ADCC can be assayed. Ths cells of interest are ~rown and labeled in vitro;
the antibody is added to the cell culture in combination with either serum complement or immune cells which may be activated by the an~i~en antibody complexes. Cytolysis of the tar~et cells is detected by the release of label from the Iysed cells. In fact, antibodies can be screened usin~ the patient's own serum as a source of complement and/or immune cells. The antibody that is capable of activatin~ complement or mediatin~ ADCC in the in vitfo test can then be used therapeutically in that panicular patient.
Antibodies of virtually any ori~in can be used for this purpose provided they bind an isolated polypeptide epitope ~ can activate complement or mediate ADCC. Monoclonal antibodies offer the advanta~e of a continuous, ample supply.
TheraDeutic and Other Uses of the Antibodies When used In vivo for therapy, the antibodies of the subject invention are administered to the patient in therapeutically effec~ive amounts li.e. amounts that restore T cell counts).
They will normally be administered parenterally. The dose and dosa~e re~imen will depend upon the de~ree of the infection, the characteristics of the particular irnmunotoxin lwhen used~, e.g., its therapeutic index, the patient, and the patient's history. Advama~eously the immunotoxin is administered continuously over a period of 1-2 weeks, intravenously to treat cells in the vasculature and subcutaneously and intraperitoneally to treat re~ional Iymph nodes. Optionally, the administration is made durin0 the course of adjunct therapy such as combined cycles of turnor necrosis factor and interferon or other immunomodulatory a0ent.
For parenteral administration the antibodies will be formulated in a unit dosageinjectable form ~solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic, and non-therapeutic.
Examples of such vehicles are water, saline, Rin~er's solution, dextrose solution, and 5%
human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicitY and chemical stabilitY, e.~., buffers and WO 91/15512 PCr/US91/02166 h V 7 ~
preservatives. The antibodies will tvpicalh~ be formula~ed in such vehicles at concentrations of about 1 m~/ml to 10 m~/ml.
Use of I~M antibodies is not currently preferred, since the anti~en is hi~hly specific for the target cells and rarely occurs on normal cells. I~G molecules by bein~ smaller may be more able than I~M molecules to localize to infacted cells.
There is evidence that cornplement activation in vivo leads to a variety of biolo~ical effects, includin~ the induction of an inflammatory response and the activation of macropha~es lUananue and Benecerraf, Texrbook of Immunoloay, 2nd Edition, Williams &
Wilkins, p. 218 ll 984~h The increased vasodilation accompanyin~ inflammation may increase the abilitv of various anti-AlDS a~ents to localize in infected cells. Therefore, anti~en-antibody cornbinations of the ~ype specified by this invention can be used therapeutically in many ways. Addilionally, purified anti~ens IHakomori, Ann. Rev. Immunol 2:103 11~84)) or anti-idiotypic antibodies INeporn et a/., Proc. Natl. Acad. Scl: 81:2864 (1985); Koprowski et al., Proc; Natl. Acad. Sci. 81:216 11984)) relatin~ ~o such anti~ens could be used to induce an active immune response in human patients. Such a tesponse includes the formation of antibodies capable of activatin~ human complement and mediatin~
ADCC and by such mechanisms cause infacted cell destruction.
The antibodies of the subject invention ate also useful in the dia~nosis of HIV in test samples. They are employed as one axis of a sandwich assay for an isolated polypeptide of HIV env, toqether with a polyclonal or monoclonal antibody directed at another stericallv-free epitope of HIV env. For use in some embodiments of sandwich assays the anti-isolated polypeptide antibody is bound to an insolubilizin~ support or is labelled with a detectable moiety followin~ conventional procedures used with other monoclonal antibodies. In another embodiment a labelled antibody, e.p. Iabelled Goat anti-murine I~G, capable of bindin~ the anti-isolated polypeptide antibody is employed to detect the isolated pol~peptide or ~IIV env bindin~ usin~ procedures previously known pef se.
The antibody compositions used in therapy are formulated and dosa~es established in a fashion consistent with ~ood rnedical practice takin~ into account the disorder to be treated, the condition of the individual patient, the site of deliverv of the composition, the method of administration and other factors known to practitioners. The antibody compositions are prepared for administration accordin~ to the description of preparation of polvPeptides for administration, Infra.
In order ~o facilitate understandin~ of the followin~ examples certain frequently occurrin~ methods and/or terms will be described.
~ Plasmids" are desi~nated by a lower case p preceded and/or followed bV capital letters and/or numbers. The startin~ plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord ,.
: : . . . : . : -WO 91/1~12 ~ .Q, ~ PCTIUS91/02166 with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
In particular, it is preferred that these plasmids have some or all of the follov~in~
characteristics~ possess a minimal number of host-organism sequences; (2) be stable in the desired host; (31 be capable of being present in a high copy numbet in the desired host;
(4) possess a re~ulatabls promoter; and (5) have at least one DNA sequence codin~ for a selectable trait present on a portion of the plasmid separate from that where the novel DNA
sequence will be inserted. Alteration of plasmids to meet the above criteria are easily performed by those of ordinary skill in the art in light of the available literature and the teachin~s herein. It is to be understood that additional clonin0 vectors maV now exist or will be discovered which have the above-identified properties and are therefore suitable for use in the present invention and these vectors are also contemplated as bein~ within the scope of this invention.
~Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, tvpically 1 ~ of plasmid or DNA fra~ment is used with about 2 units of enzyme in about 20 ~JI of buffer solution. For the purpose of isolatin~ DNA fra~ments for plasmid construction, typically 5 to 50 119 of DNA are di~ested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular r~striction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37C are ordinarily used, but may vary in accordance with the supplier's instructions. After di~estion the reaction is electrophoresed directly on a polyacrylamide ~el to isolate the desired fra~ment.
Size separation of the cleaved fra~ments is performed using 8 percent polyacrylamide ~el described by Goeddel, D. et a/., Nucleic Acids Res. 8: 4057 11980).
~PCR" lpolymerase chain reaction~ refers to a technique whereby a piece of DNA is amplified. Oli~onucleotide primers which correspond to the 3' and 5' ends Isense or antisense strand-check) of the se~ment of the DNA to be amplified are hybridized under appropriate conditions and the enzyme Taq polymerase, or eqùivalent enzyme, is used to synthesize copies of the DNA located between the primers.
"Dephosphorylation~ refers to the removal of the terminal 5' phosphates by treatment with bacterial alkaline phosphatase ~BAP). This procedure prevents the two restriction cleaved ends of a DNA fra~ment from "circularizin~ or forming a closed loop that would impede insertion of another DNA fra~ment at the restriction site. Procedures and reagents for dephosphorylation are conventional. Maniatis, T. et al., Molecular Clonino pp. 133-134 11982). Reactions usin~ BAP are carried out in 50mM Tris at 68C to suppress the activity ":: . ' . ' ', ~ , ; : ':' : , , :, :" ' : : , WO 91/15512 PCI`/U~91/ûZ166 f~ V J ~ 5 of any exonucleases which are present in the anzyme pteparations. Reactions are run for 1 hour. Followin~ the reaction the DNA fra~ment is ~el purified.
~ OIi~onucleotides~ refers to either a sin~le stranded polydeoxynucleotide or two complementarv polydeoxynucleotide strands which may be chemically synthesized. Such 5 synthetic oli~onucleotides have no 5' phosphate and thus will not li~ate to another oli~onucleotide without addin~ a phosphate with an ATP in the presence of a kinase. A
synthetic oli0onucleotide will li~ate to a fragrnent that has not been dephosphorylated.
aLi~ation~ refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T. et a/.., Id., p. 1461. Unless otherw;se provided, 10 li~ation is accomplished using known buffers and conditions with 10 units of T4 DNA li~ase ~"ligase~) per 0.5 ,u~ of approximately equimolar arnounts af the DNA fra~ments to be li~ated.
~ Filling~ or ~blunting~ refers to the procedures bV which the single stranded end in the cohesive terminus of a restriction enzyme-cleav~d nucleic acid is converted to a double strand. This eliminates the cohesive terminus and forms a blunt end. This process is a 15 vPrsatile tool for convertin~ a restriction cut end that may be cohesive with the ends created bV only one or a few other restriction enzymes into a terminus compatible with any blunt-cutting restriction endonuciease or other filled cohesive terminus. Typically, bluntin~ is accornplished by incubating 2-15 JJ~ of the tar~et DNA in 1 OmM M~CI~, 1 mM dithiothreitol, 50mM NaCI, 10mM Tris IpH 7.5) buffer at about 37C in the presence of 8 units of the 20 Klenow fra~ment of DNA polymerase I and 250 ~M of each of the four deoxynucleoside triphosphates. The incubation generally is terminated after 30 min. phenol and chloroform extraction and ethanol precipitation.
It is understood that the application of the teachings of the present invention to a specific problem or situation will be within the capabilities of one havin~ ordinary skill in the 25 art in light of the teachings contained herein. Examples of the products of the present invention and representative processes for their isolation, use, and manufacture appear below, but should not be construed to limit the invention.
EXAMPLE
We have been able to produce large amounts of two different r~pl20 fusion proteins 30 in a mammalian cell system (Laskv et a/., 1986~. This has allowed us to elucidate all nine of the disulfide bonds, the positions of the ~Iycosylation sites that are utilized and the type of oli~osaccharide moiety present at each site in r~p120 from the 1119 isolate of HIV-1 produced in CHO cells.
This sxample describes the structural characterization of the recombinant envelope 35 glycoprotein Ir~pl20) of human immunodeficienc~/ virus type 1 produced by expression in Chinese hamster ovary cells. Enzymatic cleavage of rgp120 and reversed-phase high performance liquid chromato~raphy were used to confirm the primary structure of the protein, to assign intrachain disulfide bonds and to characterize potential sites for N-glycosylation '~:
WO 91/15~12 ~ 'J PCI/US91/0216fi J
~ - 3 2 -All of the tryptic peptides identified were consistent with the primary structure predicted from the cDNA sequenc~ Tryptic mappin~ studies combined with treatment of isolat~d peptides with S. ~ureus V8 protease or with peptide: N-~lycosidase F (PNGase F) followed by endoproteinase Asp-N permitted the assi~nment of all nine intrachain disulfide bonds of r~p120. The 24 potential sites for N-~lycosylation were characterized by determinin~ the susceptibilities of the attached carbohydrate structures to PNGase F and to endo-,B-N-acetyl~lucosaminidase H. Tryptic mappin~ of enzymaticaily de~lycosYlated r~pl20 was used in conjunction with Edman de~radation and fast atom bombardment-mass spectrometry of individually treated peptides to determine which of these sites are ~Iycosylated and what types of structures are present. The results indicate that all 24 sites of ~p120 are utilized, includin~ 13 that contain complex-type oli~osaccharides as the predominant structures, and 11 ~hat contain primarily hi~h mannose-type andlor hybrid-type oli~osaccharide structures~
For convenience, complete biblio~raphic references are ~iven at the end of this Example.
EXPERIMENTAL PROCEDURES
The abbreviations used throu~hou~ this example are: AAA, amino acid analysis; AIDS, acquired immunodsficiency syndrome; amu, atomic mass unit; CHO, Chinese hamster ovary;
Drr, dithiothreitol; endo H, endo-,8-N-acetyl~lucosaminidase H; FAB-MS, fast atom bombardment-mass spectrometry; ~D1, herpes simplex type 1 Dlycoprotein D; ~p, ~Iycoprotein; HIV, human immunodeficiency virus; HPLC, hi~h performance liquid chromato~raphy; IAA, iodoacetic acid; PNGase F, peptide: N-~lycosidase F; PTH, phenylthiohydantoin; RCM, reduced and S carboxymethylated; r~p, recombinant ~Iycoprotein;
SIV, simian immunodeficiency virus; TFA, trifluoroacetic acid; TPCK, 2 5 L- 1 p-tosylamido-2-phenylethyl chloromethyl ketone~
Materials-- Recombinant ~pl 20 proteins were produced in CHO cells and purified by immunoaffinity chromato~raphy as previously described (Lasky et al., 1986). DTT, iAA, and 2-acetamido-1-,6-(L-aspartamido~-1,2-dideoxy-D-glucose ~GIcNAc-Asnl were obtained from Si~ma Chemical Company. HPLC/Spectro Grade trifluoroacetic acid tPierce), Acetonitrile UV
~American B&JI, and Milli Q" water ~Millipore) were used for reversed-phase HPLC. The enzymes used were TPCK trypsin from Worthin~ton 8iomedical Corp., endoproteinase Asp N
~"sequencin~ ~rade") obtained from Boehrin~er Mannheim t;mbH, S. aureus V8 protease from ICN ImmunoBiolo~icals, and PNGase F ~N-Glycanasen) and endo H from Genzyme.
Reduction and S-Carboxymethylation-- Recombinant ~pl 20 ~2.0 mg of CL44 ISEQ. ID NO.
121) was dialyzed a~ainst 0.36 M Tris buffer, pH 8.6, containin~ 8 M urea and 3 mM EDTA.
DTT was added to a concentration of 10 mM and the sample was incubated for 4 hours at ambient temperature. The sample was then treated with 25 mM IAA in the dark for 30 .
WO 91/15~12 PCT/l S91/02166 -33- h ~ 8 ~
minutes at ambient tsmperature. The reaction was ~uenched with excess Dl~, the sample was dialyzed auainst 0.1 M ammonium bicarbonate, and ~hen Iyophilked.
Treatment of RCM toP120 with PNGase F-- RCM r~pl20 10.5 m~) was reconstituted in 0.1 ml of 0.25 M sodium phosphate, pH 8.6, containin~ 10 mM EDTA and 0.02% NaN, to a5 concentration of 5 mg per mL. Tryptic peptides w0re reconstituted to the same molar concentration in 0.05 mM sodium phosphate, pH 7.0, con~ainin~ 0.02% NaN~. PNGase F
was arlded to the sample in the ratio of 12.5 units per mg of protein ~nd the sample was incubated overni~ht at 37C. RCM r~pl 20 treated with PNGase F was dialy~ed a~ainst 0.1 M ammonium bicarbonate.
10 Treatment of RCM r~p 120 with Endo ~/- RCM rgp120 (0.5 m~ was reconstituted in 0.1 ml of 0.05 M sodium phosphate, pH 6.0, containin~ 0.02% NaN3. Endo H (2 unitslml) was added to the sample in the ratio of 0.1 unit per m~ of protein anrJ the sample was incubated overni~ht at 37 C. RCM r~pl 20 treat0d with endo H was dialyzed a~ainst O. t M ammoniurn bicarbonate.
15 Treatment with TPCK-Trypsm- Samples of untreated, PNGase F-treated and endo H-treated RCM r~pl20 (0.5 m~ aliquots of CL44 ISEQ. ID N0. 121) in 0.1 M ammonium bicarbonate were treated at ambient temperatur~ with TPCK-trypsin by the addition of aliquots of enzyme ~enzyme to substrate rstio of 1:100 w/w~ at 0 and 6 hours of incuba~ion. The dioestion was stopped after 24 hours by freezing the samples. For disulfide determinations, a sample ot 20 r~pl 20 (0.5 m~ of 9AA [SEû. ID N0. 11 l~ was treated with TPCK-trypsin usin~ the same conditions.
Treatmenf of Trypt;c Peprides with PNG~se F Followed by Endoprotemase Asp-N - Paptides (ran~in~ ~rom 0.5 nmol to 3.7 nmol) purifi~d by reversed-phase HPLC of a 9AA tryptic di~est were reconstituted in 0.05 M sodium phosphate, pH 7.0, containin~ 0.02% NaNJ 10.05 ml).
25 PNGase F (5 units in 0.06 ml of 0.05 M sodium phosphate, pH 7.0, containin~ 0.02% NaN3) was added and the samples were incubated for 20 hours at 37C. Endoproteinase Asp-N (2 micro~raml was then added and the samples were incubated for 20 hours at 37C.
Treatment of Tryptic Peptides with S. aureus V8 Pfotease-- Peptides 13.0 nmol) purifir~d by 30 reversed-phase HPLC of a 9AA tryptic di~est were reconstituted in 0.05 M sodium phosphate, pH 7.0, containin~ 0.02% NaN3 10.04 ml). V8 protease 15 micro~ram) was added at 0 and 7 hours and the sample was incubatr~d for 24 hours at 37C.
Treatment of CL44 Peptides with Endo H Fo/lowed by PNGase F-- Peptides (tYpicallY 3 nmol) purified by reversed-phase HPLC were reconstituted in 0.05 M sodium phosphats, pH 6.0, 35 containin~ 0.02% NaN3 (0.1 ml). Endo H 10.05 unit in 0.025 ml of 0.05 M sodium phosphate, pH 6.0, containin~ 0.02% NaN3) was added and the sample was incubated for 20 hours at 37C. PNGase F 16.25 units~ and 0.5 M sodium phosphate, pH 10.3, containin~
~'` ~': ' :.' ' .
WO 91/15512 ~ ~ P~/US~1/02166 `~, 3 0.02 M EDTA and 0.02% NaN, 10.125 ml) were then added and the sample was incubated for 20 hours at 37C.
P~eversed-phase 11PLC-- Tryptic diDests were fractionated by reversed-phase HPLC on a 5 micron Vydac C18 endcapped column ~4.6 mm x 250 mm). After equilibration with 0.1%
5 aqueous TFA, the elution of tryptic peptides was carried out at 1 ml per minute with a linear ~radien~ from O to 45% acetonitrile containing 0.08% TFA in 90 minutes. The system used was a Waters gradient liquid chromato~raph consistin~ of two 6000A pumps, a 720 controller, and a WISP 71 OB injector, and a Perkin-Elmer LC75 sin~le wavelength UV detector set at 214 nm. ;
Peptides subjected to further manipulations were fractionated by reversed-phase HPLC
on a Vydac C18 column 12. 1 mm x 250 mm) equilibrated in 0. 1% aqueous TFA at a flow rate of 0.2 ml per minuts and a temperature of 40C. These peptides were eluted with a linear ~radient from O to 60% acetonitrile Icontaining 0.08% TFA) in 60 minutes. The system used was a Hewlett-Packard tO9OM liquid chromato~raph.
15 Peptjde Identific~tion-- Peptides collected from reversed-phase HPLC were identified by AAA
and/or N-terminal sequence analysis. Samples for AAA were treated with constant boilin~
IICI a~ 110C in vacuo for either 24 or 72 hours, dependin~ upon extent of elycosylation.
The extended hvdrolysis degrades ~lucosamine, which would otherwise interfere with quantitation of lle and Leu. Analysis was performed on a Beckman Model 6300 amino acid 20 analyzer with ninhydrin detection.
N-terminal sequence analysis was performed on an Applied ~iosystems Model 477At120A. The acetonitrile concentration in the equilibration buffer of the PTH analysis system was decreased from 10 to 9% to resolve the PTH derivative of GlcNAc-Asn from Drr.
25 fA~-MS-- FAB mass spectra were acquired on a JEOL HX110HF/HX110HF tandem massspectrometer operated in a normal two-sector rnode. FAB-MS was performed with 6 keV
xenon atoms 110 mA emission currentl. Data were acquired over a mass ranae of 380-4000 amu.
R E~LTS
Lasky et al. 11986) expressed ~pt 20 in CHO cells as a fusion proteln usin~ the signal peptide of the herpes simplex ~D1. Two such fusion proteins were used in this study. The recombinant ~Iycoprotein used in most of this study lCL44 lSEQ. ID NO. 121) was expressed as a 498-amino-acid fusion protein containing the first 27 residues of gD1 fused to residues 31-501 of gp120 ILasky et a/., 19861. This construction lacks the first cysteine residue of 35 mature ~p120. Disulfide assignment~ were carried out on another recombinant fusion protein 19AA ISEQ. ID NO. 111~ which conta;ns the first 9 residues of gD1 fused to residues 4-501 of gp12û. This restores the first cysteine residue, Cys 24. Carboxy-terminal analysis of CL44 ISEQ. ID NO. 1 2l usin~ carboxypeptidase digestions ind;cated that ~lutamic acid residue WO 91/15~12 PCI/US91/02166 -35~ .~
479 is the carboxy t~rminus of the fully processed rnolecule sect0ted by CH0 cells Idata not shown). The amino acid saquences of these two constructions are ~iven in Fi~ure 1.
RCM CL44 Tryptic M~p- Reversed-phase HPLC tryptic mappino was used to confirm the primary structure of the molecule, to assign intrachain disulfide bonds and to characterize potential sites for N-~lycosylation. In a~periments not intended to ~ive information about disulfides, the protein was RCM prior to di~estion with trYpsin. This treatment unfolds the protein and disrupts disulfide bonds, thereby resultin~ in smaller tryptic fra~ments than would be obtained with the native mol0cule.
Thc reversed-phass HPLC trvptic map of RCM CL44 is shown in Fi~urs 2. Tryptic ~ p 10 peptides were separated by reversed-phase HPLC usin~ an acetonitrile/water system with TFA as the ionic modifi2r. As will be discussed below, much of the pe~k heterogeneity derives from the extremely hi~h laPProximatelY 50% of total mass) carbohydrate content of the molecule. Peaks were collected and subjected to AAA for identification ITable 1). In some cases, N-terminal sequence analysis was used for confirmation Ithese peaks are 15 indicated in Table l? The peaks not assi~ned a label in Fi~ure 2 were not identified.
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WO 91/1~511 PCr/U~i~1/02166 -37~
All of th~ peptidea identified were consis~ent with the primary structu~e predicted from the cDNA sequence. Of tha 38 predict0d peptides with 3 or more amino acids, 36 were identified in the tryptic map of RCM CL44. In addition, 4 predicted peptides consisting of 2 amino acids each were alsc identified IH3, H4, T23, and T35). The tripeptide composed of residues 139-141 IVQK) was not identified in the map and was not given a label in Fi~ure 2.
The only other peptide not identified was T13 (CNNK). Aspara~ine residue 200 of peptide T13 is a potential glycosylation site and the peptide lacks hydrophobic amino acids.
Therefore, this ~Iycopeptide is likely ~o be extremely hydrophilic and poorly resolved from the salt fracticn on the reversed-phase column.
Tryptic cleava~e did not occur between peptides T5 and T6 and between peptides T8 and T9. These are designat~d in Figure 2 as two T-numbers separated by a comma ~T5,6 and T8,9). The absence of cleavage was confirmed by N-terminal sequence analysis of the peptides, In both of these cases, the asparagine residue to the C-terminal side of the cleava~e site is a potential N-~lycosylation site and it is likely that the carbohydrate moiety interferes with the action of tr~/psin. Incomplete tryptic cleavage was also observed between peptides H4 and T2' and between peptides T23 and T24 IH4,T2' and T23,241.
Several peptides arisin~ from non-tryptic cleavages were observed in the tryptic map of RCM CL44. Two of the predicted tryptic peptides were further cleaved by ~chymotrypsin-like~ cleava~es. Peptide T12 was completely cleaved af~er tyrosine residue 187 and phenylalanine residue 193 to yield peptides T12a, T12b, and T12c. Peptide T4 was partially hydrolyzed after leucine residue 95 to yield peptides T4a and T4b. Intact peptide T4 was also present.
One of the tryptic peptides, T22 ~QAHCNISRl ISEQ. ID NO.14l eluted at two different positions 132.4 minutes and 34.1 minutesl in the RCM CL44 tryptic map. Deglycosylation studies ~discussed below) with PNGase F and endo H indicated that the different retention times of the $wo forms of peptide T22 are not due to carbohydrate differQnces. It is possible that this retention time heterogeneity results from partial conversion of the N-terminal glutamine residue to pyroglutamic acid ISanger and Thompson, 19531.
Disulfide Ass/6~nments ~ p12~- Mature gp120 CQntains 18 cysteine residues lshaded in Figure 1) and therefore could contain 9 intrachain disulfide bonds. The CL44 ~SEQ. ID NO.
12] construction lacks ~ys-24, the first cysteine residue of gp120 ILasky ~t a/., 1986);
therefore, a different construction 19AA îSEQ. ID NO. t 11l, in which the first cysteine residue was restored, was expressed and purified to approximatelY the same degree as CL44 (L.
Riddle, T. Gregory and D. Dowbenko, unpublished data). Ellman's reagent (Ellman, 1959) was used to demonstrate the absence of free sulfhydryl groups in 9AA ISEQ. ID NO. 111 Idata not shown). Therefore, disulfide assignments were determined for the 9AA
construction.
WO 91/15 i l 2 ~ PCI-/US91/02166 Tryptic rnappin~ studies performed without S-carboxymethyiation of cysteine residues allowed panial assi~nment of disulfides. The tryptic map of 9AA is shown in Fi~ure 3. Peaks were identified by N-tsrminal sequence analysis ITable ll). These identitications allowed unequivocal assi~nment of three ot the nine disulfide bonds: between Cys-101 and Cys-127 ~Peak A, Table ll~; between Cys~266 and Cys-301 (Peak B, Table ll); and between Cys-24 and Cys-44 ~Peak E, Table ll).
Peptides containin~ the remainin~ cysteine residues were also identified (Table ll).
Peptide T28 contains three cysteine residues and coelutes with peptide T31, which contains one cysteine residue (Peak D, Table ll). Peptide T11 contains two cysteine residues and coelutes with peptides T3 and T4, each of which contains a sin~le cysteine residue ~Peak F, Table ll). Similarly, peptide T14 contains two cysteine residues and coelutes with peptides T12 and T13, each of which has a sin~le cysteine residue (Peaks C and E, Table ll). In each of these cases more than one disulfide bond was present in the ~roup of tryptic peptides, thereby preventin~ unambi~uous assi~nment. These tryptic peptides were further manipulated as described below to introduce selective cleava~e between cysteine residues located on a sin~le peptide.
wo 91/~5;12 39 PCr/VS91/02t66 . able 11. Identification of Cyste;ne-containing Peptides ir~ ge~ 5 T/yptic Ma~ gAA.
Cys-containing peaks trom the ~ryptic map ot 9AA were identified by N-~erminal sequence analysis. Cysteines in boxes joined by a solid line represent disulfide bond assignments. Cysteines in boxes joined by dotted lines represent dis~ulfide bonds that could not be assigned unarnbiguously in this experiment. Partial cleavages are indicated by a parenthesis. Cysteines are labelled by an amino acid number and peptides are labelled with T-numbers corresponding to the nomenclature used in Figure 1.
Peak Cys-Containing Peptides _ _ _ _ _ 101 (~5,6) A [~TDLKNDTNTNSSSGR
(GEIK)NE~3SFNISTSIR
127 ~ra _ - ~
266 (T16) B TIIVQLNQSVEIN[~3TRPNNNTR
QAH E~¦NISR
301 (T22) (T12a.~) 188 C VSFEPIPIHYl~APAGF E~NNK
/ ~ ~ ~, ' (T14a) l ~ ~
TFNGTGP[~TNVSTVQ~}THGIR
_ _ .
D QSSGGDPEIVTHSFN~GGEFFY~lNSTQLFNSTWFNSTWSTE--GSNNTEGSDTITLP R ~ _ ~ (T31) 388 E~SSNITGLLLTR
_ :
24 ~Tt) E EATTTLF~3ASDAK
AYDTEVHNVWATHA ~3VPTDPNPaEVVLVNVTENFNMWK
ITl2) 188 VSFEPIPIHY~APAGFAILK 198 (T13) / ~ _ ~ _ TFhGTGP~ITNVSTVQ~3THGlRPVVsTQLLLNGsLAErrVVlR
-- . :
NDMVEQMHEDllSLWDaSLKP~VK ~ (T4) LDllplDNDTTsyTLTs~3NTsvlTQA~3pK -.
WO 91/15~12 ~ 3~ ~ PCI/U!~9t/02166 Each of the pepsides has a potential N-linked ~IycosylaTion site located between the cysteine residues. The peptides were treated with PNGase F, which removes asparaoine-linked carbohydrate while conver~in~ the attachment aspara~ine residue to aspartic acid (Tarentino e~ a/., 1985~. The resultin~ aspartic acid residue serves as a point 5 for selective cleava~e of the peptides Witt1 endoprot~inase Asp-N ~Drapeau, 1980). The peptides were separated by reversed-phase HPLC and identified by N-terminal sequence analysis.
The HPLC chromato~ram obtained after treatment of peptides T12, T13, and T14 (Peak C, Fi~ure 3) with PNGase F follow~d by endoproteinase Asp-N is ~iven in Fi~ura 4a, and 10 the sequences of rel0vant peptides are given in Table lll. The results indicate that r~pl 20 has disulfide bonds be~ween Cys-198 and Cys-209 and between Cys-188 and Cys 217 ITable Ill). Treatment of peptides T3, T4, and T11 IPeak F, Fi~ure 3) with PNGase F followed by endoproteinase Asp-N allowed the recovery of fra~ments that demonstrated the presence of disulfide bonds between Cys-89 and Cys-175 and between Cys-96 and Cys-166 IFi~ure 4b 15 and Table lll) : : ~ :
WO 91~ 12 ~ V 7 8 ~P~/US~I/02166 Table lll. Assignment of Disu!fides from Peptides isolated in Figure 4.
. The tryptic peptides that could not be assigned unambiguously in Table li were further rnanipulated as described in Figure 4. Peaks were identified by N-terminal sequence analysis.
Peak Sequence :
.... . . .~
198 ~
1 QN ~ :
, . .
DGTG PlJT ~ :
~:
2EPIPIHY~}APAGF :~
DVSTVQ1~3THG( I R) . 3 DQSLKP~lVK ;:
DTSVITQA [~1P K
.
4 LTPL~VSLK
DDrrSYTLTS
IVTHSFN~GGE
I~SSNITGLLLTR
6FFY~NSTQLFNSTWFNSTWSTE
TITLPL~IR ~:
'' WO 91/1~512 ~ PCT/US91/02166 The last two disulfida bonds wsre assioned by treatinD peptides T28 and T31 IPeak D, Fi~ure 31 with V8 protease to cleave to tha carboxy side of the ~lutamic acid and aspartic acid residuss IDrapeau et ~/., 1972) locatad between the cystsine residues of T28. The chromato~ram obtained after V8 protease dipestior~ of T28 and T31 is ~iven in Fi~ure 4c and 5 the sequences of the relevant peptides are oiven in Table lll. The results demonstrated the presence of disulfide bonds between Cys-348 and Cys-415 and between Cys-355 and Cys-388.
Thus, th0 combined results of the tlyptic mapping analysis and the further selective deDradations permitted the assi~nment of all nine intrachain disulfid3 bonds of r~pl20.
10 Parallel experiments performed on CL44 ~SEQ. ID N0. 121 produced similar results for the 8 disulfide bonds remainin~ in that construction (not shown). The disulfide bond assi~nments of rgp120 are summarized in Fi~ure 6.
Glycosy/ation Sites of ~p t2~- Mature ~pl 20 contains 24 potential sites for N-~lycosylation, as recoanized by the sequence: Asn-Xaa-SerlThr) IKornfeld and Kornfeld, 1985~. These sites 15 are indicated by a dot above the correspondin~ aspara~ine residue in Fi~ure 1A. tn the present study, tryptic mappin~ of enzymatically de~lycosylated CL44 [SEQ. ID N0. 121 was used in conjunction with Edman de~radation and FAB-MS of individually treated peptides to determine which of the 24 potential N-~lycosylation sites are Dlycosylated and which contain Iess fully proccssed li.e. hi~h mannose-type or hybrid-type) oli~osaccharides.
The two enzymes used for de~lycosylation were PNGase F and endo H. PNGase F
releases all types of N-linked oli~osaccharide structures by cleava~e of the ~-aspartylplucosylamine linka~e ITarentino et ol., 1985). Endo H releases only hi~h mannose-type and hybrid-type oli~osaccharide structures by cleavinp between the two core N-acetyl~lucosamine residues ITai et ~/., 1977). De~lycosylation of a peptide can be 25 monitored by the increase in retention time of the peak correspondin~ to the Glycopeptide in the reversed-phase elution profile. Thus, it was possible to determine which peptides were 31ycosylated by treatment with PNGase F and, on the basis of susceptibility tn cndo H, to distin~uish those with attached high mannose~type andlor hybrid-type oli~osaccharides as the predominant structures.
The 24 potential ~Iycosylation sites of CL44 ~SEQ. ID N0. 121 are contained in 14 tr~ptic clycopeptides. Thineen of thesr~ olycopeptides were identified in the tryptic map of RCM CL44 lFiaure 2~. As mentioned above, T13 ICNNK) lSEQ. ID N0. 15~ was not identified. The tryptic maps of PNGase F-treated RCM CL44 and endo H-treated RCM CL44 are compared with the RCM CL44 tryptic map in Fi~ure 5. The peaks correspondinp to 35 ~Iycopeptides are labelled in each of thc three tryptic maps.
As would be expected for a heavily ~Iycosylated mo!ecule, treatment of RCM CL44 with PNGase F ~Fi~ure 5b~ simplified ~he tryptic map si~nificantly. Typically, the peaks correspondin~ to potential ~Iycopeptides in the RCM CL44 tryptic map IFi~ure 5a) were broad WO 91/15~1' PCI/US91/02166 -43- 7 ~
and often appeared as muitiplets. De~iycosylation resulted in sharp, sin~l~ peaks tor each pep~ide, indicatin~ that the g!ycopeptide psak multiplicity and broadness was due to carbohydrate hetero~eneity. ~ .
All of the 13 potential plvcopeptides that had been identified in the tryptic map of RCM
CL44 were shifted to later retention times in the tryptic map of PNGase F-treated material.
This dsrnonstrates that at least 13 of th0 24 potential sites are ~Iycosylated. Peptide T28 was not recover0d after de~lycosylation. This peptide contains a lar0e number of non-polar i~
amino acids and, after removal of the hydrophilic carbohydrate moieties, may bind irreversibly ~;
to the HPLC column. As described above, peptide T22 elutes at 2 positions in tha RCM GL44 tryptic map presumably as a result of conversion of the N-terminal ~lutamine to pyroglutamic acid. The retention times of both of the T22 peaks were altered in the de~lycosylated material produced by treatment with both PNGase F and endo H, confirmin~ that the difference between these forms of peptide T22 in the RCM CL44 tryptic map was not due to carbohydrate hetero~eneity. .
The ~ryptic map of endo H-treated RCM CL44 (Fi~ure 5c~ indicated that 6 of the 13 tryptic ~Iycopeptides were endo H-susceptible ~p~ptides T14, Tl 6, T22, T24, T28, and T31~.
In addition, a small amount of peptide T15 showed endo H susceptibility. For each of these ~Iycopeptides, the elution time of the endo H-treated glycopeptide was eariier than that of the correspondin~ PNGase F-treated ~Iycopeptide. This is due to the hydrophilic N-ace~yl~lucosamine residue that remains attachcd to the asparagine residue followin~ endo H treatment. Peptide Tl 6 was not identified in the tryptic map of endo H-treated RCM CL44.
This peptide contains 3 potential glycosylation sites and was poorly recovered under any circumstances.
Conclusions as to the type of ~Iycosylation present on each of the tryptic :
~Iycopeptides based on susceptibility to PNGase F and endo H are summarized in Table IV.
S0ven of the 13 ~Iycopeptides identified in the tryptic map of RCM CL44 contain only a sin~le ~Iycosylation site and thus could be characterized unambi~uously with re~ard to enzyms susceptibili~y. Peptides T2' lAsn-58), T26 ~Asn-3261, and T32 ~Asn-433) were de~lycosylated only by PNGase F and, therefore, contain attached complr~x-type oli~osaccharide structures. Peptides T22 ~Asn-302), T24 (Asn-309), and T31 ~Asn-418) were susceptible to both PNGase F and endo H and, therefore, ~arry high mannose-type and/or hybrid-typc oli~osaccharide strtJctures. Peptide T15 is only partially susceptible to endo H; therefore, Asn-246 carries primarily complex-type oli~osaccharides but must also have some attached hi~h mannose-type and/or hybrid-type oli~osaccharide structures.
WO 91~15512 ~ PCI/US91/0~166 Table IV. Assignmen~ ~I Glycosyla~ion Type ~o RCM CL44 Tryp~ic Peptides by Susceplibility to PNGase F and Endo H.
Suscep~ibili~y to PNGase F or endo H was determined by an increase in ~he retention ~ime ol a peptide in the tryptic map o~ FICM CL44 . PNGase F reieases all types ot N-linked oligosaccharide s~ructures, whereas endo H releases only high mannose and hybridoligosaccharide structures.
Tryp~ic GlycosylaVon Si~es SuscepHble Susceptible Glycosyblion Pep~ide a (Asn Residue #) To PNGase F To Endo H Type _ T2 5~ Yes No Complex T6 106,111 Yes No Complex b -T9 126,130 Yes No Complex b T11 156,167 Yes No Complex b T14 204,211,232 Yes Yes High Mannose, Hybrid, and/or Complex c T15 246 Yes Trace Complex (Trace High Mannose and/or Hybrid) T16 259,265,271Yes Yes High Mannose, Hybrid, and/or ComplexC
T22 302 Yes Yes High Mannose and/or Hybrid T24 309 Yes Yes High Mannose and/or Hybrid T26 326 Yes No Complex T28 356,362,367,376 Yes Yes High Mannose, Hybrid, and/or ComplexC
T31 418 Yes Yes High Mannose and/or Hybrid T32 433 Yes No Complex a T13 not tound.
b Eilher or both siles glycosylaled.
c Endo H susceplible glycosylalion at one or more sitels).
WO 91/1~12 . ~ 5~ Pcr/us9l/0~l~6 Peptides T6, T9, and Tl 1 each c~ntain 2 p~t~ntial ~Iycosylation sites. Each peptide was de~lycosylated by PNGase F but not by endo H indicatin~ the presence of mostly complex-type oli~osa~-charide struc~ures. In order to determine wh~th~r one or both of the potential olycosylation sites in each peptide were actually glycosylated, tha PNGase F-treated 5 olycopeptides were subjected to either FAB-MS or Edman dearadation. Treatment with PNGase F converts the attachment aspara~ine residue to aspartic acid during de~lycosylation tTarentino et ol., 1985), This conversion can be detected by FAB-MS as an increase of 1 amu in the mass of the peptide for aach site de~lycosylated tCarr and Roberts, 1986) or by Edman degradation by the appaarance of th~ PTH derivative of aspartic acid at the 10 appropriate cycles. FAB-MS of de~lYcosylated peptide T5,6 rsvealed an ion correspondin~
to the peptide mass plus 2 amu ~[MH]~ observed: m/z 1772.6; calculated: m/z 1772.7).
FAB-MS of deglycssylated pep~ide T9 gavs similar results (IMH1- observed: m/z 1301.8;
calculated: m/z 1301.5). Edman de~radation was performed instead of FAB-MS on de~lycosyla~ed peptide T11 because of its hi~h molecular wei~ht t~2000 amu). Aspartic 15 acid was observed in cycles 8 Iderived from Asn-156~ and 19 (derived from Asn-l 67). These combined results indicate the presence of complex-type oligosaccharide structures attached to Asn residues 106, 1 1 1, 126, 130, 156, and 167.
The remainin~ 3 glycopeptides identified in the tryptic map of RCM CL44 contained multiple potential glycosylation sites and were endo H-susceptible. Peptides T14, T16, and 20 T28 account for a total of 10 potential glycosylation sites. Characterization of each ~Iycosylation site was achieved by Edman degradation of HPLC-purified peptides that had been subjected to treatment with endo H followed by PNGase F.
When endo H releases the high mannose-type and hybrid-type oli~osaccharide structures, it leaves an N-acetylglucosamine residue attached to the aspara~ine residue of the 25 peptide ~Tarentino et ~/., 1974~. PNGase F will not remove this N-acetyl~lucosamine residue, but will release the remainin~ N-linked oli~osaccharide structures by cleavage of the ,B-aspar~ylglucosvlamine bond, resulting in conversion of the attachment asparagine residu0 to aspartic acid lChu, 1986). Therefore, treatment with Endo H followed by PNGase F will yield aspara~ine at an unglycosylated site, GlcNAc-Asn at a ~Iycosylation site that contained 30 primarily hi~h mannose-type and/or hybrid-type oli~osaccharide structures, and aspartic acid at a ~Iycosylation site that carried primarily complex-type oli~osaccharide structures. Paxton et ~/. It 987~ have shown that it is possible to detect the PTH derivative of GlcNAc-Asn after Edman de~radation. Using this approach, it was possible to characterize the remainder of the ~Iycosylation sites of CL44 ISEQ. ID N0. 12~. For example, treatment of ~Iycopeptide T16 35 which contains 3 potential N-glycosylation sites, with endo H followed by PNGase F resulted in the appearance of the PTH derivative of GlcNAc-Asn at cycles 7 and 13 and theappearance of PTH-Asp at cycle 19 during Edman degradation. Thus, ~Iycopeptide T16 carries primarily high mannose-type and/or hybrid-type oligosaccharides at Asn-259 and wo 91/15S12 . ~, ` PCI/US91/02166 Asn-265 and complex-type olioosaccharides at Asn-271. The rosults of shese experiments are sumrr,arized in Table V and indicate that CL44 ISEQ. ID N0. 12] contains complex-type oli~osaccharide structwes at Asn residuas 271, 367, and 376 and high mannoss-tYPe and/or hybrid-type oligosaccharide structures at Asn residues 204, 211, 232, 259, 265, 356, and 5 362.
~ ' ' ~ ' ' I . ' . ' ' , ' ' ' ' ' ' ~ ' , ' . , : " ' ' , ' ' `,`: ' ~ ' '.' ' ' ~ ' ' ' ' ~ . .
WO 91/1~;512 PCr/US91/02166 Table V. Assignment of Glycosylation Type ~o RCM CL44 Tryptic Glycopeptides Containing Multiple Potential Glycosylation Sites.
Characterization of multiple potential glycosylation sites on RCM
CL44 tryptic glycopeptides was achieved by Edman degradation of HPLC purified peptides subjected to treatment wi~h endo H followed by PNGase F. Edman degradation of deglycosylated peptides shows either an Asn residue at an unglycosylated site, a GlcNAc-Asn at a glycosylation site to which had been attached high mannose or hybrid oligosaccharide structures, or an Asp residue at a glycosylation site which had carried complex type oligosacc~aride structures.
Tryptic Asn ~esidue Peptide Residue # Observed Gtycosylation Type . ~ ..... . _ .. .. ....
T14 204 GlcNAc-Asn High Mannose and/or Hybrid 211 GlcNAc-Asn High M~nnose and/or Hybrid 232 GlcNAc-Asn High Mannose and/or Hybrid T16 259 GlcNAc-Asn High Mannose and/or Hybrid 265 GlcNAc-Asn High Mannose and/or Hybrid 271 Asp Complex T28 356 GlcNAc-Asn High Mannose and/or Hybrid 362 GlcNAc-Asn High Mannose and/or Hybrid 367 Asp Complex 376 Asp Complex .. , . : . , ~. . .. . - ., , , ~ , , . . .. . . . . , ,., , . , ~ .; . ... .... . .
wo 91/15~12 ~ ) PCI/US91/02166 -~8-Peptide T13, which con~ains the remainin~ ~Iycosylation site, was not identified in any of the tryptic maps presented in this paper. However, FAB-MS data obtained from the void peak of a tryptic map of RCM CL44 treated with endo H followed by PNGase F revealed an ion correspondin~ to MH~ for that peptide containin~ an attached N-acetyl~lucosamine residue lobserved: m/z 740.1; calculated: m/z 740.4). The prescnce of peptide T13 in the void peak was further confirmed by AAA. Therefore, we conclude that Asn-200 is ~Iycosylated and carries primarily hi~h mannose-type and/or hybrid-type oli~osaccharide structures.
The data presented here demonstrate that all 24 potential ~Iycosylation sites of ~p120 10 are utilized, that 13 sites contain primarily complex-type oli~osaccharide struc~ures while 11 sites contain primarily hi~h mannose-type and/or hybrid-type oli~osaccharide structures. The type of glvcosylation at aach site is summarized in Figure 6.
DISCU~SION
We have determined the disulfide bondin~ pattern and the attachment positions of15 oli~osaccharide moieties of r~p120 from the 11i8 isolate of HIV-1. A schematic representation of this information is presented in Fi~ure ô [SEQ. ID N0. 10]. The r~p120 molerules from which the structural data were obtained possess the functional properties attributed to ~p120 produced by HIV-1 virions includin~ hiqh-affinity CD4 bindinq ~Lasky et ~1., 1987), and HIV-1 neutralizin~ anti~enicity tLasky et a/., 1986). We therefore conclude that the CH0-expressed 20 gp120 is properly folded and that the disulfide-bonded domains reported here for the recombinant molecules are representative of those occurrin~ in gp120 produced by HIV-1 virions.
functiona/Aspectsofop120Structute--Theqp120moleculecomprisesfivedisulfide-bondedloop structures. The first and fourth are simple loops formed by sinqle disulfide bonds while 25 the second, third and fifth are more complex arrays of loops formed by nested disulfide bonds. The fourth disulfide-bonded domain kesidues 266-301) has been shown to contain siqnificant type-sp0cific neutralizin~q epitopes lMatsushita et ~/., 1988; Rusche et a/., 1988;
Goudsmit et ~/., 1988; Javaherian et ~/., 1989~ and the fifth disulfide-bonded domain Iresidues 348-415) has been shown to be important for CD4 bindin~ ILaskY et a/., 1987;
30 Kowalski et a/., 1987). No direct functional correlates have been described for the other three disulfide bonded domains. The amino acid sequence of ~p120 varies to a larqe extent between different viral isolates but the majority of the variability is localized in hypervariable re~ions which punctuate the otherwise relatively conserved sequences ~Willey et al., 1986;
Modrow et a/. 1987~. Modrow et ~ 1987) have identified five hypervariable re~ions which 35 are characterized by sequence variation, insertions and deletions. Four of these hypervariable reaions correspond to well delineated loops as indicated in Fi~ure 6. With the excep~ion o~
the third hypervariable loop ~disulfide-bonded domain IV~ the functional si~nificance of these re~ions is unknown.
WO 91/1~512 PC~/US91/02166 The positions ot the :ystein~ residues and, presumably, the disulfids bondino pattern in op120 are hi~h~y conserved b~twe~n isolat~s. Amon~ HIV-1 isolates, the only ~xception to this c~nservation is the Z3 isolate ~Will~y ~t ~J., 1986) which has an additional pair of cysteine residues in the fourth hypervariable domain ~residues 363-384i. These residues most likely forrn a tanth disulfide bond in the ~p120 from this isolate. The presence of this extra bond in such a hypervariable region probably has no more effect on the structure and function of the molecule than the other sequence variations that occur in that re~ion.
As shown in Fi~. 7 in HIV-2 lSEQ. ID NO. 13], and similarly in SIV ~da~a not shown) the positions of the cystaine r~sidues in disulfide-bonded dorr~ains 1, Il, IV and V are conserved IHuman ~etroviruses and AIDS ~1989). G. Myeres, A. P~abson, S. Josephs, T.
Smith, J. ~erzofsky and F. Won~-Stahl, Editors. U.S. Government Printin~ Offica, Los Alamos National Laboratory, Los Alamos, New Mexico, LA-UR, 89-743). In domain lll there are two additional pairs of cysteine residues ~three in SIV isolate MM142) which are presumed to be disulfide bonded within a fin~er-like domain lll structure analo~ous to that illustrated in Figure 6. Another major differenc0 between HIY-1, IIIV-2 and SIV is that hypervariabls reDion V2 is reduced to five amino acids in HIV-2 and SIV. Ths functional si~nificance of the differences between HIV-1, HIV-2 and SIV is unknown at this time.
One of the most important functions of gp120 is its ability to bind to CD4 and thereby mediate the attachment of virions to susceptible cells IKlatzman et al., 1984; Dal~10ish et ~1., 1984). The CD4-bindin~ function has been locilized by mutagenesis and sttustural studies ILasky et ~1., 1987; Kowalski et al., 1987~ to the r0~ion between residues 320 and 450, which includes the fifth disulfide-bonded domain. Lasky et a/. (1987~ showed that deletion of residues 396 to 407 and muta~enesis of Ala-402 to Asp abolished CD4 binding. They also mapped the epitope of a monoclonal antibody that bloclcs ~pl20-CD4 bindin~ to residues 392-402. Kowalski et ~/. 11987~ identified three re~ions as bein~ involved with CD4 bindin~
Insertions between residues 333-334, 388-390 and 442-443 abolished CD4 bindin~. In addition, a deletion of residues 441-479 abolished CD4 bindin~ while deletion of residues 362-369 within the fourth hypervariable re~ion had no effect on bindin~. Cordonnier et ~1 11989) have shown that muta~enesis of Trp-397 to Tyr or Phe dacreases CD4 binding and chan~es to Ser, Gly, Val or Ar~ abolish bindin~. Nygren et ~ 1988) have teported that a protflolytic fra~ment of ~p120 from residue 322 to near the C-terminus retains the ability to bind to CD4. The results of these studies indicate that the CD4 bindin~ capacity of ~pl 20 is localized to the re~ion between residues 320 and 450 and more specifically to the residues around 333-334, 442-443 and the sequence between 388 and 407.
In the course of efforts to map the epitope of monoclonal antibody 5C2-E5 which blocks ~pl 20-CD4 bindina, Lasky et a/. (19871 treated r~pl 20 (CL44 lSEQ. ID NO 1 2l~ with acetic acid to cleave the protein at aspartic acid residues (In~ram, 1963~ and isolated the peptide fra~ment 383-426 from a column of immobili~ed anti-~p120 monoclonal antibody WO ~/15~12 ~ ') P~/US~ 166 ` ~
,. 50-5C2 E5. Dior~stion of reduc~d rDpl20 yielded the sarre fra~m~nt. ConssquentlY~ il was concludad that a disulfide bond existed between Cys residues 388 and 415. In the analysis reported here we hava failed to find this disulfide bond and, instead, have consistentlY found the disulfide bonds betwe0n Cys 355 and Cys 388, and between Cys 348 and Cys 415 as 5 summarized in Fi~ure 6. We beliflve that the true disulfide bond assi~nment is as indicated in Figure 6 and that the acetic acid dioestion produced some disulfide bond rearran~ement (Ryle and San~er, 1955) in the earlier work.
The 01igosacchat;des of oP12~ Approximately 50% of the apparent molecular mass of ~pl 20 is carbohydrate. The struçtures of the oli~osacchari~e moieties released by 10 hydrazinolysis of CL44 ISEQ. ID N0.
121 r~pl 20 have been exhaustively analyzed (Mizuochi et a/., 1 988a; Mizuochi et ~/., 1 988b).
These authors found that 33% of the N linked oli~osaccharides were of the high mannose type, 4% were of the hybrid type, and 63% were of the complex type. Of the complex olioosacchatides 90% were fucosylated and 94% were sialylated. The complex 15 structures were approximately 4% monoantennary, 61% biantennary, 19% triantennary and 16% tetraantennary. ND 0-linked oligosaccharides were found. Geyer er a/. 11988) have analyzed the oli~osaccharides of ~pl20 from the lll~ isolate of HlV-1 infected human cells.
They found that high mannose type oli~osaccharides accounted for approximately 50% of the carbohydrate structures. The remainin~ structures were fucosylated, partially sialylated 20 bi, tri, and tetraantennary complex type oli~osaccharides. No novel carbohydrate structures, or moieties that would be expected to act as heterophile anti~ens in man, have been isolated from ~pl20 from either source.
We have shown here that all 24 glycosylation sites are utilized, and that 13 of the 24 :
sites contain complex type oli~osaccharides as the predominant structures while 11 contain 25 primarily hybrid and/or hi~h mannose structures. The demonstration of endo H susceptible structures at 11 of the 24 sites is consistent with the earlier results of Mizuochi et al.
~1988a,1988b~ who determined that nearly 40% of the total oli~osaccharide structures released from r~pl 20 were hybrid and/or hi~h mannose type oli~osaccharides.
The 24 potential N linked ~Iycosylation sites in the ~pl 20 sequence are conserved to 30 a lar~e extent between different viral isolates IWilley et a/., 1986; Modrow et a/., 1987~.
Based on the ~pl20 sequence comparisons in these references, 13 of the sites on ~pl20 from the ill9 isolate of HIV 1 are absolutely conserved; these include 8 of the 11 sites that carry predominantly hybrid type and/or hi~h mannose type oli~osaccharides. Thus, the less fully processed li.e. Endo H susceptible) oli~osaccharides of ~p120 are found preferentially 35 at the most conserved ~Iycosylation sites. The remainin~ sites ~8 complex and 3 hybrid/hi~h mannose~ are relatively conserved, even thou~h many of them occur in the hypervariable re~ions. The positions of these sites may shift or be deleted, but there is always one or more new sitels) within 5 to 10 residues of the reference 1119 site. Studies by Willey et a/. 11988) ` ' :
WO 91/15~12 PCrlUS91/02166 -51 h ~ S
d~monstrated that muta~n~sis of Asn-232 to Gln decreased the inf~c~ivity of virions containing th~ mutant ~pl 20 molr~cules without aff~r,tin~ CD4 bindin~ or syncytium formation. At this tim~, no particular functional si~nificanc~ can be attributed to the type of oli~osaccharide structur~ at any of the sitr~s.
The rol~ of th~ carbohydrate moi~ties on ~p120 in CD4 bindin~ has been investi~ated by several authors ILifson et a/., 1986; Matthews e~ a/., 1987; Fenouillet et a/., 1989).
Those that smployed enzymatic dr~lycosylation in the presence of deter~ents ILifson et ~I., 1986; Matthews er ~/., 1987) have conclud~d that the carboh~/drates ar~ not directly involved with the bindin~, but that th~y are required to maintain the conformation o~ ~p120 10 necessary for bindin~. In contrast, Fenouillet et ~/. 11989) enzymatically de~lycosylated ~pl 20 without deter~ent and demonstrated that the CD4 bindin~ affinity was preserved. It ~herefore appears that the carbohydrate moie~ies of ~p120 are not required for its bindin~ to CD4 but that the conformational stabiiity of ~p120 to deter~ents is lost after de~lycosylation.
The r~pl20 used for these determinasions is functionally and structurally equivalent 15 to gp120 produc~d by HIV-1 infected cells.
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.. ' "' ''' ' . . : ' :
WO 91/tS512 ~ PCr/US91/02166 ~ SEQUENCE LISTING
(1) sENERAL INFORMATION:
~i) APPLICANT: G~n~nt~ch, Inc.
(ii) TITLE OF INVENTION: HIV Envelope Polypeptide~
(iii) NUM~ER OF SEQUENCES: 15 ~iv) CORRESPONDENC~ ADDRESS:
~A) ADDRESSEE: Genentech, Inc.
(B) STREET: 460 Polnt San Bruno Blvd : :
~C) CITY: South S~ Fr~n~i~co (D) 5TATE: California -~
(E) COUNTRY: USA
(F) ZIP: 94080 (v) COMPUTER READABLE FORM: .
(A) MEDIUM TYPE: 5.25 lnch, 360 Xb ~loppy di~k (~) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOSIMS-DOS
(D) SOFTWARE: patin (Genentech) (v' ) CURRENT APPLICAT$ON DATA: .
~A) APPLICATION NUMBER:
~B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPEICATION NUMBER: U.S.S.N. 07/504,772 (8) FILING DATE: 03-APRIL-1990 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Adler, Carolyn R.
(B) REGISTRATION NUMBER: 32,324 (c) REFERENCE/DOCKET NUMBER: 639 ..
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415/266-2614 (B) TELEFAX: 415/952-9881 ~ `
(C) TELEX: 910/371-7168 ~`
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS: ~ .
(A) LENGTH: 18 amino acid~
(B) TYPE: amino acid ~D) TOPOLOGY: lLnear txi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Cys Val Lys Leu Thr Pro Leu Cy8 Cya Asn Thr Ser Val Ile Thr Gln Ala Cy~
WO 91/15512 PCr/US91/02166 -55~ 3 4 ~
t2) INFORMATION POR SEQ ID NO:2:
(i) SEQUENCE CHARAC~ERIS~ICS:
(A) LENGTH: 40 amLno ~cid~
( B ) TYPE: ~mino acid (D) TOPOLOCY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 Pro Ile Hi~ Tyr Cy~ Pro Al~ Gly Phe Ala Ile Leu Ly~ Cy~
A~n Asn Lyu Thr Ph~ A~n Cly Thr Gly Pro Cy8 Thr A~n Val 5er -~
20 25 30 ~:
Thr Val Gln Cy8 Thr His Gly Ile Arg Pro ~:
- 35 40 .- :
:
(2) INFO~MATION FOR SEQ ID NO:3: ,.
`
(i) SEQUENCE CHARACTERISTICS~
~A) LENGTH: 12 amino acids ~) TYPE: amino acid : : :
~D) TOPOLOGY: linear :
~5 ~xi) SEQUENCE DESCRIP~ION: SEQ ID NO:3: ~:
Cy~ Asn Asn Ly~ Thr Phe A~n Gly Thr Gly Pro CYD
30~
~2) INFORMATION FOR SEQ ID NO:4:
.
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 ~mino ~cids ~B) TYPE: amino acid ~D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
40 Cys Ala Pro Ala Gly Phe Ala Ile Leu Ly~ Cy8 Cy5 Thr A~n Val Ser Thr Val Gln Cy~
~2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS: ~ .
(A) LENaTH: 12 amino acida ~:
50 . ~ S ) TYPE: amino acid :~
(D) TOPOLOGY: linear (xl) SEQU~NCE DESCRIPTION: SEQ ID NO:5: ~
55Pro Ile His Tyr Cys Cy~ Thr Hia Gly Ile Arg Pro ~:
1 5 . 10 12 ~.
:
, .:
WO 91/15~12~ PCI/U591/02166 ~
2) INFOF~ATION FOR SEQ ID NO:6:
(i) SEQUFNC~ C~A~ACTERISTICS:
(A) LENGTH: 58 amino acid~
~B) TYPE: ~mlno acid (D) TOPOLOCY: line~r (xi) SEQUENCE DESCRIPTSON: SEQ ID NO:6: ~i Gly Gly ABP Pro Glu Ile Val Thr H$~ Ser Phe A~n Cys Gly Gly Glu Ph~ Ph~ Tyr Cy~ Asn Ser Leu Pro Cy~ Arg Ile Ly~ Gln Phe 20 25 30 ~.`
Ile Aqn Met Trp Gln Glu Yal Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser G-y Gln Ile Arg Cy~ Ser Ser A~n Ile Thr aly 58 .
(2) INFORMATION FOR SEQ ID NO:7:
~i) SEQUENCE C~ARACTERISTICS:
~A) LENGT~: 36 amino acids tB) TYPE: amino acid ~D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Cy~ Gly Gly Glu Phe Phe Tyr Cy~ Cy~ Arg Ile Ly~ Gln Phe Ile 1 5 10 15 : :
A~n Met Trp Cln Glu Val Gly Lyn Ala ~et Tyr Ala Pro Pro Ile : .
Ser Gly Gln Ile Arg Cy~
(2) INFORMATION FOR SEQ ID NO.8: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 ~mino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:B:
Cys Ala Ser Asp Ala Ly~ Ala Tyr Asp Thr Glu Val Hi~ A3n Val Trp Ala T~r Hl~ Ala Cy~
~2) INFORMATION FOR SEQ ID NO:9:
.-.
W O 91/1~512 PCT/USg1/02166 (1) SEQUENCE CHAR~cTERISTICS~
(A) LBNGTH: 32 amino aeid~
(~) TYPE: ~Imino acid ~:~
tD~ TOPOLOGY: 1i~e~r (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Ly~ Ala Tyr Asp Thr ,~ -Glu Val Hi~ A~n Val Trp Ala Thr Hi~ Ala Cy~ Val Pro Thr Asp Pro A~n - .
(2) INFORMATI0N FOR SEQ ID N0:10:
( i ) SEQUENCE CHZ~CTERISTICS: ;-20(A) LENGTB: 479 amino ac~ d~
(B) TYPE: amino alcid (D) TOPOLOGY: lin~ar (Xi) SEQUENCE DESCRIP2ION: SEQ ID NO:10:
Thr Glu Lyl3 Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp 5 10 15 : .
Lyl3 Glu Ala Thr Thr Thr Leu Phe Cya Ala Ser Aap Ala Ly~ Ala :.
3020 25 30 ~, Tyr A~p Thr Clu Val Hi~ A~n Val Trp Ala Thr Hi~ Ala Cy~ Val 35 40 45 ~ -35Pro Thr A~p Pro Asn Pro Gln Glu Val Val Leu Val Asn Val Thr Glu A~n Phe A~n Met Trp Lys Asn A~p Met VB1 Glu Glrl Met Hi~
. 65 70 75 Glu A~p Ile Ile Ser Leu Trp A~p Gln Ser Leu Ly~ Pro Cy5 Val 80 85 90 ' Ly~ Leu Thr Pro Leu Cy~ Val Ser Leu Ly~ Cy~ Thr A~p Leu LyfJ
A~n A~p Thr A~n Thr A~n Ser Ser Ser Gly Arg Met Ile Me'c Glu 50Ly~ Gly Glu Ile Ly~ Asn Cy3 Ser Phe A~n Ile Ser Thr Ser Ile ~:
Arg Gly Ly~ Val Gln Lys Glu Tyr Ala Phe Phe Tyr Ly~ Leu Asp Ile Ile Pro Ile A-p A-n Asp Thr Thr Ser Tyr Thr Leu Thr Ser ~, .
WO ~5~12,. '~ ~', PCI/US9t/0~166 Cy~ A~n Thr Ser Val Ile Thr Gln Ala Cy~ Pro Ly~ Val Ser Phe G}u Pro Ile Pro Ile Hi~ Tyr Cy~ Ala Pro Ala Gly Phe Ala Ile Leu Ly~ CYB Aan A~n LyE~ Thr Phe A~n Gly ~hr Gly Pro Cyl3 Thr Asn Val Ser Thr Val Cln Cy~ Thr Hi~ Cly Il~ Arg Pro Val Val 15Ser Thr Gln LBU Leu Leu A~n Gly Ser Leu Ala Glu Glu Glu Val 230 ~35 240 Val Ile ~rg Ser Ala AE~n Phe Thr A~3p A~n Ala Lyn Thr Ile Ile Val Gln Leu Asn Gln Ser Val Glu Ile A~n Cys Thr Arg Pxo A~n A~n Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Ly~ Ile Gly A~n Met Arg Gln Ala Hi~
30Cy~ Asn Ile Ser Arg Ala Ly~ Trp Asn Asn Thr L~u Lys Gln Ile Asp Ser Lya Leu Arg Glu Gln Phe Gly Asn Asn Ly~ Thr Ile Ile Phe LYB Gln Ser Ser Gly Gly A~p Pro Glu Ile Val Thr Hi~ Ser Phe Asn Cy~ Gly Gly Glu Phe Phe Tyr Cy~ Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Phe A~n Ser Thr Trp ser Thr Glu Gly Ser 45Asn Asn Thr Glu Gly Ser Asp Thr Ile Thr Leu Pro Cy~ Arg Ile LYB Gln Phe Ile Asn Met Trp Gln Glu Val Gly Ly~ Ala Het Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cy~ Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arq A~p Gly Gly A~n A~n A~n Asn Clu Ser ~, .: , ,"" "" ~
WO 91/15;12 PCI/US!Jl/02166 -5~ 7 ~
Glu Il~3 Phe Arg Pro Gly Gly Gly Asp M~t Arg A~p A3n Trp Arg Ser Clu Leu Tyr Ly~ Tyr Ly0 Val Val Ly~ Ile Glu Pro Leu Gly ~;
Val Ala Pro Thr Ly~ Ala Ly~ Arg Arg Val Val Cln Arg Glu ( 2 ) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE C~ACTERISl`ICS: ~, (A) LENGTH: 9 amino Acid ~ B ) TYPE: amino acid (D) TOPOLOGY: linear ~
(xi) SEQVENCE DESCRIPTION: SEQ ID NO:11: ; ;
Ly~ Tyr Ala Leu Ala A~p Ala Ser Leu ~ . .
1 5 9 ~ .
(2) INFORMP~TION FOR SEQ ID NO:12 ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 nmino ~Icid~
( B ) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ly~ Tyr Ala Leu Ala A~p Ala Ser Leu Ly~ Met Ala Asp Pro A~n Arg Phe Arg Cly Lys A~p L~u Pro Vai Leu A~p Gln 27 ~
(2) INFORMATION FOR SEQ ID NO:13: ~ ;
( i ) SEQUENCE CHMACTERISTICS:
(A) LENGTH: 481 amino ac~ds ( B ) TYPE: amino acid ( D ) TOPOLWY: l inear :
(xi) SEQVENCE DESCRIPTION: SEQ ID NO:13:
Thr Gln Tyr Val Thr Val Phe Tyr Gly Val Pro Thr T~p Lys Asn Ala Thr Ile Pro Leu Phe Cys Ala Thr Arg Asn Arg Asp Thr Trp Gly Thr Ile Gln Cy~ Leu Pro Asp Asn Asp Asp Tyr Gln Glu Ile 55 Thr Leu Asn Val Thr Glu Ala Phe Asp Ala Trp Asn Asn Thr Val ':
,~
WO ')1/1551~ P~/US91/V2166 Thr Glu Cln Ala Ile Glu Asp Val Trp Hi~ Leu Phe Glu Thr Ser I1Q Ly~ Pro Cy~ Val Ly~ Leu Thr Pro Leu Cy8 Val Ala M~t L~
Cys Ser Ser Thr Glu sQr Ser Thr Cly Asn A~n Thr Thr Ser Ly~
10Ser Thr Ser Thr Thr Thr Thr Thr Pro Thr A3p Gln Glu Gln Glu 110 llS 120 Ile Ser Clu A~p Thr Pro Cy~ Ala Arg Ala A3p A~n Cy~ 5er Gly Leu Gly Glu Glu Glu Thr Ile Asn Cy~ Cln Phe Asn 2let Thr G,ly Leu Glu Arg Asp Lys LYR Lys Gln Tyr Asn Glu Thr Trp Tyr S6!r Ly~ A~p Val Val Cy~ Glu $hr A~n Asn Ser Thr Asn Gln Thr Gln 170 175 lB0 25Cys Tyr Mat Asn His Cy~ AEln Thr Ser Val Ile Thr Glu Ser Cy~
Asp Lyn Hi~ Tyr Trp Asp Ala Ile Arg Phe Arg Tyr Cy~ Ala Pro Pro Gly Tyr Ala Leu Leu Arg Cy8 Asn Asp Thr Asn Tyr Ser Gly Phe Ala Pro Asn CYB S~r Lys Val Val Ala Ser Thr Cys Thr Arg Met Met Glu Thr Gln Thr Ser Thr Trp Phe Gly Phe Asn Gly Thr 40Arg Ala Glu Asn Arg Thr Tyr Ile Tyr Trp Hi3 Gly Arg Asp Asn Arg Thr Ile Ile Ser Leu Asn Lys Tyr Tyr A~n Leu Ser Leu Hi~
Cys Lyu Arg Pro Gly A~n Ly~ Ile V~ll Ly~ Gln Ile Met Leu Met Ser Gly His Val Phe His Ser Hil3 Gln Pro Ile Asn Lys Arg Pro Arg Gln Ala Trp CYB Trp Phe Lys Gly Lys Trp Lys Asp Ala ~let 55Gln Glu Val Lys Glu Thr Leu Ala LYB His Pro Arg Tyr Arg Gly W ~ ~ItlS~12 PCTtUS91/~21~6 -61~ 5 Thr A~n A Jp Th~ Arg Asn Il~ ser Phe Aln Ala Pro Gly Ly~ Gly Ser A~p Pro GIu Val Ala Tyr Met Trp Thr A~n Cy~ Arg Gly Glu Ph~ Leu Tyr Cy~ A~n Met Thr Trp Ph~ Leu Asn Trp Ile Glu Aan 3~0 385 390 10Ly~ Thr ~ia Arg A~n Tyr Ala Pro Cyn ~l~ Ile Ly~ Gln Ile Ile Asn Shr Trp Hi~ Ly~ Val Gly ~rg A~n VA1 Tyr L~u Pro Pro Arg Glu Gly Glu Leu Ser Cy~ Asn Ser ~hr Val Thr Ser Ile Ile Ala A~n Ile Asp Trp Gln Aan A~n A~n Gln Thr Asn Ile Thr Phe Ser Ala Glu Val Ala Glu Leu Tyr Arg Leu Glu Leu Gly Asp Tyr Ly~
4~5 460 465 25 L~u Val Glu Ile Thr Pro Ile Gly Phe Ala Pro Thr Ly~ Glu LYB
Arg (2) INFORMATION FOR SEQ ID NO~14:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear txi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Gln Ala HiD Cy A~n Ile Ser Arg l 5 B
~2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids ~) TYPE: amino acid ~D) TOPOLOGY: linear .,.
~0(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Cy~ Asn Asn Ly~
l 4 58 `
......... . . . . . . . . .. . . .
. . . ~ . : . : , , ~
Claims (17)
1. An isolated cyclized polypeptide sequence comprising the amino acid residues selected from the group consisting of:
a) C V K L T P L C C N T S V I T Q A C [SEQ. ID NO.1] and containing less than about 28 amino acid residues;
b) P I H Y C A P A G F A I L K C N N K T F N G T G P C T N V S T V Q C T H G
I R P [SEQ. ID NO. 2] and containing less than about 45 amino acid residues;
c) C N N K T F N G T G P C [SEQ. ID NO. 3] and containing less than about 22 amino acid residues;
d) C A P A G F A I L K C C T N V S T V Q C [SEQ. ID NO.4] and containing less than about 30 amino acid residues;
e) P I H Y C C T H G I R P [SEQ. ID NO. 5] and containing less than about 22 amino acid residues;
f) G G D P E I V T H S F N C G G E F F Y C N S L P C R I K Q F I N M W Q E V G
K A M Y A P P I S G Q I R C S S N I T G [SEQ. ID NO. 6] and containing less than about 65 amino acid residues;
g) C G G E F F Y C C R I K Q F I N M W Q E V G K A M Y A P P I S G Q I R C
[SEQ. ID NO. 7] and containing less than about 45 amino acid residues;
h) C A S D A K A Y D T E V H N V W A T H A C [SEQ. ID NO. 8] and containing less than about 30 amino acid residues; and i) T T T L F C A S D A K A Y D T E V H N V W A T H A C V P T D P N [SEQ. ID
NO. 9] and containing less than about 50 amino acid residues.
a) C V K L T P L C C N T S V I T Q A C [SEQ. ID NO.1] and containing less than about 28 amino acid residues;
b) P I H Y C A P A G F A I L K C N N K T F N G T G P C T N V S T V Q C T H G
I R P [SEQ. ID NO. 2] and containing less than about 45 amino acid residues;
c) C N N K T F N G T G P C [SEQ. ID NO. 3] and containing less than about 22 amino acid residues;
d) C A P A G F A I L K C C T N V S T V Q C [SEQ. ID NO.4] and containing less than about 30 amino acid residues;
e) P I H Y C C T H G I R P [SEQ. ID NO. 5] and containing less than about 22 amino acid residues;
f) G G D P E I V T H S F N C G G E F F Y C N S L P C R I K Q F I N M W Q E V G
K A M Y A P P I S G Q I R C S S N I T G [SEQ. ID NO. 6] and containing less than about 65 amino acid residues;
g) C G G E F F Y C C R I K Q F I N M W Q E V G K A M Y A P P I S G Q I R C
[SEQ. ID NO. 7] and containing less than about 45 amino acid residues;
h) C A S D A K A Y D T E V H N V W A T H A C [SEQ. ID NO. 8] and containing less than about 30 amino acid residues; and i) T T T L F C A S D A K A Y D T E V H N V W A T H A C V P T D P N [SEQ. ID
NO. 9] and containing less than about 50 amino acid residues.
2. A method for the prophylaxis or treatment of HIV infection comprising administering a therapeutically effective dose of a sterile composition comprising the cyclized peptide of claim 1 and an pharmaceutically acceptable vehicle to a patient having or at risk of having HIV infection.
3. The method of claim 2 wherein the therapeutic dose is about from 0.5 x 104 to 5 x 104 molar.
4. The method of claim 2 wherein the composition further contains an adjuvant.
5. An antibody which is directed to an antigenic determinant comprised by the isolated cyclized polypeptide of claim 1.
6. The antibody of claim 5 which is conjugated to a cytotoxin.
7. The antibody of claim 5 which is covalently bound to a detectable marker or a water-insoluble matrix.
8. The antibody of claim 5 in a sterile pharmaceutically acceptable vehicle.
9. An isolated polypeptide having an antigenic determinant or determinants immunologically cross-reactive with a determinant of an HIV env polypeptide having an amino acid sequence selected from the group consisting of a) residues 1-80;
b) residues 8- 180;
c) residues 165-260;
d) residues 160-260;
e) residues 260-310; and f) residues 320-479.
b) residues 8- 180;
c) residues 165-260;
d) residues 160-260;
e) residues 260-310; and f) residues 320-479.
10. An antibody directed to an isolated polypeptide having an antigenic determinant or determinants immunologically cross-reactive with a determinant of the HIV env polypeptide of strain HTLV-IIIB having an amino acid sequence selected from the group consisting of:
a) residues 1-80;
b) residues 8-180;
c) residues 165-250;
d) residues 160-260;
e) residues 260-310: and f) residues 320-479.
a) residues 1-80;
b) residues 8-180;
c) residues 165-250;
d) residues 160-260;
e) residues 260-310: and f) residues 320-479.
11. The antibody of claim 10 which is conjugated to a cytotoxin.
12. The antibody of claim 10 which is covalently bound to a detectable marker or a water-insoluble matrix.
13. The antibody of claim 10 in a sterile, pharmaceutically acceptable vehicle.
14. A method for the prophylaxis or treatment of HIV infection comprising administering a therapeutically effective dose of a sterile composition comprising the antibody of claim 5 and an pharmaceutically acceptable vehicle to a patient having or at risk of having HIV infection.
15. The method of claim 14, wherein said antibody is conjugated to a cytotoxin.
16. A method for the prophylaxis or treatment of HIV infection comprising administering a therapeutically effective dose of a sterile composition comprising the antibody of claim 10 and an pharmaceutically acceptable vehicle to a patient having or at risk of having HIV infection.
17. The method o{ claim 16, wherein said antibody is conjugated to a cytotoxin.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US50477290A | 1990-04-03 | 1990-04-03 | |
US07/504,772 | 1990-04-03 |
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CA2078545A1 true CA2078545A1 (en) | 1991-10-04 |
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ID=24007676
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CA002078545A Abandoned CA2078545A1 (en) | 1990-04-03 | 1991-04-01 | Hiv envelope polypeptides |
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EP (1) | EP0527789A1 (en) |
JP (1) | JPH05506221A (en) |
AU (1) | AU7676891A (en) |
CA (1) | CA2078545A1 (en) |
WO (1) | WO1991015512A2 (en) |
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ES2194836T3 (en) * | 1990-09-27 | 2003-12-01 | Tripep Ab | PEPTIDES FOR USE IN VACCINATION AND INDUCTION OF NEUTRALIZING ANTIBODIES AGAINST THE VIRUS OF HUMAN IMMUNODEFICIENCY. |
DK0601108T3 (en) * | 1991-08-29 | 2000-04-17 | Us Gov Health & Human Serv | Multideterminant peptide antigens that stimulate T helper lymphocyte response to HIV in a group of people |
US5869624A (en) * | 1993-03-26 | 1999-02-09 | Progenics Pharmaceuticals, Inc. | HIV-1 vaccines, antibody compositions related thereto, and therapeutic and prophylactic uses thereof |
CA2164505A1 (en) * | 1993-06-07 | 1994-12-22 | Phillip W. Berman | Hiv envelope polypeptides |
DE4405810A1 (en) | 1994-02-23 | 1995-08-24 | Behringwerke Ag | Peptides derived from a retrovirus from the HIV group and their use |
US6585979B1 (en) | 1996-07-08 | 2003-07-01 | Genentech, Inc. | HIV envelope polypeptides and immunogenic composition |
ZA975889B (en) * | 1996-07-08 | 1998-02-23 | Genentech Inc | HIV envelope polypeptides and vaccine. |
GB9615533D0 (en) * | 1996-07-24 | 1996-09-04 | Peptide Therapeutics Ltd | Diagnostic test for herpes simplex virus type-2 |
ATE355300T1 (en) * | 1996-11-06 | 2006-03-15 | Genentech Inc | STRAINED, HELIX-FORMING PEPTIDES AND PROCESSES FOR PRODUCING THEM |
US6271198B1 (en) | 1996-11-06 | 2001-08-07 | Genentech, Inc. | Constrained helical peptides and methods of making same |
PT1141315E (en) | 1998-12-31 | 2008-05-05 | Novartis Vaccines & Diagnostic | Modified hiv env polypeptides |
CA2358385C (en) | 1998-12-31 | 2013-08-06 | Chiron Corporation | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
EP1411770A4 (en) | 2001-07-05 | 2006-05-10 | Chiron Corp | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
US20030170614A1 (en) | 2001-08-31 | 2003-09-11 | Megede Jan Zur | Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof |
WO2011008863A2 (en) * | 2009-07-14 | 2011-01-20 | Lucia Irene Gonzalez | Stereoisomer peptides and their polymer conjugates for hiv disease |
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ATE151638T1 (en) * | 1987-02-20 | 1997-05-15 | Genentech Inc | METHODS AND PREPARATIONS FOR USE OF HIV ENV POLYPEPTIDES AND ANTIBODIES |
AU2914889A (en) * | 1987-08-28 | 1989-04-17 | Board Of Regents, The University Of Texas System | Prophylaxis and therapy of acquired immunodeficiency syndrome |
SE8704185L (en) * | 1987-10-28 | 1989-04-29 | Ferring Ab | NEW PEPTIDES, ARTIFICIAL ANTIGENS AND IMMUNO ANALYSIS TEST RATES |
WO1989005821A1 (en) * | 1987-12-21 | 1989-06-29 | Arch Development Corporation | Hiv-related antigens and antibodies |
IL88963A (en) * | 1988-01-27 | 1996-05-14 | Iaf Biochem Int | Cyclic synthetic peptides and mixtures thereof for detecting hiv antibodies and producing vaccines |
CA1341285C (en) * | 1988-02-12 | 2001-08-14 | Chang Yi Wang | Synthetic peptides for the detection of antibodies to hiv gp120 envelope protein for diagnosis of aids and pre-aids conditions and as vaccines |
-
1991
- 1991-04-01 CA CA002078545A patent/CA2078545A1/en not_active Abandoned
- 1991-04-01 WO PCT/US1991/002166 patent/WO1991015512A2/en not_active Application Discontinuation
- 1991-04-01 EP EP91908106A patent/EP0527789A1/en not_active Withdrawn
- 1991-04-01 JP JP91507619A patent/JPH05506221A/en active Pending
- 1991-04-01 AU AU76768/91A patent/AU7676891A/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
EP0527789A1 (en) | 1993-02-24 |
JPH05506221A (en) | 1993-09-16 |
WO1991015512A3 (en) | 1991-12-12 |
WO1991015512A2 (en) | 1991-10-17 |
AU7676891A (en) | 1991-10-30 |
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Date | Code | Title | Description |
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FZDE | Discontinued |