Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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
2-
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.
WO 91/15512 PCI/US91/02166
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
-4 -
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 .
~' ~
1/15512 PCI/US91/02166
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~ -
8-
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 ~
-13-
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
-14-
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
-15
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
-17-
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
1 8-
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
-21 -
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
22-
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'
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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
-26-
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
-28-
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
-30-
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.
.. - .: : .
:- . .. . .. . .. : ., : .
WO91/15~12 ~ ~, PCI/US91/02~66
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WO 91/15512 PCI/US91/02166
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wo 91/15~12 PCIJUS91/02166
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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
-38-
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
209 21 7
_ _ .
D QSSGGDPEIVTHSFN~GGEFFY~lNSTQLFNSTWFNSTWSTE-
-GSNNTEGSDTITLP R ~ _ ~ (T31)
388 E~SSNITGLLLTR
415
_ :
24 ~Tt)
E EATTTLF~3ASDAK
AYDTEVHNVWATHA ~3VPTDPNPaEVVLVNVTENFNMWK
ITl2) 188
VSFEPIPIHY~APAGFAILK 198 (T13)
/ ~ _ ~ _
TFhGTGP~ITNVSTVQ~3THGlRPVVsTQLLLNGsLAErrVVlR
209 217
-- . :
NDMVEQMHEDllSLWDaSLKP~VK ~ (T4)
LDllplDNDTTsyTLTs~3NTsvlTQA~3pK -.
WO 91/15~12 ~ 3~ ~ PCI/U!~9t/02166
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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
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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 ~ :
209
~:
188
2EPIPIHY~}APAGF :~
DVSTVQ1~3THG( I R)
217
89
. 3 DQSLKP~lVK ;:
DTSVITQA [~1P K
175
.
4 LTPL~VSLK
DDrrSYTLTS
166
348
IVTHSFN~GGE
I~SSNITGLLLTR
41
355
6FFY~NSTQLFNSTWFNSTWSTE
TITLPL~IR ~:
388
''
WO 91/1~512 ~ PCT/US91/02166
42-
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
44-
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
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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
-46-
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
-47
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
.. , . : . , ~. . .. . - ., , , ~ ,
, . . .. . . . . , ,., , . , ~ .; . ... .... . .
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-~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
-49-
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.
REFERENCES
Allan, J.S., Coli~an, J.E., Barin, F., McLane, M.F., Sodroski, J.G., Rosen, C.A., Haseltine,
W.A., Lee, T.H. and Essex, M. 11985). Sctence 228, 1091-1094.
Arthur, LØ, Pyle, S.W., Nara, P.L., Bess, J.W. Jr., Gonda, M.A., Kelliher, J.C., Gilden, R.V.,
20 Robey, W.G., Bolognesi, D.P., Gallo, R.C. and Fischin~er, P.J. l1987). Proc. Natl. Acad. Sci.
USA 84, 8583-8587.
Barre-Sinoussi, F., Chermann, J.C., Rey, F., Nu~eyre, M.T., Chamaret, S., Gruest, J.,
Dau~uet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Monta~nier,
L. 11983). Science 220, 868-871.
25 Berman, P.W., Groopman, J.E., Gregory, T.J., Weiss, R.A., Clapham, P.R., Ferriani, R.,
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.: . . . . . . .. : ...
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5~-
Fenouillet, E., Cl~rge~-Raslain, B., Gluckman, J.C., Guetard, D., Monta~nlet, L. and Bahraoui,
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.. ' "' ''' ' . . : ' :
WO 91/tS512 ~ PCr/US91/02166
54-
~ 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
1 5 10 15
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~
1 5 10 15
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
1 S 10 12
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
1 5 10 15
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
~
-56-
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
1 5 10 15
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
35 40 45
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~
36
(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
1 5 10 15
Trp Ala T~r Hl~ Ala Cy~
21
~2) INFORMATION FOR SEQ ID NO:9:
.-.
W O 91/1~512 PCT/USg1/02166
57 V
(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 - .
1532
(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
50 55 60
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
4595 100 105
A~n A~p Thr A~n Thr A~n Ser Ser Ser Gly Arg Met Ile Me'c Glu
110 115 120
50Ly~ Gly Glu Ile Ly~ Asn Cy3 Ser Phe A~n Ile Ser Thr Ser Ile ~:
125 130 135
Arg Gly Ly~ Val Gln Lys Glu Tyr Ala Phe Phe Tyr Ly~ Leu Asp
140 145 150
Ile Ile Pro Ile A-p A-n Asp Thr Thr Ser Tyr Thr Leu Thr Ser
~,
.
WO ~5~12,. '~ ~', PCI/US9t/0~166
58-
155 160 165
Cy~ A~n Thr Ser Val Ile Thr Gln Ala Cy~ Pro Ly~ Val Ser Phe
170 175 180
G}u Pro Ile Pro Ile Hi~ Tyr Cy~ Ala Pro Ala Gly Phe Ala Ile
185 190 195
Leu Ly~ CYB Aan A~n LyE~ Thr Phe A~n Gly ~hr Gly Pro Cyl3 Thr
10200 205 210
Asn Val Ser Thr Val Cln Cy~ Thr Hi~ Cly Il~ Arg Pro Val Val
215 220 225
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
245 250 255
Val Gln Leu Asn Gln Ser Val Glu Ile A~n Cys Thr Arg Pxo A~n
260 265 270
A~n Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg
25275 280 285
Ala Phe Val Thr Ile Gly Ly~ Ile Gly A~n Met Arg Gln Ala Hi~
290 295 300
30Cy~ Asn Ile Ser Arg Ala Ly~ Trp Asn Asn Thr L~u Lys Gln Ile
305 310 315
Asp Ser Lya Leu Arg Glu Gln Phe Gly Asn Asn Ly~ Thr Ile Ile
320 325 330
Phe LYB Gln Ser Ser Gly Gly A~p Pro Glu Ile Val Thr Hi~ Ser
335 340 345
Phe Asn Cy~ Gly Gly Glu Phe Phe Tyr Cy~ Asn Ser Thr Gln Leu
40350 355 360
Phe Asn Ser Thr Trp Phe A~n Ser Thr Trp ser Thr Glu Gly Ser
365 370 375
45Asn Asn Thr Glu Gly Ser Asp Thr Ile Thr Leu Pro Cy~ Arg Ile
380 385 390
LYB Gln Phe Ile Asn Met Trp Gln Glu Val Gly Ly~ Ala Het Tyr
395 400 405
Ala Pro Pro Ile Ser Gly Gln Ile Arg Cy~ Ser Ser Asn Ile Thr
410 415 420
Gly Leu Leu Leu Thr Arq A~p Gly Gly A~n A~n A~n Asn Clu Ser
55425 430 435
~,
.: ,
,"" "" ~
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
440 445 450
Ser Clu Leu Tyr Ly~ Tyr Ly0 Val Val Ly~ Ile Glu Pro Leu Gly ~;
455 460 465
Val Ala Pro Thr Ly~ Ala Ly~ Arg Arg Val Val Cln Arg Glu
470 475 479
( 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
5 10 15
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
5 10 15
Ala Thr Ile Pro Leu Phe Cys Ala Thr Arg Asn Arg Asp Thr Trp
50 20 25 30
Gly Thr Ile Gln Cy~ Leu Pro Asp Asn Asp Asp Tyr Gln Glu Ile
3S 40 45
55 Thr Leu Asn Val Thr Glu Ala Phe Asp Ala Trp Asn Asn Thr Val
50 5 5 60
':
,~
WO ')1/1551~ P~/US91/V2166
60-
Thr Glu Cln Ala Ile Glu Asp Val Trp Hi~ Leu Phe Glu Thr Ser
65 70 75
I1Q Ly~ Pro Cy~ Val Ly~ Leu Thr Pro Leu Cy8 Val Ala M~t L~
580 85 90
Cys Ser Ser Thr Glu sQr Ser Thr Cly Asn A~n Thr Thr Ser Ly~
100 105
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
125 130 135
Leu Gly Glu Glu Glu Thr Ile Asn Cy~ Cln Phe Asn 2let Thr G,ly
140 145 150
Leu Glu Arg Asp Lys LYR Lys Gln Tyr Asn Glu Thr Trp Tyr S6!r
20155 160 165
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~
185 190 195
Asp Lyn Hi~ Tyr Trp Asp Ala Ile Arg Phe Arg Tyr Cy~ Ala Pro
200 205 210
Pro Gly Tyr Ala Leu Leu Arg Cy8 Asn Asp Thr Asn Tyr Ser Gly
215 220 225
Phe Ala Pro Asn CYB S~r Lys Val Val Ala Ser Thr Cys Thr Arg
35230 235 240
Met Met Glu Thr Gln Thr Ser Thr Trp Phe Gly Phe Asn Gly Thr
245 250 255
40Arg Ala Glu Asn Arg Thr Tyr Ile Tyr Trp Hi3 Gly Arg Asp Asn
260 265 270
Arg Thr Ile Ile Ser Leu Asn Lys Tyr Tyr A~n Leu Ser Leu Hi~
275 280 285
Cys Lyu Arg Pro Gly A~n Ly~ Ile V~ll Ly~ Gln Ile Met Leu Met
290 295 300
Ser Gly His Val Phe His Ser Hil3 Gln Pro Ile Asn Lys Arg Pro
50305 310 315
Arg Gln Ala Trp CYB Trp Phe Lys Gly Lys Trp Lys Asp Ala ~let
320 325 330
55Gln Glu Val Lys Glu Thr Leu Ala LYB His Pro Arg Tyr Arg Gly
335 340 345
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
350 355 360
Ser A~p Pro GIu Val Ala Tyr Met Trp Thr A~n Cy~ Arg Gly Glu
5365 370 375
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
395 400 405
Asn Shr Trp Hi~ Ly~ Val Gly ~rg A~n VA1 Tyr L~u Pro Pro Arg
410 415 420
Glu Gly Glu Leu Ser Cy~ Asn Ser ~hr Val Thr Ser Ile Ile Ala
425 430 435
A~n Ile Asp Trp Gln Aan A~n A~n Gln Thr Asn Ile Thr Phe Ser
20440 445 450
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
470 475 480
Arg
481
(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 `
......... . . . . . . . . .. . . .
. . . ~ . : . : ,
, ~
