Note: Descriptions are shown in the official language in which they were submitted.
3 j~ 'J ~
~90~09805 PCT/US90/01132
~UMAN MONO~QNA~ ANTI~ODIES TO HUMAN ~MMUNODEFICIENCY VIRUS
BACKGROy~D OF~ INVENTION
The goverr~ent has rights to this invention by virtue
of funding from grant AI-72658 from the National Institutes of
~ealth.
The human immunodeficiency virus (aIv) has been
implicated as the causative agent of acquired immune deficiency
syndrome (AIDS). Two different serotype~ of the virus have
been identified to date: ~IV-l and ~IV-2. It i~ currently
believed that the majority of individuals that become infected
with ~IV eventually will develop AIDS and are likely to succumb
to fatal infections and/or malignancies. At this time it is
estimated that approxi~tely l.5 million individuals have been
infected by ~IV in the United States alone~
Several avenues have been explored to treat individu~
afflicted with AIDS or ~IV infections. The antiviral ~t ~
azidothymidine (AZT) has been found to produce both clinic.ll
and immunological improvements upon short term administratio
to patients afflicted with AIDS and ARC (AIDS Related Comp1e~-
a prodrome of the disease) and iecrease the mortality r~
and frequency of opportunistic ~ctions. Although clinical
benefitfi are achieved with A it is costly. A furtheL
d~8~b~ that s~ifica~t drug toxi~ity oft~n accompanies
administration of AZT. This may necessitate blood transfusions
and/or reduction of the AZT dosage, or in some instances,
discontinuance of AZT therapy altogether. Nonetheless, AZT is
'O90/09805 PCTtUS90/01l32
the only drug currently authorized for the treatment of AIDS.
An alternative treatment that is currently under
evaluation involves a~inistration of one or more lymphokines~
Interferon (particularly gamma-interferons) and interleukin-2
are currently being studied for possible use in the treatment
of HIV infections. ~owever, the prelimlnary results of early
clinical trials are not promising. Patients receiving lym-
phokine therapy often suffer serious side effects including low
blood pressure, nausea and diarrhea.
It has been proposed to use monoclonal antibodies of
defined specificities directed against ~IV proteins expressed
in infected ind~viduals as therapeutic agents. These proteins
are part of the virus particles and are expressed by ~IV
infected cells and are designated inter alia as p24 and gp41.
The identification and isolation of gp41 is described in ~.S.
Patent No. 4,725,66~ of M. Essex, issued February 16, 1988 as
is its use in the treatment and diagnosis of AIDS. The
identification of p24 has been described in Allan, J.S. et al.,
Science 228: 1091, 1985. However, the use of monoclonal
antibodies for the treatment of ~IY infections has been
hampered because only a limited number of murine and rat
monoclonal antibodies to ~IV proteins are available. In
addition, none of the currently available monoclonal antibodies
directed against ~IV proteins are of human origin. Administra-
tion of murine antibodies to humans can cause dangerous lifethreatening immunologic reactions, and such antibodies may not
be effective in binding to the target ~IV proteins in humans.
Stable hum~n cell lines which produce monoclonal
antibodies directed against ~IV would be useful for treating
and/or diagnosing individuals infected with the virus.
~owever, human monoclonal antibodies and particularly those
directed against ~IV have proven to be far more difficult to
produce than those of either rat or mouse origin. Amongst the
explanations for this problem are: (a) the most available
source of lymphocytes from humans, the peripheral blood,
normally contains few antibody producing cells and in some
instances, none at all; (b) transformation of antibody produc-
'090/09805 PCT/US90/01132
ing cells can be achieved using Epstein-Barr virus (E~V), but
the level of antibody production hy these transformed cells is
often low and unstable; ~c) stability of antibody production
can be enhanced, as can levels of antibody production, by
fusion of E~V-transformed lines to mouse myeloma cells but,
these 'heterohybridomas' readily delete human chromosomes and
immunoglobulin production i6 often lost; and (d) fusion of
normal or transformed B cell5 to human lymphoblastoid lines or
to heteromyelomas stabilizes antibody production but, until
recently, few satisfactory parent lines of this cell type have
been available.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide
human lymphoblastoid cell lines producing monoclonal antibodies
directed against protein components of HIV.
Another object of the present invention is to provide
diagnostic and therapeutic agents comprising human monoclonal
antibodies directed against ~IV proteins which have a low non-
specific toxicity for use in the diagnosis and treatment of in-
dividuals infected with ~IV.
A further object of the present invention is to provide
a method for treating individuals uffering from ~IV infections
by administering human monoclonal antibodies directed against
protein components of ~IV to said individual~
A still further object of the present invention is to
provide pharmaceutical formulations for treating individuals
suffering from ~IV infections.
These and other objects of the present invention will
be apparent to those of ordinary skill in the art in light of
the following specification.
SUMMARY OF THE INVENTION
The present inventors have discovered new monoclonal
antibodies for the treatment, prophylaxis and diagnosis of
human immunodeficiency virus (~IV) infections. These are human
monoclonal antibodies directed against ~IV proteins gp4l and
6~ . d -- ~ ~
`'O90/09805 PCT/US90/01132
p24 which are expressed by infected cells. The human monoclo-
nal antibodies of the present in~ention may be used as diagnos-
tic agents, directly as therapeutic agents, as the basis for
vaccines or to form conjugates by covalent coupling with
cytotoxic agents, specific anti-~IV drugs or radionuclides (the
antibody/toxin conjugates are alternatively referred to herein
as immunotoxins) for use in the diagnosis and treatment of in-
dividuals that have been exposed to or infected with HIV. The
present invention provides stable human lymphoblastoid cell
lines which secrete human monoclonal antibodies directed
against ~IV proteins gp4l and p24. The invention also provides
human monoclonal antibodies directed against ~IV proteins p24
and gp4l.
Another aspect of the present invention comprises a
method for treating a mammal infected with HIV comprising
administering to a mammal in need of such treatment an effec-
tive amount of a human monoclonal antibody directed against
HIV.
In a further aspect, the present invention comprises
pharmaceutical f ormulations comprising an effective amount of a
human monoclonal antibody to ~IV proteins.
~RIEF_DESCRIPTION OF TBE DRAWINGS
Figure 1 is a radio-immunoprecipitation assay of
l25tI]-labelled ~IV lysate with serum from an HIV-infected
subject or with antibodies from a sub~et of the human
monoclonal antibody producing cell lines of the present
invention.
Figure 2 is a Western blot analysis of human sera and
an anti-p24 monoclonal antibody of the present invention.
Figure 3 (a-g) are graphs of the growth kinetics and
immunoglobulin production of a subset of the human lymphoblas-
toid cell lines of the present invention which produce
monoclonal antibodies directed against ~IV.
Figure 4 is a graph showing the inhibition testing of
monoclonal antibodies 120-l6 and 71-3l.
~090J09X~5 PCT/USgO/01132
DETAILED DESCRIPTION OF TH~ INVENT~ON
All literature references and patents cited in this
specification are hereby incorporated by reference in their
entirety.
The pre6ent inventor~ have isolated fourteen novel
stable human lymphoblastoid cell lines producing monoclonal
antibodies directed against ~IV encoded proteins. The an-
tibodies of the invention are useful in the prophylaxis of ~IV
and in the diagnosis, and treatment of humans suffering from
~IV infections. These human monoclonal antibodie~ are directed
against ~IV protein gp41 and protein p24 (and the precursors
and decomposition products of such E~roteins).
gp41 is a viral membrane glycoprotein expressed on the
surface of infected cells and is a product of the env gene of
~IV (as described in E~sex, M., U.S. Patent No. 4,725,669
i~sued February 16, 1988). p24 is a viral core protein and is
a product of the HIV aag gene (as described in Allan, J.S. et
al., supra~).
The human monoclonal antibodies of the present inven-
tion may be employed as the antibody component in the conven-
tional diagnostic assays of the type used to determine if a
patient has been exposed to, or infected with, ~IV. Example 6
below illustrate~ the use of the antibodies of the invention in
a diagnostie as~ay. Admini~tered to human~, the antibodies can
provide passive immunization of HIV-infected individuals. In
addition, the antibodies of the invention can serve prophylac-
tically for administration to non-infected, high-risk in-
dividuals tsuch as health care workers who have been exposed
via a needle stick to ~IV). The anti~odies of the invention
also can serve as research tools for epitope mapping of ~IV
proteins gp41 and p24.
A particularly important use of the human monoclonal
antibodies of the present invention is for administration to
~IV infected expectant mothers. All of the antibodies of the
present invention are of the IgG serotype (see below)-. Since
IgG's can pass through the placenta and reach the fetus in
utero, passive a~;nistration of the antibodies of the present
~ ~ 3 ~
-`'090~09805 PCT/US90/01t32
invention to ~IV-infected pregnant women would provide effec-
tive therapy for the fetus.
The human monoclonal antibodies may be conjugated to
cytotoxic agents and used as immunotoxins (as described in
Vitetta, E.S. et al., Science 238: 1098-1104, 1987) or incor-
porated onto the surface of liposomes containing anti-~IV drugs
or toxins to specifically target such drugs or toxins to
infected cells. As employed herein the term "immunotoxin
refers to a conjugate of an antibody with one or more toxins,
drugs, radionuclides or cytotoxic agents. Among the cytotoxic
agents that may be conjugated to the antibodies of the present
invention are ricin, diphtheria toxin and radionuclides. ~icin
is an extremely po-tent toxin produced by the beans of the plant
Ricinus communis. In a typical treatment employing the human
monoclonal antibodies of the present invention as immunotoxins,
the antibody (which binds to a protein that is expressed by
HIV-infected cells) is conjugated to a toxin (e.g. ricin) that
is toxic to the ~IV-infected cell (and $o non-infected cells as
well). By coupling the cytotoxic agent to the antibody, a high
level of toxic efficacy can be achieved against the target cell
with a markedly lower level of non-specific toxicity. The use
of the toxic agent is possible because the human monoclonal
antibodies to which the agent is coupled will carry the agent
directly to the target (in this case, HIV-infected cells),
thereby sparing non-infected cells from the toxin. Techniques
that may be employed to conjugate human monoclonal antibodies,
including those of the present invention, to cytotoxic agents
are described in detail in Vitetta et al., supra and in
European Patent Application Serial No. 279,668, published
August 24, 1988 of Genentech, Inc
The human lymphoblastoid cell lines (which produce the
monoclonal antibodies of the present invention) were formed by
immortalizing lymphocytes obtained from ~IV-seropositive
patients by infecting such lymphocytes with Epstein Barr Virus
(EBV) in vitro. Initially, blood was obtained from 58 ~IV-
seropositive individuals, peripheral blood mononuclear cells
were obtained and incubated overnight with EBV. The EBV
O90/09805 PCT/US90/01l32
infected cells were cultured at 80,Q00 cells per well in
microtiter wells for 3-4 weeks and assayed for anti-~IV
antibody production using a non-commercial ELISA (see below)
and a commercial ELISA employing HIV-coated beads. The
specificity of each positive reaction obtained by the ELISA was
confirmed by te6ting for their non-reactivity on identical
beads coated with bovine serum albumin (BSA).
Approximately 9% of the lymphoblastoid cell cultures
te~ted positively in the non-commercial ~LISA. After expansion
the positive well6 were cultured for two more weeks. It was
found that 2.4~ tested positively for ~IV proteins by ELISA and
0.67% proved to be specific for HIV by virtue of their non-
reactivity on the BSA beads. The anti-~IV antibodies produced
were found to be directed again~t gp41 or p24 and had suffi-
cient avidity to show reactivity by ELISA, Western blot, radio-
immunoprecipitation and~or i~munofluorescence. Therefore/ all
of these monoclonal antibodies would be useful in diagnostic
assays for HIV. The stable clones were then ~ubcultured 1 to 2
times at 10 or 100 cells per well with irradiated human
lymphoblastoid feeder cells and expanded into tissue culture
flasks.
In a ~econd round of immortalization/selection,
peripheral blood mononuclear cells from another 36 HIV-
seropositive individuals were obtained, immortalized by EBV
infection and as~ayed for anti-~IV production as above. Four
stable lymphoblastoid cell lines were obtained producing
monoclonal antibodies: 120-16, 126-6, 126-50, 167-7 and 191-3
against gp41, and 134-F6 against p24.
In addition, one cell line, 98-4.3, producing
monoclonal antibodies against p24, was obtained from positive
cultures derived from the first group of 58 patients de~cribed
above. The culture was subcultured twice after several
unsuccessful trials and is presently stable in culture.
The characteristics of the human monoclonal antibodies
produced by the lymphoblastoid cell lines of the present
invention are described in Table III of Example 5 below.
The present inventors have performed some epitope
~O90/09805 PCT/US90/01l32
mapping of the human monoclonal antibodies of the present
invention. For example, it can be seen from the data presented
in Example 5, Table III below that monoclonal antibodies 50-69
and 98-43 bind to the same epitope cluster (i.e. amino acids
falling between residues 579 and 613) Whereas 98-6 binds to to
a different re~ion (amino acids falling between residues 642
and 692~. ~owever, monoclonal antibodies 50-69 and 98-q3
differ in their epitope ~pecificity a5 demonstrated by the fact
that 50-69 binds to peptide 599-613 whereas 98-43 binds to
pepticle 579-604. Three of the anti-p24 antibodies have been
tested (i.e. 71-31, 91-5 and 91-6). All bind to the same
region of p24 (131-198).
All of the human monoclonal antibodies of the present
invention directed against gp41 (i.e. 50-69, 98-6, 98-43, 120-
16, 126-6, 126-50, 167-7 and 191-3) mediated antibody dependent
cellular cytotoxicity (ADCC) as shown in Table III below. This
is a most important finding in that serotherapy (e.g. passive
immunization) and seroprophylaxis in animal retroviral models
have been shown to be mediated by ADCC (Plata, F. et al., Cell
48: 231, 1987; Weinhold, K.J. et al, J. Natl. Cancer Inst. 75:
717, 1985). The ADCC immune response is directed by specific
antibodies and involves mobilization of effector cells
(cytotoxic T-cells, monocytes, and killer cells against
speci~ic targets. It i8 believed that the ability to mount an
ADCC response will be important for serotherapy and
seroprophylaxis in HIV infections also.
The fourteen lymphoblastoid cell lines thus obtained
are stable in culture and produce human monoclonal antibodies
directed against targets on BIV which are expressed in vivo in
infected patients.
The human monoclonal antibodies of the present inven-
tion are all of-the IgG serotype and may be recovered from the
~upernatants of monoclonal antibody producing lymphoblastoid
cell cultures and purified by conventional methods known in the
art for the purification of IgG. Such methods include Protein-
A Sepharose chromatography, a combination of Affigel Blue (Bio-
Rad, Richmond, CA) and Protein-A Sepharose chromatography, or
'O90/09805 PCT/US90/01132
~igh Performance Liquid Chromatography (~PLC).
The eleven stable lymphoblastoid cell lines described
in Examples 1-6 below produce human monoclonal antibodies which
are directed against unique epitopes which are expressed in
~IV-infected patients. Although some epitope mapping has been
performed (see Table III in Example 4 below), further epitope
mapping will determine the exact specificity of each of the
monoclonal antibodies and may reveal targets on the ~IV gp41
and p24 protein molecules which can be candidates for vaccine
production.
The human monoclonal antibodies of the present inven-
tion are directed against either immunodominant (common) or
non-dominant epitopes of the gp41 and p24 viral proteins. As
employed in this specification, the term "immunodominant"
refers to an antigenic determinant that most patients respond
by the production of antibodies. Antibodies 50-69 and 120-16,
directed against gp41, are to immunodominant epitopes. These
two antibodies may be employed for passive immunizations and/or
diagnostic reagents. Antibodies 71-31 and 91-5, directed
~0 against p24, are to non-dominant epitopes.
Lymphoblastoid cell lines 91-5 and 120-16 (producing
human monoclonal antibodies directed against p24 and gp41,
respectively) have been deposited with the American Type
Culture (ATCC, Rockville, MD) and have received Accession
Numbers CRL lgO38 and CRL 10037, respectively.
The classification of the antibodies of the invention
into an immunodominant or non-dominant grouping was ac-
complished by inhibition testing as describe~ in Example 6
below. In this assay, sera collected from HIV seropositive
individual~ were used to inhibit the binding of biotinylated
monoclonal antibodies directed against ~IV proteins to their
re~pective antigens.
Of the monocional antibodies to p24 and gp41, only
those to gp41 mediated antibody-dependent cellular cytotoxicity
(ADCC). In vitro cytotoxicity studies revealed that the most
active induced significant levels of killing was at minimum
concentrations of 15-250 ng Ab/ml. ADCC was assayed using
`~090t09805 PCT~US90/01132
peripheral blood mononuclear cells (MNC) as effectors and the
CEM.NKR cell line, infected with ~TLV-IIIB, MN and RF (viral
strains), as targets.
Two monoclonal antibodies to gp4l and one monoclonal
antibody to p24 were purified and coupled to the deglycosylated
A chain of ricin. The anti-p24 immunotoxin (IT) did not kill
infected or uninfected H9 or U937 cells at concentrations up to
ug Ab/ml. IT made with monoclonal antibody to gp4l,
however, reduced protein synthesis in infected ~9 cells by so%
(IC50) at concentrations of 500 ng/ml. The IC50 of anti-gp41
IT for infected U937 cells was lO00 ng/ml. In the presence of
chloroquine, the IC50 of these immunotoxins (ITs) was 5-lO
ng/ml.
When employed to treat individuals infected by HIV or
suffering from AIDS, the human monoclonal antibodies of the
present invention (having a specificity and a binding affinity
for HIV proteins p24 and gp41) may be administered as passive
immunization agents in effective amounts broadly ranging
between about 200 mg and about 15 grams and preferably between
50 mg and l gram. The antibodies o~ the invention are adminis-
tered parenterally, and preferably via the intravenous route.
A typical treatment regimen would comprise administration of an
effective amount of antibody administered over between about
one wee~ and about 6 months. The number of treatments required
to control a patient's disease will vary from individual to
individual, depending upon the severity and stage of the
illness and the individual characteristics of each patient
being treated. The total dose required for each treatment may
be administered by multiple doses or in a single dose. The
human monoclonal antibodies may be administered alone or in
conjunction with other HIV treatment~, such as AZT, in order to
control a patient's disease. The anti-HIV treatment may be
administered one or two times a week or more as determined by
the patient's condition and the stage of the patient's disease.
The human monoclonal antibodies of the present inven-
tion can be incorporated into conventional pharmaceutical
formulations for use in treating individuals that are afflicted
`'090/09805 PCT/US90/01132
11
with ~IV or for prophylaxis in individuals at risk for such
infections. The pharmaceutical formulations of the invention
comprising an anti-~IV effective amount, range between about
200 mg and about 15 grams, of the human monoclonal antibodies
of the present invention identified in Examples 1-4 below. The
quantity of effective dose applied by each injection is
relatively unimportant since the total dosage can be reached by
administration of one or a plurality of injections. In addi-
tion, such formulations may comprise pharmaceutically-accep-
table carriers, diluents, saltS and other materials well-known
in the art. Isotonic saline, sterile water, 10% maltose, human
serum albumin, glycine or other pharmaceutically-acceptable
materials may be u~ed as diluents, carriers or solvents in
preparing the pharmaceutical formulations comprising the human
monoclonal antibodies of the present invention.
The present invention is described below in specific
working examples which are intended to illustrate the invention
without limiting its scope.
EXAMPLE 1: IMMORTALIZATION OF UUMAN ~-CELLS
Blood was obtained from 58 HIV-seropositive individuals
who were intravenous drug users or homosexuals. The presence
of antibody to ~IV in the blood was determined using a commer-
cial enzyme-linked immunosorbent assay (ELISA) (Organon-Teknika
Bio-Enzabead UTLV-III ELISA, Durham, NC) and confirmed by
Western blot (Novapath I~1~unoblot Assay, Bio-Rad, ~ercules, CA
and Biotech/DuPont, DuPont, Wilmington, DE). The disease
status of patients was established on the basis of an im-
munologic staging system as described by Zolla-Pazner et al.
(Proc. Nat. Acad. Sci. USA 84: 5404, 1987):
Scale Score T4/T8 ratio #T~lmm #lymphocytes/mm
0 > 1.0 > 500~ 1500
1 < 1.0 > 500> 1500
2 < 1.0 < 500> 1500
3 < 1.0 < 500< 1500
Peripheral blood mononuclear cells collected from the
58 patients were obtained by centrifugation of heparinized
~O90/09805 PCTtUS90/01132
12
blood, diluted 1:1 with RPMI-1640 and centrifuged on Histopa~ue
(Sigma, St. Louis, MO) at 300 x g for 30 minutes. Cells at the
medium/Histopaque interface were recovered, washed three times
and incubated overnight at a density of 2 x 106 cells/ml with
the filtered supernatant from the EBV-transformed marmoset cell
line B95-8 (Proc. Nat. Acad. Sci. ~a 70: 190, 1973, available
under Accession Number CRL 1612 from the American Type Culture
Collection, ATCC, Rockville, MD). Lymphocytes were then washed
once and cultured in RPMI-1640 medi~m ~M.A. Bioproducts,
Walkersville, MD) supplemented with 10% fetal calf serum
(~yclone Labs, Logan, UT) 2mM L-glutamine, 100U/ml penicillin,
and 100 micrograms/ml streptomycin (complete medium) for four
weeks in 96-well plates (Costar, Cambridge, MA) at 80,000 cells
per well.
EXAMPLE 2: ISOLATION AND SCREENING OF LYMPHOBLASTOID
CELL LINES FOR ANTI-~IV ANTIBODY PRODUCTION
After screening for anti-HIV antibody production by a
non-commercial ELISA (see below), positive cultures were
expanded into wells of 24-well tissue culture plates (Costar)
and cultured for two more weeks. All initial and expanded
cultures were fed at weekly intervals with complete medium.
Cultures with supernatants showing specific reactivity to ~IV
were then subcultured one to two times at doubling dilution on
feeder layers of irradiated GK-5 human lymphoblastoid cells
(derived from a variant of GM1500; Satoh, J. et al., N. Engl.
J. Med. 309: 217, 1983) which had been exposed to 3000 Rads of
gamma-radiation. Stable clones were then subcultured one to
three times at 10 to 100 cells per well on feeder cells and
then expanded into flasks.
Thus, initial cultures of immortalized B-cells
(hereinafter referred to as lymphoblastoid cell lines) were
established and further characterized as described below.
The screening of the initial cultures in 96-well plates
was performed using a non-commercial ELISA. Immulon 2 plates
2 ~
O90/09805 PCTtUS90/01132
13
(Dynatech, Chantilly, VI) were coated overnight at 4C with 4
micrograms/ml of ~TLV-IIIg lysate (purchased from Electro-
Nucleonics, Inc., Silver Spring, MD) diluted in carbonate
buffer, pH 9.8. Plates were washed three times with phosphate
buffered saline, p~ 7.2, containing 0.05% Tween 20 (PBS-Tween).
The culture supernatants to be assayed (0.1 ml per well) were
then added and incubated for 90 minutes at 37C. Subsequently,
plates were washed with PBS-Tween and incubated with goat anti-
human immunoglobulin conjugated to alkaline phosphatase
(Organon Teknika-Cappel, Malvern, PA) for another 90 minutes at
37C. The plates were washed again with PBS-Tween and the
substrate, p-nitrophenyl phosphate in 10% diethanolamine, was
added for 30 minutes. The reaction was terminated with 25
microliters of lN NaO~ and the ab~orbance was read at 405 nm in
an automated ELISA reader (MR 600 Microplate Reader, Dynatech).
The specificity of the antibody bindinq was assessed by
testing the supernatants for reacti~ity against ~IV-coated
beads (Bio-Enza~ead) and against uncoated beads (obtained from
Organon Teknika Cappel~ which were then coated with bovine
serum albumin (BSA, Sigma Chemical Co.) by incubating the beads
in 1.25% BSA diluted in PBS for 1 hour at room temperature.
Reactivity with ~IV-coated beads, but not with BSA-coated
beads, was used as a criterion f or specificity. Further
analysis of the specificity o~ the monoclonal antibodies was
then carried out by Western blot using a commercially-available
kit ~Aio-Rad, Richmond, CA) and by radioimmunoprecipitation
(RIP). RIP assay~ were carried out using the method of Pinter
and ~onnen (J. Immunol. Methods, in press). Briefly, 30
micrograms of ~TLV-IIIB lysate (purchased from Organon-
Teknika), was labeled with 125[I] using the E3Olton-~unter
reagent (New England Nuclear, Boston, MA). Bound label was
separated from free label on a Bio~Gel P-4 column (Bio-Rad).
Fif ty microliters of culture supernatant were incubated with 5
x 106 cpm of the labeled lysate for 1 hour at 37~C, then 50
microliters of 10% fixed Staphlococcus aureus (Pansorbin,
Calbiochem, La Jolla, CA) was added~ The immunoprecipitate was
washed three times by centrifugation and the air-dried pellet
YO90/~9805 PCTJUS90/01l32
14
was resuspended in buffer, boiled for 3 minutes and
electrophoresed on a 10% SDS polyacrylamide gel. The gels were
dried and exposed for one to three days to X-Omat S film
(Kodak, Rochester, NY).
The class and light chain type of anti-~IV antibody was
determined by ELIS~. For these assays, microtiter plates
(Immulon 2) were coated with 4 micrograms/ml of HIV lysate
tElectro-Nucleonics~ and then incubated with culture super-
natants. The type of antibody binding to ~IV was determined
using the following alkaline phosphatase-coupled antibodies:
goat anti-human IgG (gamma specific), goat anti-human kappa
chain and goat anti-human lambda chain (Organon Teknika-Cappel,
Malvern, PA). The subtype of the monoclonal antibody was also
analyzed by ELISA using alkaline phosphatase-labeled mouse
monoclonal antibodies against the four subclasses of human IgG
(Zymed, San Francisco, CA).
Immunoglobulin quantitation was also performed by
ELISA. Immulon 2 plates were coated with goat anti-human IgG
(gamma specific) and incubated with culture supernatants.
Bound IgG was detected with alkaline phosphatase-labeled goat
anti-human IgG (gamm~ specific). Affinity-purified human IgG
~Cappel) was used to produce standard curves.
A total of 14,329 culturec in microtitre wells were
established using cells derived from the 58 subjects. Ap-
proximately half of these cultures were derived from three
serial bleeds from a single subject (with a scale score of 1)
over a period of three months. The remaining wells were
established using cells derived from 57 subjects whose scale
scores ranged from O to 3. The results of this procedure are
shown in Table I below.
'~ $ ?~ 3J d
'O90/09805 PCT/US90/01132
TABLE I
QUANTITATIVE RESULTS OF T~E PROCEDURE VSED TO
PRODUCE ~UNAN MONOCLONAL ANTIBODIES TO HIV
No. of % positive
wells wells
l. Infection of PBMC with EBV14,329 (l00)
1 4 weeks
Screen for anti-~IV by
Non-commercial ELISA 1,290 9.O
2. Expand positive wells
¦ 2 weeks
~r
Screen for anti-HIV by:
Non-commercial ELISA 573 4,0
Commercial ELISA 340 2.4
Screen for specificity (reactive
HIV, unreactive with BSA) 97 0.67
Screen for reactivity by RIP57 0.40
3Q
3. Subculture positive wells by doubling
dilution (l0,000-l0 cells/well)
4-6 weeks
r
Screen by commercial ELISA16* 0.ll*
4. Subculture positive wells at
(l00 and l0 cells/well)
1 4-6 weeks
Screen by commercial ELISA 7* 0.05*
* No. and ~ of positive plates from ~ubcultures of individual
wells which contain at lea~t one antibody positive well.
~ 3f~ .
WO90/09805 PCT/US90/01132
16
After four weeks, 9~ of the wells displayed antibody
production as revealed by the non-commercial ELISA (Table II).
After expansion, only 2.4% of the original cultures continued
to produce antibody reactive with a commercial ELISA kit and
only one-quarter of these ~0.67~ of the original 14,329
cultures) were producing antibodies which reacted specifically
with ~IV.
To determine whether the severity of disease in the
cell donor affected the number of cultures able to produce
antibodies and the specificity of antibody produced, the
seropositive cell donors were categorized with respect to
disease statuR using the m~unologic staging sy~tem of Zolla-
Pazner et al. (supra) and the results are shown in Table II
below.
2-32li3~ 2 ~
`vo90/0980s PCT/US90/01132
17
TABLE II
C~ARACTERISTICS OF CELL CULTURES DERIVED FROM
PATIENTS AT DIFFERENT STAGBS OF ~IV INFECTION
No. of
positive
wells No. of
(Includes wells
HIV-specific with HIV- No. of
Scale No. of No. of & non-specific specific clumps
Score patients wells reactivity) antibody per well+
_
BIV-seronegative patients:
3 637 0 0 7.3
~IV-~eropositive patients:
0 4725 18 ~2.4%) 6 (0.8%) 0.92
1 138,789 180 (2%)66 (0.7%) 0.88
2 202,792 54 (1.9%~ 16 (0.5%) 0.65
3 212,023 88 ~4.3%) 9 (0.4%) 0.22
5814,329 340 2.4%97 0.67% 0.66
* Specificity of anti-HIV antibodies were assessed by commer-
cial ELISA using BIV-coated and BSA-coated beads.
~ B cells transformed by EBV stick together and create
characteristic clumps of cells which were quantitated
microscopically.
2 ~
WO90/09805 PCT/US90/01132
18
The results, shown in Table II, revealed that cultures
obtained from patients with a scale score of 3 (severe im-
munodeficiency) gave a higher percentage of antibody producing
wells than patients with lesser scale scores. However, only
10% (9/88) of reactive wells from the cells of stage 3 patients
were specifically reactive with HIV whereas 30-37% of wells
from cells of patients with scale scores of 0-2 were specifi-
cally reactive with BIV. Thus, after six weeks of culture,
cells from patient~ with lower scale scores produced a higher
percentage of wells containing ~IV-specific antibody.
Analysis of antibodies from ELISA-positive expanded
cultures was carried out by RIP. Only 59~ of supernatants from
these cultures were also positive on RIP analysis~ RIP
analysis demonstrated that, out of 57 supernatants, 44 showed
reactivity to env-e~coded proteins, ll to 9~ proteins and 2 to
reverse transcriptase.
Therefore, specific lymphoblastoid cell lines were
isolated and further cloned as described below.
EXAMPLE 3: SPBCIFICITY AND REACTIVITY OF THE ~UMAN MONOCLONAL
ANTIBODIES OF A SUBSET OF T~E PRESENT INVENTION
The 57 cell lines mentioned above were then cloned by
doubling dilution from lO,OOO to lO cells per well. Wells with
the lowest cell concentration which were producing antibodies
were then picked and cloned at lOO or lO cells per well. Using
this procedure, seven cell lines, 3 producing anti-gp4l
antibodies and 4 producing anti-p24 antibodies were established
which have been cloned from one to three times at lOO or lO
cells per well. The reactivities of the antibodies from these
lines are shown in Figures l and 2.
All seven of the cell lines of this Example produced
antibodies of the IgG subtype as shown in Figure l. In Fig1~re
l, all lines received [l25I]-labeled ~IV lysate reacted Wi~ll
lane l, serum from an HIV-infected subject, lane 2, with
antibody from cell line 50-69, lane 3, with antibody from cell
line 98-6, lane 4, with antibody from cell line 98-43, lane 5,
with antibody from cell line 71-31 lane 6, with antibody from
V090/09805 PCT/US90/01132
19
cell 91-5, lane 7, with antibody from cell and line 91-6 and
lane 8, with antibody from 98-4.9. The molecular weights of
the major viral proteins are shown on the left in kilodaltons.
Antibody from three of the cell lines bound to env-encoded
protein gp41 (lines 50-69, 98-6 and 98-43, lanes 2-4 respec-
tively). Antibodies from four of the cell lines bound to qag
encoded protein p24 (lines 71-31, 91-5, 91-6 and 98-4.9, lanes
5-7 respectively). By RIP, antibodies from cell lines 71-31,
91-5 and 91-6 reacted with p24. Antibodies from 98-4.9 were
unreactive by RIP since IgG3, the subtype of this antibody,
does not bind to Protein A and is therefore not precipitated.
Antibody from all 3 of these anti-gag cell lines were also
tested by Western blot ~Figure 2).
In Figure 2, Western ~lot strips were used to show the
reactivity o~ serum from a normal control (lane 1), an HIV-
infected subject (lane 2), and of supernatant from cell lines
71-31 (lane 3), 91-51 (lane 4), 91-6 (lane 5), and 98-4.9 (lane
6j. Western blot analysis showed all 3 monoclonal antibodies
reacted with p24 and with known aag precursors p55 and p40.
All four antibodies also reacted with a breakdown product of
p24 which migrated with a mobility of approximately 22 kilodal-
ton~ (kd); three of the four anti-~ monoclonal antibodies
also reacted with additional intermediate precursors which were
noted with a mouse monoclonal antibody and which had mobility
of approximately 37, 31 and 28 kd.
Studies of the growth characteristics and level of
antibody production o~ each line was performed. Each cell line
was initially cultured in replicate wells at 0.5 x 106 cells/ml
for 1-8 days. The number of cells and the amount of ;~
munoglobulin produced is shown in Figure 3. In Figure 3,
each time point, separate wells were used to test for viahl-
cell number (closed cir~les) l for secreted human IgG (o~-~n
circles). Cell lines ~u~ied ~re 50-69(a), 98.6(b), sn
~ d~, g~-5~e~, 9~-~(f) and 9~-4 9(g)-
Peak cell density was noted at 4 days with maximum
densities of 1.0 to 2.4 x 106 cells/ml. The doubling time of
cells in log phase ranged from 40-61 hours. The concentration
2 ~
WO90/09805 PCT/US90/01132
of immunoglobulin produced varied widely, generally peaking at
day 5 of culture and ranging from 9-112 micrograms/ml.
It should be noted that cell line 98-4.9 recently lost
the ability to produce human monoclonal antibodies.
EXAMPLE 4. GENERATION OF ADDITIONAL LYMP~OBLASTOID CELL LINES
PRODUCING ~UMAN MONOCLONAL ANTIBODIES TO HIV
Peripheral blood mononuclear cells were obtained from
another 39 ~IV-seropositive individuals, the cells immortalized
by EBV infection, screened and selected as in Examples 1-3
above. Positive cultures were expanded, subcultured by
doubling dilution and again subcultured one to three times at
10 to 100 cells per well. Four stable lymphoblastoid cell
lines producing human monoclonal antibodies were obtained as
follows: 120-16, 126-6 and 126-50 directed against gp41; and
134-F6 directed against p24.
Each of the monoclonal antibodies of this Example were
tested for their specificities by means of commercial ELISA
(the 6upernatants were reactive with HIV-coated beads and
unreactive with BSA-coated beads),~ by radioLmmunoprecipitation
and by Western Blot as in Example 3 above. The characteristics
of these human monoclonal antibodies are shown in Table III
below.
EXAMPLE 5: C~ARACTERIZATION OF T~E ~UMAN MONOCLONAL
ANTIBODIES OF T~E PRESENT INVENTION
The specificity and biological activities of the human
monoclonal antibodie~ of the present invention are summarized
in Table III below.
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-"O90/09805 PCT/US90/01132
22
In Table III, epitope mapping was performed using
recombinant antigens using a Western slOt format. viral
Neutralization and enhancement assa~s were performed as in J.
Ciin. Micro. 26: 231, 1988. ADCC assays were performed
according to Lyerly, H.K. et al. AIDS and Human Retroviruses
3: 409-422, 1987. Western Blot analysis was performed using
recombinant p24 or gp41 (supplied by Organon-Teknica).
Elisa's were performed using cloned gp41 antigens.
ENV9 is a cloned gp41 protein encompassing residues 461 to 761
(obtained from DuPont, Wilmington, DE). PE3 is a 286 amino
acid sequence from gpl20 (obtained from DuPont, Wilmington,
DE~. pl21 contains residues 561-649 of gp41 (obtained from
Centocor, Malvern, PA).
As can be ~een in Table III above, all of the anti-gp41
human monoclonal antibodies of the present invention were o~
the IgG serotype and mediated antibody dependent cellular
cytotoxicity (ADCC).
In addition, epitope mapping showed that five of the
anti-gp41 antibodies bound to different epitopes on the viral
protein. Monoclonal antibody 50-69 bound to residues 599-613;
monoclonal antibody 98-6 was directed against a peptide
encompassing residuec 642-692; monoclonal antibody 98-43 bound
to a peptide encompassing gp41 residues 579-604; monoclonal
antibody 120-16 bound residues 644-663; and monoclonal antibody
116-7 bound to residues 661-683. The numberinq system for the
gp41 peptide is according to J. Virol. 61: 570, 1987.
All three of the anti-p24 monoclonal antibodies tested
(71-~1, 91-5 and 91-6) bound to a HIV p24 peptide encompassing
residues (131-198 (the numbering system according to Wain-
Hobson et al. Cell 40: 9, 1987~ (Table III).
Finally, none of the monoclonal antibodies of the
present invention were able to neutralize the infectivity of
aIV and one (120-16) enhanced viral pathoqenicity.
EXAMPLE 6: INHIBITION TESTING OF T~E MONOCLONAL
ANTIBODIES_OF T~E PRESENT INVENTION
Presented below is an example of the use of the
`'O~0/09805 PCT/US90/01132
23
monoclonal antibodies of the present invention in a diagnostic
assay for HIV.
Immulon 2 plates ~Dynatech) were coated with 0.5
micrograms/well of an HIV lysate diluted in 0.05M bicarbonate
buffer, pH 9.6 for 2 hours at 37C, and overnight at 4C.
After washing the plates with phosphate buffered saline, pH
7.4, containing 0.05% Tween (PBS-Tween), samples of human sera,
obtained from HIV seropositive or seronegative individuals were
added to each well at 0.5 micrograms/well, diluted l:10 to
1:1000. The plates were incubated at room temperature over-
night and washed three times with PBS-Tween. A predetermined
dilution of biotinylated monoclonal anti~HIV antibodies (see
below) was then added in a volume of lO0 microliters and the
plates incubated for 2 hours at 37C. The wells were washed
three times with PBS-Tween and the reaction developed by adding
an avidin-biotinylated-horseradish peroxidase complex (Vector
Labs) and incubated for 2 hours at 37C. After washing 5 times
with PBS-Tween, 2,2'-aæino-di-[3-ethylbenzthiazoline sulfonate]
(ABTS) was added as substrate and incuhated for 30 minutes at
room temperature. The optical density of each well was read in
an ELISA reader at 410nm.
~iotinylation of the monoclonal antibodies to ~IV was
performed as follows. Each monoclonal antibody was partially
purified by ammonium ~ulfate precipitation and/or chromatog-
raphy on Protein A-Sepharose. After dialysis against O.lM
sodium bicarbonate 75 microliters of N-hydroxyl-suc-
cini m; dobiotin (5 mg in 1 ml of DMSO~ was added to 1 ml of the
antibody at a concentration of 5 mg/ml. The reaction was
allowed to proceed at room temperature with haking for 3 hours
and then dialyzed against phosphate buffered saline, pH 7.4.
The biotinylated monoclonal antibodies were stored at -25C in
50% glycerine before use.
The results of the inhibition tests are shown in Figure
4. In Figure 4, the seronegative sera is designated t-) and
the seropositive (+).
The data presented in Figure 4 shows that antibodies
120-16 and 71-31 are directed against immunodominant epitopes
2 ~
~090~09805 PCT/US90/0113
24
and non-dominant epitopes respectively. Thus, seropositive
sera are a~le to compete for the binding of labeled antibody
120-16 to the ~IV lysate defining the epitope of this
monoclonal antibody as immunodominant. Non-dominance was
established for the epitope with which 71-31 reacts as a result
of the inability of most seropositive sera to compete with this
antibody for binding to thè ~IV lysate.
