Note: Descriptions are shown in the official language in which they were submitted.
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WO 92/05799 ~, PCI~/US91/0{iO79
~ 1
2 ~ 2 ~ 3
10M13T~IOD FOR IN~IBITING THE INFECTIVITY
OF HUM~N IMMUNODEFICIEMCY VIRUS
Field of the Invention
This application is a continuation-in part application
15of co-pending U.S. Patent Application Serial No. 287,270 filed
December 20, 1988.
The government has rights to this invention by virtue
of funding from U.S. Pu~lic Service Grant No. ROl-AI28194.
One aspect of this invention relates to methods and
compositions for influencing the immunogenicity of human im-
munodeficiency virus ~HIV) antigens and more specifically to
methods and compositions for raising antibodies that inhibit
the propagation of HIV infection.
Another aspect of this invention relates to antibodies
that have the ability to inhibit such propagation.
Back~round of the_Invention
Acquired Immunodeficiency Syndrome (AIDS) is believed
to be caused by a retrovirus called human immunodeficiency
virus t~IV), also known as ~TLV III or hAV. This syndrome is
considered responsible for a variety of immunologic abnor-
malities including but not limited to the depletion and/or
selective infection by this vixus of helper/inducer (T4)
lymphocytes, which results in impairment of the helper/inducer
T cell function in affected individuals and in eventual
inhibition of normal immune response in such individuals.
The viral envelope (~IV env) includes a population of
glycoproteins tcalled gp 160) anchored in the viral cell
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W092/05799 PCT/US9l/06079
2 ~ 2 !~
membrane bilayer via their C-terminal region. Each
glycoprotein contains two segments: the N-terminal segment,
called gpl20, which protrudes from the membrane into the
surrounding medium; and the C-terminal segment, gp 41, which
spans the membrane.
It has been reported that ~IV infects CD4+ T lym-
phocytes by a sequence of events beginning with attachment of
gpl20 to its cellular receptor CD4, a nonpolymorphic surface
glycoprotein described in Maddon, P.G. et al, Cell, 42:93-104,
1985; Clark, S., et al, P.N.A.S. (USA), 84:1649, 1987 and
Germain, R.N., Cell, 54:441-444, l9R8. It is believed that the
binding of gpl20 to CD4 then tri~gers anchorage of gp 41 to the
lymphocyte membrane, an event in turn followed by cell fusion
between the virion and the target lymphocyte; Kowalski, M. et
al, Science, 237:1351-1355, 1987; Gallaher, W.R., Cell, 50:327-
328, 1987 and Gonzalez~Scarano, F. et al, AIDS Research & Human
Retroviruses, 3(3):245-252, 1987.
In addition, HIV infection is propagated by direct
lymphocyte-lymphocyte fusion between virus-infected cells
(which have been shown to express gpl20 and gp 41 on their
surface) and uninfected CD4~ cells. This fusion takes place
even in the absence of free HIV in the surrounding medium.
Lifson, J.D. et al, Science, 232:1123-1127, 1986; Sodroski, J.
et al, Nature, 322:470-474, 1986; and Lifson, J.D. et al,
Nature, 323:725-728, 1986.
The properties and isolation of gpl20 from HIV par-
ticles and its sequencing from different HI~ isolates are well-
known and have been extensively described e.g. in the foregoing
references. The preparation of gpl20 via reco~binant DNA
techniques has been described in Lasky, L.A. et al, Science,
233:209, 1986 and in published European patent application of
Genentech, Inc. published on August 24, 1988, Serial No.
279,688 (based on U.S.S.N. 155,336 ~iled 02/12~88) naming
~erman, P.W.; Gregory, T.J.; Lasky, L.A.; Nakamura, G.R; et al.
as inventors, as well as in Landau, N.R., et al., Nakure,
334:159-162, 1988; and Lasky, L.A., et al., Cell, 30:975-985,
1987. Finally, isolation of gpl20 (whether native or recom-
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W092/05799 PCT/US91/06079
binant) has been described in Essex, U.S. Patent NoO 4,725,669
(2/16/88).
CD4, the cellular receptor of gpl20l has been isolated
from lymphocytes. Synthetic (soluble) and recombinant CD4 have
been described in Smith, D.H. et al, Science, 238:1704-1707,
1937. Other methods as well as properties of CD4 have been
descri~ed in Jameson, B.A. et al, Science, 240:1335-1339, 1988;
Fisher, R.A. et al, Nature, 331:76-78, 1988; ~ussey, R.E. et
al, Nature, 331:78-81, l9B8; Deen, K.C. et al, Nature, 331:82-
84, 1988; Traunecker, A. et al., Nature, 331:84-86, 1988; and
Lison/ J.D. et al, Science, 241:712-716, 1988.
Two regions of HIV env (gp 160) exhibit the highest
immunogenicity that has been observed against this protein:
The first region has been placed between residues 307
and 330 of gpl20 and represents an immunodominant epitope since
animals immunized with whole gp 160 or gpl20 (or with fragments
of gpl20 containing the epitope) produce high titers of HIV-
neutralizing antibodies (i.e., antibodies that inhibit virion-
lymphocyte fusion). This immunodominant epitope is situated in
a highly variable segment of gpl20 that varies from isolate to
isolate and, as a result, the antibodies are also isolate-
specific. This limits their utility in immunological studies
and in therapy against (or prevention of) HIV infection. Also,
sera from HIV-infected humans do not contain high titers of
these antibodies.
Instead, sera from infected humans contain HIV-neutra-
lizing antibodie~ (specifically antibodies that inhibit the
binding of viral gpl20 to lymphocytic CD4) directed to other
epitopes of gpl20 which have not been precisely identified
(though it is thought to be proximal to the CD4-binding site of
gpl20); Lasky, L.A. et al, C~ll, 50:975-935, 1987. The
antibodies are group- and not isolate-specific, which indicates
that this second epitope is located in a more conserved domain
of gpl20. However, animals immunized with gpl20, gp 160 or
various fragments of gpl20 have not produced HIV-neutralizing
antibodies. Thus, these other epitopes of gpl2a appears to be
less immunogenic.
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W092/057~9 PCT/US91/06~79
~2 a ~ 4
Accordingly, one object of this invention is to provide
significant amounts of antibodies that neutralize the infec-
tivity of HIV virus ti.e./ inhibit its ability to invade T4
lymphocytes).
Another object of this invention is to provide an-
tibodies that prevent HIV-induced cell fuc;ion between healthy
T4 lymphocytes and lymphocytes infected with HIV.
Yet another object of this invention is to use such
antibodies to improve the understanding of the pathogenesis of
HIV and to inhibit propagation of ~IV infection in human T4
lymphocytes.
Still another object is to provide compositions and
methods for raising such antibodies.
Further objects of this invention include use of such
antibodies in research to elucidate the structure and function
of HIV components and the mechanism of HIV infectivity and use
of such antibodies in the passive or active immunization of
humans for propylactic or therapeutic purposes.
These and other objects of the invention will be
apparent to persons skilled in the art in light of the present
specification, accompanying claims and appended drawings in
which:
B ef Descrl~tion of the Drawinqs
Figure l A-C is a series of plots comparing the
magnitude and cell fusion-blocking ability of various antibody
titers in successive bleedings of animals immunized with CD4,
gpl20 and a combination of CD4 and gpl20.
Figure 2 is a series of graphs illustrating the time
course of an experiment with three groups of mice respectively
injected with CD4 (A,D,G), CD4-gpl20 complex (B,E.H) and gpl20
(G,H,I). The weekly serum samples were assayed individually
for gpl20-binding antibodies (A,B,C), CD4-binding antibodies
(D,E,F), and syncytia-blocking capacity (G,H,I). The mice
immunized with the complex showed a somewhat higher anti-gpl20
response than those immunized with gpl20 alone (panels B ~ersus
C); a markedly lower titer of CD4 binding as compared to those
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W092/0~799 PCT/USg1/~079
~ 2~2~3
, 5
receiving CD4 alone (panel E versus D); and a significantly
higher syncytia-blockin~ response (panel El versus G).
Figure 3 is a graph showing titration of 11-week serum
from 4 mice injected with CD4-gpl20 complex for IIIB and RF
syncytia-blocking capacity. The parallel behavior of in-
dividual sera in the two tests suggests that the antibodies are
directed at group-specific determinants.
Figure 4 is a graph showing the effect of antibodies on
rCD4-phosphatase binding to solid phase rgpl20. In panel A the
antibody is OKT4A; in panel B, 94; in panel C, OK~4, in panel
D, 55. CD4-phosphatase concentrations: closed circles = 10
micrograms/ml; open circles - 3 micrograms/ml~ Ordinate:
OD405/60 min. Abscissa: antibody concentration, from left to
right, 0, 0.3, 1, 3, 10 micrograms/ml.
Figure 5 is a graph showing effect of rgpl20 on rCD4-
phosphatase binding to various antibodies captured on solid
phase goat anti-mouse Ig. In panel A the antibody is OKT4A; in
panel B, 94; in panel C, OKT4; in panel D, 55. Concentration
of rCD4-phosphatase: closed circles = 10 micrograms/ml; open
circles - 1 microgram/ml; squares = 0.1 microgram/ml. Or-
dinate: OD40s/60 min (A,B) or OD40s/120 min (C,D)- Abscissa:
concentration of gpl20 (0, 0.4, 4, 40 micrograms/ml).
Figure 6 is a graph showing the effect of rgpl20 on
phosphatase-labeled antibody binding to solid phase rCD4. In
panel A, the antibody is 94; in panel B, 55. Open circles = no
rgpl20; closed circles = rgpl20 at 0.1 microgram/ml; squares =
rgpl20 at 1.0 microgram/ml. Ordinate: OD40s/60 min. Abscis-
sa: antibody concentrations (0.1, 1, 3, 10 micrograms/ml).
Summary of the Invention
It has now been discovered that antibodies raised
pursuant to immunization with a complex of (a) the HIV antigen
gpl20 and (b) CD4-Immunoadhesin have the ability to inhibit
propagation of HIV infection to healthy human T-lymphocytes (by
inhibiting lymphocyte-lymphocyte fusion and in~asion of
lymphocytes by HIV).
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W092l05799 PCT/US91/06079
~2~ ~2~ 6
Detailed Desçription of the Invention
As used in this disclosure, each of the following terms
shall have the mea~ing ascribed to it below.
"gpl20" shall mean not only gpl20 itself b~lt also any
other molecule that binds with CD4 in a si;milar manner and that
when so bound has the same conformation. For example, the te~n
will include fragments of gpl20 that bind to CD4 as well as
analogs and derivatives of gpl20 that possess the ability to
bind CD4 and to generate anti~odies with the cell-fusion
blocking ability of the antibodies of the present invention7
In addition, this definition of gpl20 shall include gpl20 from
any HIV isolate since methods for sequencing this protein are
known and are independent of the particular isolate of ~IV from
which the native protein is derived.
"CD4" shall mean CD4 and/or fragments, derivatives or
analogs containing the gpl20-binding site of CD4, such as CD4
isolated from the lymphocytic surface and CD4 or CD4 deriva-
tives (such as CD4-Immunoadhesin and analogs (e.g. soluble CD4)
produced by synthetic (including but not limited to recombinant
DNA) techniques.
"gpl20/CD4 complex" shall mean a bimolecular (i.e.,
noncovalent) conjugate or complex between gpl20 and CD4 (or
between gpl20 and antiidiotypic antibody bearing a CD4 internal
image). A simple mixture of gpl20 and CD4 contains this
complex because of the high affinity between gpl20 and its
cellular receptor~ It is not necessary that the complex be
made of isolated gpl20 and CD4. For example, whole lymphocytes
having gpl20 bound to the CD4 on thPir surface are envisioned
as a possible form of an immunogen encompassing the gpl20/CD4
complex of the present invention. The native lymphocytes of an
HIV-infected human would not act as such an immunogen in that
human because of the ability of autologous CD4+ lymphocytes to
act as antigen-presenting cells (APC), as reported by Lanzavec-
chia, A. et al/ Nature, 334:530-532, 1988. (Consequently, the
gpl20/CD4 complex even if it exists on the surface of these
human lymphocytes could not act as an immunogen.) ~owever,
antibodies of the type of the present invention could be
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W092t~799 PCT/US9~/06079
~ 7 2 ~
induced in a human by immunization with the foregoing complex
in one or more of its forms contemplated herein.
"Antibodies of the present inven~ion" or ~the present
antibodies" shall mean (a) polyclonal, group~specific an-
tibodies raised by immunization with the qpl20/CD4 complex andcapable of inhibiting lymphocyte fUSiOII; and/or (b) monoclonal
anti~odies raised against this immunogen and possessing the
same fusion inhibiting property.
"Neutralization of HIV" shall meaLn inhibition of the
lQ ability o~ ~IV to bind to and invade a susceptible lymphocyte.
"Cell fusion inhibition" shall mean inhibition of the
ability of HIV-infected lymphocytes to for~ syncytia with
healthy CD4+ lymphocytes (i.e. lymphocytes possessing surface
CD4) in the absence of free HIV.
As stated above, although HIV-infected humans do
produce antibodies to gpl20 that inhibit the binding of HIV to
CD4 and hence prevent invasion of CD4~ T lymphocytes (i.e.,
lymphocytes possessing surface CD4) by the virus, the titers of
such antibodies are not very high either in absolute terms or
by comparison to these individuals' titers of nonneutralizing
antibodies.
The majority of sera from seropositive indi~iduals
contain some HIV-neutralizing antibodies directed against an
epitope located within a more conserved area of gpl20, and
distinct from the immunodominant epitope. Such antibodies are
group-specific and constitute useful investigative tools in the
pathogenesis of HIV and in research efforts to produce abate-
ment or prevention of ~IV infection. However, even though
these human group-specific antibodies neutralized HIV in vitro
and inhibited lymphocyte fusion in vitro, they failed to
inhibit lymphocyte invasion by free ~IV introduced in the serum
of primates passively immunized with the human antibodies.
This f~ilure has been attributed to the fact that these
antibodies are not directed towards an immunodominant epitope
and/or to the insufficient affinity of these antibodies for
their antigenic determinant compared with the extremely high
affinity of gpl20 for its lymphocytic receptor CD4 (the
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WOg2/~5799 PCT/US9~/~079
29~2~3 8 ~-
equilibrium constant of the latter is of the order of 10-9 M
which is significantly higher than the affinity of most
antigens for their antibodies). Also, nonprimate mammalian
immune systems do not recognize this epitope of gpl20 and
consequently animals do not produce such antibodies. There-
fore, the need still exists for different antibodies that (a)
are group-specific, (b) neutralize the virus and inhibit cell
fusion and (c) do not compete with the high-affinity binding of
gpl20 to CD4.
In accordance with the present invention, comparative
experiments were conducted in which animals were immunized with
(a) CD4, (b) gpl20, and (c) a mixture of CD4 and gpl20 which is
believed to result in a bimolecular (noncovalent) conjugate or
complex between CD4 and gpl20 because of the high affinity (10-
9 ~) of gpl20 for its receptor.
Animals immunized with CD4 alone exhibited a very high
titer of anti-CD4 antibodies. ~owever, only a relatively very
small portion of these antibodies ~nhibited the ability of HIV
infection to spread among T4 lymphocytes as measured by a cell
fusion assay which tests the ability of HIV-infected lym-
phocytes to fuse with healthy lymphocytes bearing CD4.
In parallel, another group of animals were immunized
with gpl20 alone. The anti-gpl20 immune response was sig-
nificant (although not nearly as high as the anti-CD4 response)
but the anti-gpl20 had no HIV-neutralizing ability (as measured
by the same assay).
A third group of animals were immunized with a mixture
of CD4 and gpl20. The immune response showed the presence of
anti-CD4 antibodies ~although the titer was significantly lower
compared to the anti-CD4 elicited by immunization with CD4
alone) and anti-gpl20 antibodies (in amounts comparable to
thoss elicited by immunization with gpl20 alone). However, the
sera showed a very high titer of cell-fusion inhibiting
antibodies.
The antibodies of the present invention are not simply
anti (CD4), since the cell-fusion inhibiting titer does not
correlate with the titer obtained by the immunization with CD4
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W092/05799 PCT/US91/06079
~ 2~`7 ~3
alone and since anti CD4 do not possess the cell-fusion
inhibiting ability of the antibodies of the present invention.
For the same reasons, the antibodies of the present invention
do not appear to be simply anti gpl20. Furthermore, the
present antibodies are not the same as previously observed
antibodies which inhibit the event of binding between CD4 and
gpl20 because the titers of the present antibodies do not
correlate with the titers of the previously observed binding-
inhibiting antibodies. Finally, the antibodies of the present
invention are not elicited except in the presence of the
gpl20~CD4 complex, as will be illustrated below, and therefore
constitute novel and distinct entities.
The HIV-neutralizing ability of the antibodies of the
present invention has been measured by a cell-fusion assay
developed by Skinner, M.A. et al, J. Virol., 62:4195, 1988.
This assay exploits the ability of HIV-infected lymphocytes to
form syncytia (fused cells) with healthy but HIV-susceptible
(CD4~) lymphocytes (in the absence of free HIV), a process that
starts by the expression (on the surface o infected lym-
phocytes) of gpl20, which then binds to the CD4 of heal-thy
cells. The assay thus compares the ability of HIv-infected
lymphocytes to form such syncytia under experimental conditions
with the ability of the infected cells to form syncytia under
control conditions, i.e. in the absence of a potential fusion-
inhibitor.
This assay is a stringent indicator of HIV-infection
inhibition ability by a given inhibitor and in particular by an
antibody. Another fusion assay is available that measures the
a~ility of free ~IV to invade lymphocytes ~i.e., the fusion
takes place between the virion and the lymphocytes). However,
although many antibodies are available that can inhibit
lymphocyte infection by free virus, very few antibodies also
inhibit lymphocyte-lymphocyte fusion in the absence of free
virus. On the other hand, most, if not all, antibodies that
inhibit cell fusion also inhibit infection by Eree virus.
For these reasons, the performance of the present
antibodies in the lymphocyte-lymphocyte cell fusion assay
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W092/05799 PCT/US91/06079
2a~2~'~ 10 ~
constitutes good evidence of the ~IV-inhibiting ability of such
lymphocytes.
The antibodies of the present invention may be thus
used to inhibit both invasion of lymphocytes by ~IV and spread
of HIV infection via lymphocyte fusion and hence constitute
good candidates for passive immunization (both prophylactic and
therapeutic). Such passive immunization may be combined with
cytotoxic agents or coadministered with other HIV inhibitors,
such as Iicin toxin A chain which when linked to recombinant
CD4 has been shown to be selectively toxic to 'nfected T-
lymphocytes. Till, M.A., et al, Science, 242:1166-1168, 1988.
In addit;on, immunization of susceptible mammals with a
combination of gpl20/CD4 is expected to improve the effective-
ness of the immune response of these mammals against HIV infec-
tion both preventively and therapeutically.
Other uses for the antibodies of the present invention
include use in screening tests for the presence of the
gpl20/CD4 complex; as research tools to identify new epitopes
of gpl20 and specifically epitopes that are available only by
changes in the conformation of gpl20 by CD4 binding and/or vice
versa. Monoclonal antibodies in accordance with the invention
and especially human monoclonal antibodies represent a
preferred form of the present invention and can be used for
passive immuniæation in humans.
In addition, many other uses are contemplated as will
be apparent to those of ordinary skill in the art.
Amounts used for immunization in mammals can generally
vary from a~out 10 to about 100 micrograms CD4/kg body weight
and from about 13 to about 130 micrograms of gpl20/kg body
weight. The foregoing amounts are based on the assumption that
equivalent amounts of CD4 and gpl20 will be used, which is
preferred but not necessary. It will be appreciated of course
by those of ordinary skill in the field that an excess of one
or the other constituent of the complex (i.e., an amount in
addition to that sufficient to form a complex with the avail-
able amount of the other constituent) is not fatal to the
operability of the present invention but an equimolar mixture
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W092/05799 PCT/US91/06079
~ 2 c~ 3
of CD4 and gpl20 is preferred.
Well-known immunization protocols may be used with or
without adjuvant. One preferred protocol involved use of the
complex in complete Freund's adjuvant as s,et forth in Example 1
(of course any other well-known immunization adjuvant can be
used or adjuvant can be omitted altogether).
A sin~le immunization is sufficient, but immunization
may be repeated 4 weeks after the first injection with an
additional optional booster 4 weeks after the second injection
in incomplete adjuvant (or without adjuvant). In addition, the
immunogenic ability o~ the complex of the present invention can
be boosted by use of carriers such as tetanus toxoid, keyhole
limpet hemocyanin, vaccinia virus, diphtheria toxoid, etc., as
i5 well known in the art.
Concentrations of the antibodies of the present
invention effective in inhibiting lymphocyte-lymphocyte fusion
should be at least sufficient to prevent successful carrying
out of the sequence of events that lead to either invasion of
the lymphocytes by free HIV or lymphocyte-lymphocyte fusion
between any available gpl20 (whether on the viral envelope or
on the surface of an infected lymphocyte) and CD4~ lymphocytes.
The upper limit of the effective concentration is irrelevant ln
vitro. In vivo, the upper limit of the effective antibody
concentration may be limited by factors outside the binding
mechanism, such as on immune response of the host against the
antibodies.
The antibodies of the present invention (whether
monoclonal or polyclonal) may be purified by well-known techni-
ques for purification of immunoglobulins, including but not
limited to use of precipitation techniques (such as ammonium
sulfate precipitation) and/or immunoaffinity chromatography
methods (with an antigen as the adsorbent) wherein the desired
antibody is preferentially bound to the column or excluded in
the eluant; protein A sepharose chromatography; Affigel-blue
chromatography; high performance liquid chromatography and
combinations of these techniques.
Monoclonal antibodies to the complex of the present
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WOg2/05799 PCT/US91/~079
20'~12~'~ 12 ~
invention may be raised according to well-known techniques,
such as those described in Kohler and Milstein, Nature,
256:495, 1975 and in Goding, infra.
In addition, it is believed that human monoclonal
antibodies may be raised from immortalized human l~mphocytes
sensitized against the complex o~ the present invention ln
vitro (as described in Reading, C.L., 1982, J. Immunol. Meth.,
53:261; Hoffman, M.K. et al in Engleman, E.G. et al (Eds) Human
Hybridomas and Monoclonal Antibodies, Plenum Press, New York,
1985, p. 466; Borrebaeck, C.A.K., Trends in BiotechnoloqY,
4:147, 1986) or in viv_ ~by immunizing humans with the complex
as described herein and selecting ~-cells with the appropriate
specificity). ~uman C3D+ peripheral blood mononuclear lym-
phocytes can be exposed to Epstein-Barr virus for immortaliza-
tion. Epstein-Barr virus (EBV) can be obtained from the
filtered supernatant of the marmoset cell line B95-8 (Miller,
G., and Lipman, M., 1973, PNAS (US~) 70:190) available from the
ATCC under Accession No. CRL-1612. Infected lymphocytes are
then washed and cultured in RPMI-1640 medium in the presence of
fetal bovine serum, glutamine, penicillin and streptomycin in
96-well plates at 104 cells per well. After screening for
antibody production, positive cultures can be expanded and
cultured further. Cultures with supernatants showing specific
reactivity to the complex of the present invention can be
subcultured on feeder layers of GK5 human lymphoblastoid cells
(derived from GM1500 as described in Kearnyl J., N. Enql~ J-
Med./ 309:217, 1983) irradiated with 3000 rads of gamma-
radiation. Stable clones can then be subcultured at low
densities (10-100 cells) on feeder cells and the subcultures
can be expanded. The specificity of the antibodies can be
tested by the Skinner et al assay referenced and described
herein.
Antiidiotype antibodies can be obtained by immunizing
syngenic mice with anti-CD4 monoclonal antibody. A most
efficient protocol of immunization is to inject the monoclonal
antibodies four times a day at weekly intervals. The total
amount injected is 13 doses of 20 micrograms per mouse at each
W092/057g9 ~ 9 ~ 1 2 ~ 3 PcT/us91/o6o79
"il 13
time. In particular, on day 1, the antibody is injected
coupled to RLH (keyhole limpet hemocyanln) in complete Freund's
adjuvant; on day 6 the antibody is injected co~pled to KLH in
incomplete Freund's adjuvant ~IFA); on day 13 the antibody is
injected coupled to KLH in saline; on day 27 the antibody is
injected alone in incomplete Freund's adjuvant. To obtain the
monoclonal antibodies, the animals are boosted after 4 to 12
weeks after the last injection by injecting again the
monoclona~ antibody in incomplete Freund's adjuvant in saline.
Spleen fusion with an appropriate myeloma partner is performed
3 days after the boost. Fusion and hybrid growth and ~election
are then performed in accordance with well-known techniques.
The antiidiotypic antibodies bearing CD4 internal image can he
identified by testing for binding to gpl20 (e.g., by radioim-
muno assay, enzyme-linked immunosorbent assay, etc.) and
purified using well-known methods including immunoaffinity
chromatography using gpl20 as the adsorbent.
Again, the complex of the present invention when
antiidiotype antihodies ("AA") are used can be formed by mixing
gpl20 and AA preferably in equimolar amounts and waiting for
the two constituents to complex with each other.
The materials used in the present invention may be
purified from natural sources or synthesized by well-known
Irecombinank and other~ techniques as described above. In
addition, recombinant CD4 and CD4-Immunoadhesin are available
from Genentech, Inc. and so is recombinant gpl20. If not
already obtained in purified form, these materials should
preferably be purified prior to use in immunization. Techni~
ques for purification are well known; see, e.g., U.S. Patent
No. 4,725,669 of Es~ex et al. issued February 16, 1988.
To be effective in inhibiting cell invasion by HIV and
cell fusion, the antibodies of the present invention must be
present in the vicinity of infected T4 lymphocytes (or ~IV-
susceptible uninfect~d T4 lymphocytes) before the gpl20 ex-
pressed on the surface of infected T4 lymphocytes ~or the gpl20
on the surface of the virion) binds to its receptor (CD4) on
the surface of an uninfected lymphocyte (or on the surface of a
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W092/05799 PCT/US91/06079
2~ 3 14 ~
susceptible lymphocyte in the case of infection by free ~irus).
Preferably, the antibodies of the present invention will be
present in such vicinity prior to encounter between the gpl20-
bearing infected lymphocyte (or virus) and the target CD4+
lymphocyte.
The inYention is further described in the Examples that
follow. The purpose of these examples is to illustrate the
present invention and not to limit its scope.
Example 1: Immunization of Mice and Antibody Titers
Three groups of 4 mice (each weighing 25 g) were im-
munized as follows:
The first group was injected once intraperitoneally
li.p.) with 10 micrograms of CD4 in 0.2 ml of complete Freund's
adjuvant.
The second group was injected once i.p. with 12.5
micrograms of gpl20 in 0.2 ml of complete Freund's adjuvant.
The third group was injected once i.p. with a mixture
of 10 micrograms of CD4 and 12.5 micrograms of gpl20
(previously incubated together for 30 minutes) in 0.4 ml of
complete Freund's adjuvant.
The mice were bled weekly over a three-month period and
the sera were monitored for CD4- and gpl20-binding titers. In
addition, the sera were monitored for their ability to block
lymphocyte-lymphocyte fusion, according to an assay described
in Example 2 belvw, and for their ability to inhibit binding
between CD4 and gpl20, according to an assay described in
Example 3.
CD4 and gpl20 titers were determined as follows:
Binding antibodies were assayed by ELISA. Microtiter plates
were coated with the appropriate antigen (CD4 or gpl20,
respectively~ at a concentration of 3 micrograms/ml in car-
bonate buffer 0.1 M, pH 9.6 overnight at 4C, washed and
blocked by incubating them with 1% bovine serum albumin (BSA)
in phosphate buffer saline (PBS) for 45 min. at room tempera-
ture. A 0.1 ml sample of test serum at various dilutions in
PBS were then added and incubated for two hours at room
,
. . . :
W092/0~799 2 ~ 2 ~ 3 PCT/US9~/~6079
~; 15
temperature. The plates were then treatecl with goat-antimouse
antibodies labelled with alkaline phosphatase and finally
incubated with PNPP (disodium p-nitrophenyl phosphate) at a
concentration of 1 mg/ml in diethanolamine buffer, pH 9.8.
Each step was followed by a wash in PBS-Tween 20 buffer.
Absorption at OD405 was determined 1 hour after incubation with
substrate in a Titertek Multiskan MCC 340. The OD readings
were converted to micrograms/ml of undiluted serum by inter-
polation to OD-0.5 and assuming a l:l ratio between bound mouse
antibody and ~oat and mouse tracer.
Figure lA is a plot of the anti-gpl20 titer (in
microgramstml serum) of sera elicited by immunization of mice
with gpl20 (solid line), CD4 (broken line) and the gpl20/CD4
complex (- - - -) for each weekly bleeding.
As evident from Figure lA, immunization with gpl20
alone elicited anti-gpl20 antibodies; immunization with CD4
alone elicited no anti-gpl20 antibodies, immunization with the
complex also elicited anti-gpl20 antibodies (i.e. the complex
generated formation of antibodies against gpl20 alone).
Figure lB is a plot of the anti-CD4 titer (in
micrograms/ml serum) of sera elicited by immunization with CD4
alone (broken line), gpl20 alone (solid line) and complex (- -
_ ) .
In Figure lB, immunization with CD4 alone elicited a
very high ant~-CD4 immune response; immuni~ation with the
complex elicited a lower but significant anti-CD4 response; and
immunization with gpl20 alone elicited no anti-CD4 response
except after week 7 when a small antiidiotype (anti-(anti
gpl20)) response was observed.
Example 2: Iymphocyte Fusion Assay
The assay to determine the immune serum capacity to
block lymphocyte-lymphocyte fusion was described in Skinner,
supra. Ten microliters of different dilutions (at least 1:10)
of the test serum were distributed in Costar 96 A/2 (half~well~
plates.
5x103 or 10x103 infected cell partners (from CBJIIIB or
. .
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, ' '
-
W092/0~799 PCT/US91/06079
2~9:~2~ 16 ~
CEM/RF lymphocyte cell line publicly available from National
Institute of Allergy and Infectious Disease, Reagent Program
respectively infected with ~TLV IIIB or HTLV RF isolate
available from Dr. Gallo at the National Institute of ~ealth)
were added to each well contained in 40 microliters of culture
m~dium. Then, 7x104 uninfected CD4-~ human lymphocytes (Molt4
available from American Type Culture Collection, Rockville, MD,
Accession No. CRL-1582) in 40 microliters of culture medium
containing fetal bovine serum were ~dded to the wells and the
plates were incubated at 37OC in a 5% CO2 atmosphe~e for 20-24
hours. The plates were then read for the occurrence and the
number of lymphocyte syncytia in an inverted microscope at a
40-fold enlargement. Giant cells having a size of at least
five times the area of normal cells were scored as syncytia
produced by cell fusion. In the absence of any antiserum, the
syncytia were 50-80/well (control). Fusion-blocking units
(FBV) were calculated hy converting the percent decrease in
syncytia scored relative to the control value and taking into
account the serum dilution (1 FBU is defined as the amount of
antibody that reduces the number of syncytia to 50% of the
control value). The results, in FBU, are plotted in Figure lC.
Again, the solid line represents FBU achieved by immune sera of
mice immunized with gpl20 alone. In Figure lC, this value is
essentially the same as the control. The broken line
represents the fusion-blocking ability of immune sera elicited
by immunization with CD4 alone. In Figure lC, this value is
positive but not very high. This is attributable to the fact
that anti-CD4 will bind some of the CD4 on the uninfected lym-
phocyte surfaces and thus prevent the gpl20 of the infected
lymphocytes from binding to the CD4.
In Figure lC, the line ----- - represents the fusion-
blocking ability of immune sera elicited by immunization with
gpl20/CD4 complex. The FB~ of this sera starts at about that
of the anti-CD4 sera in week 1 and extends to about 50 times
that of the anti-CD4 sera.
W092/05799 PCT/US91/0607
f
-` 17 2~912~3
ExamPle 3- Monoclonal_Antibodies
Monoclonal antibodias will be raised by immunizing mice
with complex in accordance with the method of Ex~mple 1 ~except
that two injections can be used spaced 4 weeks apart) optional-
ly with a booster using incomplete Freundi's adjuvant 4 weeks
after the second injection. Three days after the last im-
muni~ation, spleen cells will be obtained, purified and fused
with myeloma cells. The spleen of the mouse with the highest
fusion-blocking ability will be excised using well-known
dissertion techniques. A single-cell suspension ~ill be made
up by teasing the spleen as described in Goding, J.W.,
Monoclonal Antibodies: Principles and Practice, Academic Press,
Inc., New York 1983, pp. 50-97 and specifically on p. 64. The
spleen cells will be harvested by centrifugation (e.g. 400xg
for 5 min.) and wash~d. Erythrocytes will be removed by
ammonium chloride lysis followed by centrifugation. The spleen
cells will be counted and approximately 108 cells will be used
for fusion with commercially available mouse myeloma cells
te.g. SP 20 from American Type Culture Collection under
Accession No. CRL-1581). (2 - 3)x107 myeloma cells will be
mixed with the spleen cells in serumi-free media and centrifuged
at 400xg for 5 min. Any remaining medium will be removed by
suction. The cell pellet will be suspended in 0.5-1 ml of warm
fusion medium containing 10 g of 50% w/v PEG (m~WO 1500) and 10
ml Dulbecco's modified Eagles' Minimum Essential Medium, pH
7.6. The mixture will be stirred, centrifuged and resuspended
in fetal bovine serumi-containing medium using normal spleen
cells as feeders. The cells will then be exposed to XAT
selective media and grown in such media in an atmosphere
containing 5% CO2. The cultures will be pulled and fed.
Hybrids will be growing and screenable at 10-15 days after
incubation begins. Positive clones (i.e. clones secreting
fusion-blocking antibody) will be identified and recloned until
their secretion of the desirable immunoglobulin is steady and
reliable. Monoclonals will thus be obtained. The Skinner et
al assay can be used to determine specificity of the desired
antibody.
'
.
W092/05799 PCT/US91/~079
2C~3 18
Difficulty in obtaining such monoclo~als is not
expected because the titers of fusion-blocking antibody are
relatively high as demonstrated in previous Examples. If
desired, spleen cells se~reting anti-CD4 and/or anti-gpl20 will
be separated first before fusion to maximize the probability of
obtaining fusion-blocking monoclonals~
Example 4:
It is known that the HIV infection of CD4+ cells can be
prevented by antibodies specific for the gpl20 binding site on
CD4, defined as the V1 domains of CD4 involved in the initial
CD4-gpl20 binding event, i.e., the region homologous to CDR-2,
amino and residues 41-52 (Peterson, A., et al., Cell. 54:65,
1988; Landau, N.R. et al., Nature. 334:159, 1988; Clayton,
L.X., Na~ . 335:363, 1988; Jameson, B.A. et al., Science.
240:1355, 1988), and in part CDR-3, amino and residues 83-92
(Nara, P~L., et al., Proc. Natl. Acad. Sci. USA. 86:7139, 1989;
Sattentau, Q.J., et al., J. Exp. Med. 170:131~, 1989). These
antibodies prevent infection by sterically interfering with the
20 binding site.
Poly- and monoclonal antibodies from mice immunized
with CD4 complexed to gpl20, their binding characteristics and
capacity to prevent the formation of ~IV-dependent syncytia,
are described below. The data presented below demonstrate that
there are epitopes on CD4, unrelated to the binding site of
gpl20, which antibodies can recognize, thus affecting post-
virus-binding events that usually lead to infection.
The following materials and methods were used as
described below.
Recombinant molecules. Solllble recombinant CD4 (rCD4), CD4
immunoadhesin (containing V1 and V2 domains of CD4 spliced to
the CH2 and CH3 domains of human IgG as described in Byrn,
R.A., et al., Nature 344: 667, 1990, and recombinant gpl20 were
obtained from Genentech Inc., South San Francisco, CA.
35 Animals. Balb-c female mice 10-15 weeks old (Jackson Labs, Bar
~arber, ME) were used both for immuni~ation and for production
of monoclonal antibodies.
W092/05799 PCTtUS91/06079
~ 19 ~ 2~
Immunizations. Mice were injected intraperitoneally (i.p.)
with antigen emulsified in Complete Freund's Adjuvant (CFA,
Difco, Detroit, MI). The antigens and doses were a) CD4, 16
micrograms/mouse; b) gpl20, 12.5 micrograms/mouse, c) CD4-
gpl20, 16 micrograms CD4 and 12.5 micrograms gpl20, thoroughly
mixed and incubated for 20 min, and then emulsified in CFA.
The mice were bled prior to immuni~ation and every week after,
for 13 weeks. Serum samples were stored at -20Co
Enzymes and substrates. Alkaline phosphatase and glutaral-
dehyde, used to label antibodies for Enzyme Linked Immunosor-
bent Assay (ELISA) tests, and substrate paranitrophanyl-
phosphate (PNPP) were acquired from Sigma Chemicals (St. Louis,
MO).
Monoclonal antibodies. The monoclonal antibodies (mAbs)
mentioned below are F-91-36, F-91-55 and F-91-94, hereinafter
called 36, 55 and 94 respectively. They were derived from a
fusion of mouse 91, immunized with CD4-gpl20 complex as
described above. All these mAbs axe IgGl subclass.
Antibodv bindinq of qpl20 and CD4. ELISA tests were performed
by coating plates with 10 micrograms/ml gpl20 or, respectively,
with 3 micrograms/ml rCD4 and incubating them with serial
dilutions of the mouse sera starting at 1:100 dilution.
Phosphatase-labeled host anti-mouse IgG (obtained from Sigma
Chemicals) was used to reveal bound antibodies. Readings were
performed in a Titertek automated photometer.
Inhibition of qpL20 bindinq to solid phase CD4. After coating
the plates with 3-10 micrograms/ml rCD4, 50 micrograms of gpl20
(5 micrograms/ml) were added and incubated for 1 hour, +/-
serial two-fold dilution of test antiserum or antibody starting
at 100 micrograms/ml concentration. After washing,
phosphatase-labeled monoclonal anti-gpl20 was applied and
incubated 1 hr. The phosphatase activity was then measured by
the rate of PNPP hydrolysisO The inhibition caused by the test
antibody was expressed in percent decrease from the contxol
gpl20 binding.
Effect of mAbs on CD4 bindinq_to solid phase qpl20. Plates
were coated with 3 micrograms~ml gpl20. CD4-phosphatase at 10
- ~ . . .
, ~ ..
.
' . ' ', ' ' ': .. ~: ~ ~ '' ,, :
:. . --- ~
:
W~92/0579g PCT/US9l/OfiO79
2 ~ 12~ 20 ~
micrograms/ml and 3 micrograms~ml were incubated separately for
one hour with PTH or with the test mAbs at concentrations 0~3,
1, 3 and lO micrograms/ml. The mixtures were then adcled to the
coated wells and incubated l hr. After washing, the amount of
bound CD4 was revealed by PNPP.
Effect of qpl20 on CD4 bindinq_to solid-phase captured mAbs.
In separate 96-well plates rgpl20 and phosphatase-labeled rCD4
were serially titrated by 10-fold dilution from 80 and 40
micrograms/ml respectively, in different directions on each
plate. Equal volumes of each were then combined and incubated
2 hr at room te~peratures ~RT). Simultaneously with the above
incubation, the test monoclonals were added to a goat anti-
mouse IgG coated plate for 2 hrs at RT, then washed of excess
sample. Fifty microliters of the titrated complex was trans-
ferred to the captured mAb-GAM plate and incubated it RT for 2
hrs, then washed. PNPP substrate was added and color
developed.
Effect of gpl20_on mAbs bindinq to solid-phase CD4. P 1 a t e s
were coated with 3/micrograms/ml rCD4; 25 microliters of 0.1-
1.0 micrograms/ml rgpl20 and 25 microliters of scalar con-
centrations (0.3-10.0 micrograms/ml) of test mAb were added
together to the wells and incubated for 2 hr. After washing,
the bound mAb was revealed using goat anti-mouse Ig
phosphatase-labeled antibody.
Cross-inhibltion of CD4 bindinq. Competition mapping of mAb
specificities was performed by coating plates with rCD4 and
layering alkaline phosphatase-labeled mAbs in the absence and
presence of graded concentrations of unlabeled test mAbs whose
specific binding sites are known from the literature. OKT4A
(binding in Vl~ and OXT4 (binding in V4) were obtained from
Ortho (Diagnostics Systems, Raritan, NJ), anti-Leu3a (binding
in V1), L83 (V1-V2), L88 tVl-V2), L120 (V4) were a gift from
Dr. David Buck, Becton-Dickinson Laboratories, Mountain View,
CA.
Inhibition_of sYnC~ ia formation. The cell fusion among CD4+
cell lines acutely infected by the virus requires gpl20-CD4
specific binding. The test w~s performed according to Matthews
W092/05799 21 ` 3
et al. (Proc. Natl. Acad. Sci USA 84:5424, 1987). Briefly, GEM
cells chronically infected with either HThV3-:~IIB or HTLV3-RF
were used for each determination. Sera diluted 1:10 were
distributed in 96-well A/2 plates (Costar). Five to 10 x 103
HIV-infected cells were added. 70 x 103 uninfected Molt 4
cells were admixed and the plates were inc:ubated overnight at
37C, after which the number of giant cells (>5 times the size
of the parental cells) were counted at 40 X magnification. The
mouse serum control varied from 50 to 85 giant cells per well.
Results
1. Polyclonal responses to in-iection of complexed sCD4-rq~120
in mice.
Three groups of four mice were injected once, either
with rCD4 alone (Fig. 2, panels A,D,G), with sCD4-rgpl20
complex (Fig. 2, panels B,E,H), or with rgpl20 alone (Fig.2,
panels C,F,I). The weekly bleedings were titrated over a 3-
month period for capacity to bind CD4 (panels D-F~ and gpl20
(panels A-C), and for capacity to inhibit the formation of
syncytia (panels G-I). The overall results were strikingly
different among the three groups. Figure 2 displays the
individual titers for each parameter and for each group. There
were often multiple peaks during the response (panel D) and
mice of a single group showed different timings for their peaks
(panels D and H). When the timing and magnitude of the binding
and neutralizing responses were examined, (a) there was no
clear correlation between the titers of gpl20 binding a~d
syncytia blocking (R versus H); (b) there was inverse relation-
ship between rCD4 and rCD4-rgpl20 immunized groups when rCD4
~inding titers and syncytia blocking were compared (D vs~ G and
E vs. H), ~c) there was a small CD4 binding response in the
group immunized with rgpl20, which could be attributed to anti-
idiotypes (panel F).
2. Polvclonal syncytia-blockinq responses by mice receivinq
CD4-qpl20 comDlexed are not type-specific.
The antisera from mice injected with CD4-gpl20 effi-
ciently block syncytia formation caused by such widely dif-
ferent HIV isolates as HTLV3-IIIB and ~TLV3-RF. Figure 3 shows
. , ~ . .: . .
.
' .'
W092/0~799 PCTtUS91/06079
3 22 ~
individual titrations of the sera from these mice, revealing a
similar rank order o~ the individual responses against the two
isolates (93>94>91>92)~
3. The sync~tia-blockin~ capacity of CD4-q~120 responders_ is
absorbed by CD4 and not by qpl20.
To determine whether the high capacity of sera from
complex-immunized mice to inhibit syncytia formation was due to
anti-CD4 or anti-gpl20 antibodies (or to a cooperative action
of the two) a series of absorption experiments was performed
using c~oluble and solid phase bound antigens. The samples were
subsequently monitored for changes in binding or syncytia-
blocking titers. The results are se-t forth in Table 1 below.
W092/US799 PCT/US91/06079
~ ~3 ~ r9 1 2 ~ 3
able 1
Absorption of pooled sera from mice immunized with CD4-
gpl20, demonstrating that syncytia blocking is associ.ated with
anti-CD4.
.
CD4 binding gpl20 binding # syncytia**
Absorbent O.D.*_ (~) O.D.*~) IIIB RF
none 1.216 (100) .940 (100) 0 3-
gpl20 1.180 (97) .188 (20) 0 5
CD4 .420 (34) .936 (99) 11 24
no antiserum - - - - 52 60
*O.D. 405/60 min (color developed by phosphatase hydrolysis of
PNPP).
**Syncytia blocking assay as described above.
The results unequivocally attributed the syncytia-
blocking capacity to antibodies that recognize the CD4 moiety
of the complex rather than anti-gpl20, since absorbed anti-
gpl20 serum showed unaltered syncytia blocking, while the
decrease of CD4 binding was accompanied by a significant
decrease of syncytia blocking with both isolates tested (Table
1) .
4. The_ca~acit~ to inhibit CD4-~pl20 bindinq in vitro corre-
lates with the CD4-bindinq titer and not with the syncytia-
blockinq titer.
Since the syncytia-blocking capacity observed was
mediated by anti-CD4, the inverse relationship of binding and
syncytia-blocking titers in mice receiving CD4 versus CD4-gpl20
had to be attributed to a difference in the fine speciicity of
the anti-CD4 antibodies in the two groups. To further charac-
terize the anti-CD4 antibodies, one test was to compare their
relative capacity to inhibit gpl20 binding to CD4. The results
are set forth in Table 2 below.
. :
.
:,,
W092/05799 P~T/US9~06079
2~ 2~ ~
~3~ Difference in fine specificity distribution
among polyclonal anti-CD4 anti~odies.
CD4 binding
(microgram ~locking gpl20 binding Fusion blocking
Ab/ml~ ~Uso/ml)* _ us0/ml~
Pool #9
(immuni~ed
CD4~gpl202.4 80.0 45.0
Pool #18
(immunized
CD4) 31.0 890.0 7.0
*One blocking U50 is the amount of antibody that
reduces to 50~ the amount of gpl20 bound to CD4-coated
wells or, respectively, the number of syncytia in the
fusion test. U5p/ml was calculated by multiplying 1
U50 unit by the dllution factor for each test.
The results (Table 2) showed high inhibition titers in
all mice immunized with CD4 alone and low titers in those
immunized with the CD4-gpl20 complex, indicating that the
blocking of syncytia by the latter appeared to be mediated by a
mechanism other than prevention of binding of HIV to its
receptor on the cPll surface.
5. Study of anti sCD4-rpl20 res~onses usina monoclonal anti-
bodies.
In order to dissect the polyclonal response of mice im-
munized with the CD4-gpl20 complex, hybridomas were produced
from one of these animals, and the resulting antihodies were
characterized for capacity to bind CD4, to bind gpl20 and to
block syncytia formation. The results are set forth in Table 3
below.
W092/05799 P~T/US911~6~79
~ 2~
Table 3 List of hybridomas obtained from fusion 91,
hierarchically ordered according to their
supernatant's capacity to bind CD4, and
tested for binding gpl20 and blocking
syncytia formation.
Binding Bindiny # SyncytiaSyncytia
Clone CD4 spl20 at 1/2 Blocking
Desiqnation IO-D.)* (O.D.)* dilution** (%)
1 135 1.363 0
144 1.208 0
148 1.149 .007
1.059 0
215 1.049 0
145 .982 0
84 .932 0
142 .930 .012
140 .937 o ~ .
210 .724 .015
185 .672 0
143 .5g9 .OOg
201 .482 0
J .437 .006
203 .398 .006
o385 .010
156 .372 ~028
58 .313 .011
224 .308 0
146 .255
.182 0 9 80
165 .157 0
36 .153 .041 0 100
172 .145 .006
9~ .136 0 0 100
223 .125 0
59 .121 0
V . 109 0
R .106 .012
48 .106 0
0 .361
0 .358
M (o023) .176
68 0 .116
116 (.012 0 16 65
0 0 18 60
32 0 0 18 60
[control 46]
17Q 0 ~ _ .
*O.D. 405/120 min when the supernatant was tested in ELISA on
plates ooated with CD4 (respectively, gpl20~. See methods
W092/05799 PCT/US91/06079
26
above 2~1253
** Lower than the control are shown.
Table 3 shows an early test of 170 wells with hybridoma
clones, ordered according to their CD4 binding capacity~
Thirty (i.e~, the majority) of the positive clones produced
anti-CD; only four produced anti-gpl20, and the remainder were
negative for both. Three of the anti-CD4 clones (and none of
the anti-gpl20) exhibited capacity to block syncytia. All
borderline positives (i.e. those showing less than 0.100 in the
gpl20 binding) and those with partial blocking of syncytia
(clones 116, 95 and 32) became negative af-ter subcloning.
Table 4 below shows a further characterization of the
hybridomas when the inhibition of gpl20-CD4 binding test was
performed.
Table 4
Classification of mA~s from mice immunized with CD4-
gpl20 complex.
Inhibiting
Binding ~inding CD4-gpl20 Group-Specific
qpl20 CD4Bindinq* Syncitia Blockinq
mAbs:
48, 35, 40 +
68
135, 144, 148 - +
75, 215, 145
210, 185, 143
94, 36 _ + +
- + _ +
*Cut-off for a positive response was 25% blocking at
greater than or equal to a 1:2 dilution.
The anti-CD4 mAbs can be divided into three categories:
a) those that do not inhibit gpl20 binding and do not block
syncytia; b) those that do not inhibit gpl20 binding and block
syncytia; and c) those that inhibit gpl20 binding and block
syncytiaO MAbs 55 and 94 were further studied, as representa-
tives of the latter two categories, respectiv~ly. Both were of
IgG1 isotype.
W092/0~799 ~ 3 P~T/US~1/06079
~; 27
6. Preliminary_ma~pinq experiments. The binding site of both
mAbs 55 and 94 was localized within the first two domains (V1-
V2) of CD4 by binding experiments using the CD4 IgG immunoad-
hesin (Genentech) which contains the two external domains of
CD4 spliced to an immunoglobulin constant region (data not
shown). A cross-inhibition experiment using labeled mAbs 94
and 55, was performed. The binding of these m~bs -to solid
phase CD4 was tested in the presence of a s~ries of anti-CD4
monoclonal antibodies whose epitopes and binding characteris-
tics are shown or partially known from the literature. Theresults are set forth in Table 5 below.
Table 5
Cross-inhibition of CD4 bindina bv mAbs 55 and 94.
_ Inhibitor mAbs
OKT4A LEU-3A L83 L88 L92 L120 OKT4 F91-55 F91-94 F91-3G
mAb 55 - - +/- - - - - + - +
mAb 94 - - - - _ _ _ _ ~ _
*Cut-off point for positivity was 25% or larger
decrease in hinding when the inhibitor was 6 times more
concentrated than the test mAb.
The two mAbs were not inhibited by any of the tested
antibodies with the exception of L83, which produced a partial
but consistent competition (Table 5). These results do not
provide a precise mapping of the binding sites of the two mAbs.
OKT4A and anti-Leu3a bind in the first and second domains of
CD4 and the test showed that neither binding site overlaps with
mAbs 5~ and 94. L83 recognizes a conformational determinant
which is affected by mutations both in region 8-40 ~Vl) and
region 119-188 (V2). These data taken together indicated that
the fine specificity of mAbs 55 and 94 are different from most
studied antibodies and different from each other.
7. Interference of qP120 with CD4 bindinq by mAbs. Preliminary
experiments had shown that mAb 94 blocked gpl20 binding to CD4,
while mAb 55 did not. In order to be able to detect both
li .
.
,
~ ' . .
W092/0~799 PCTt~S9~/06079
2~ 28 ~ ~
inhibition and possible cooperativity between antibody and
gpl20, a series of three ELISA tests were performed by which
the ternary intera~tion of CD4, antibody and gpl20 was examined
by keeping in turn one of the reactants in solid phase, and
varying the concentrations of the other two. The results are
shown in Figures 4, 5 and 6, which also include curves obtained
with reference antibodies OKT4A twhich competitively interferes
with CD4-gpl20 binding) and OKT4A (which does not). In Figure
4 thP binding of labeled CD4 to gpl20 was s:Lightly enhanced or
non-significantly changed in the presence of increasing
concentrations of 55, while it was progressively inhibited by
94. In Figure 5 the binding labeled CD4 to solid phase-
captured antibody was increased 40% by gpl20 in the case of 55
at the highest concentration of CD4, but was unaffected or
slightly decreased at lower concentrations of CDq. In the case
of 94 there was a progressive decrease of CD4 binding in the
presence of increased gpl20 concentrations, more evident when
CD4 was limiting. In Figure 6, the binding of labeled 55 to
solid phase CD4 was moderately enhanced in the presence of 0.1
or 1.0 micrograms/ml gpl20 while the binding of 94 was
depressed in these condi.tions.
The conclusions of this series of experiments are that
a) mAb 94 behaves always as an inhibitor/competitor of the CD4-
gpl20 binding, and b) mAb 55 in certain conditions does not
interfere with the binding in a way similar to OKT4, while in
other conditions it showed a degree of cooperativity with CD4-
gpl20 binding.
Example 5
The ef f ect of immunizing mice with CD4 im~unoadhesin
(12.5 micrograms), rgpl20 (16.5 micrograms) and the combination
of CD4 and rgpl20 (12.5 micrograms and 16.5 micrograms,
respectively) was studied. Three groups of f our mice were
immunized as described in Example 4 above, and their antibody
response to the immunogens was examine~ 30 days post-immuniza-
tion using the techniques described in Example 4 above. The
results are set forth in Table 6 below.
W092/05799 PCT/US91/n6079
2~ ~ 2~j3
29
I M M U N I Z A T I O N
16.5 microgram 12.5 microgram 16.5 microgram gpl20
gpl20 Immunoadhesin 12.5 microgram Immunoadhesin
51 1 3.652 1 053 1 1.7
2 3.0 2 0 2 3.3
3 3.6 3 0 3 3.7
4 3.~ 4 0 4 3.4
gpl20 Binding
55 1 ~.357 1 056 1 4.8
2 2.8 2 0 2 3.6
3 3.8 3 0 3 ~.0
4 4.3 4 0 4 3.3
.
51 1 0 52 1 2.053 1 2.0
2 0 2 3.9 2 2.0
3 0 3 1.7 3 2.4
4 0 4 2.0 4 2.3 ,
CD4 Binding
1 0 57 1 3.35~ 1 3.8
2 0 2 4.0 2 3.7
3 0 3 4.0 3 3.9
4 0 4 3.9 4 3.6
51 1 0 52 1 053 1 1.0
~ 0 2 0 2 1.9 -
3 0 3 0 3 2.2
4 0 4 0 4 1.6
Syncitia Blocking
55 1 0 57 1 056 1 1.6
2 0 2 0 2 1.3
3 0 3 0 3 0
4 ~ 4 0 4 1.0
As can be seen in the date set forth in Table 6 above,
mice receiving the complex Immunoadhesin-rgpl20 responded to
gpl20 with antibody titers similar to those receiving only
rgpl20 while these mice responded to CD4 with antibody titers
sLmilar to those receiving Immunoadhesin alone. ~owever, mice
which were immunized with Immunoadhesin-rgpl20 were the only
group of mice which showed significant-titers of syncitia-
blocking antibodies.
All cited literature and patents or patent applications
are incorporated by reference in their entirety.
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W092/05799 PCT/US91/06079
2~ %~3 30
App-endix of Materials Sources
CD4, CD4-Immunoadhesin Genentech Inc., South San Francisco, CA
Freund's Adjuvant Difco Laboratories, Surrey, England
goat antimouse
immunoqlobulin Sigma Chemical Co., St. Louis, Missouri
gpl20 Genentech Inc., South San Francisco, CA
microtiter plates Linbro available from Fisher Scientific,
lS Springfield, N.J.
96A half-well plates Cos-tar available from Fisher Scientific
Springfield, N.J.
PNPP Sigma Chem~cal Co., St. Louis~ Missouri
Tween Sigma Chemical Co., St. Louis, Missouri
Titertrek Multiskan
Apparatus Flow Laboratories, McLean, Virginia
RPMI 1640 GIBCO, Grand Island, N.Y.
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