Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~YO 91/10742 2 ~ 7 3 ~ 6 0 PCI`/US91/00319
MONOCLONA:~J ANTIBODY SPECIFIC FOR NON~
IMMUNODOMINANT EPITOPE OF HIV PROTEINS
Backqround of the Invention
This invention relates to antibodies specific for
Human Immunodeficiency Virus (HIV).
HIV is the proposed causative agent of Acquired
I~mune Deficiency Syndrome (AIDS). (Popovic et al.,
1984, Science ~24:497). It is a pathogenic human
retrovirus whose genome is capable of encoding at least
six gene products. The env gene encodes a 160 kDa
glycosylated protein (gpl60) that is processed by
proteolysis into a 120 kD external glycoprotein (gpl20)
and a 41 kD transmembrane protein tgp41). gpl20 is
anchored to the virion by noncovalent interactions with
gp41. gp120 and gp41 are present on the surface of both
virion particles and virus-infected cells.
Different strains of HIV vary in the amino`acid
sequences of proteins encoded by the viral genome,
particularly in the amino acid sequence of the external
envelope glycoprotein gpl20 (Starcich, 1986, Cell 45:637;
Hahn et al., 1986, Science 232:1548). Over its entire
length, the gpl20 polypeptide sequence varies from one
HIV variant to the next by approximately 20-25%. The
extent o~ variation is not constant over the whole
envelope protein. There is a pattern ~f conserved and
variable regions, which suggests that the protein is
divided into regions responsible for distinct functions.
A num~er of different regions have been identified; for
example, the CD4 binding domain, the principal
neutralizing determinant/ and cytotoxic T-cell
recognition determinants.
~ he use of antibodies to target cells has
previously been used in the treatment of cancer and other
disorders. Zarling et al. (EPO 308 936) disclose
... '
~BSTIT~Jl E S~IE~T : '
_._
WO 9}/10742 2 ~ 7 3 ~ ~ ) PCT/US91/00319
antibo~y heterocon~ugates specific for the gpl20
principle neutralizing domain which selectively kill HIV-
infected cells. Pincus et al. (J. Immunol. (1989)
142;3070) describe antibody-toxin conjugates that also
5 recognize an immunodominant region of gpl20, and Till et ~
al. (Proc. Nat. Aca Sci., 1989, 86;1981) disclose anti- - ?
gp41-toxin conjugates.
Summary of the Invention
The invention features an antibody capable of
10 recognizi~g a non-immunodominant epitope of the envelope
protein of HIV, wherein the binding of the antibody to
the envelope protein is not blocked by serum from an HIV-
infected patient. As used herein, "antibody" refers to a
whole antibody molecule, or to a fragment ox to a
15 modification of an antibody, e.g. a fragment of an
antibody bay be the Fab2 fragment of the molecule, the
Fabl fragment , or the heavy or light chain alone; and a
modification, for example, may be a linear polypeptide
molecule which includes both the heavy and light chains,
as described in Huston et al., WO B8/09344 and Ladner et
al., WO 88/01649. As used herein, "non-immunodominantll
epitope means an amino acid ~equence within the natural
confor~ation of a protein that is not significantly -
immunogenic; i.e., does not e~icit an antibody response ~;
~5 in at least 75% of human patients. An antibody directed
toward a non-im~unodominant region of the HIV envelope ;
protein can, according to the invention, bind that region
due to the absence or low level of potentially
competitive circulating antibodies. In contrast, an
antibody directed toward~ an immunodominant region of the
envelope protein will be partially or completely blocked
from binding the targeted envelope protein region due to
the presence of antibodies in the patient due to the
patient's natural immune response to HIV infection. The ;
35 non-immunodominant envelope region may be`within the ~ ~
: '
SUE~STITVTE SHEET
.,` . ': :.:. , ' ,,.' ,': ' ' : ' ' '' :: ,, `` ` . ' ' ' ': :``: ' : ,` . :` ' . ' ': ' : ' ` .
~091/10742 2 0 7 3 ~ ~ ~ PCT/USg1/003~9
3 --
gpl20 or the gp41 portion of the Pnvelope protein. Non-
immunodominance can be measured in vitro by binding the
antibody to a target antigen in the presence of HIV-
positive human serum; an antibody specific for a non-
immunodominant epitope of the target antigen willdemonstrate a comparable binding efficiency to the target
antigen in the presence or absence of HIV positive serum.
The binding efficiency of the antibody to the target
antigen in the presence of HIV-positive serum is at least
80% of its binding efficiency in the absence of HIV-
positive serum.
In preferred embodiments, the non-immunodominant
epitope recognized by the antibody is group common. As
used herein, "group common determinant" means an
antigenic portion of a protein encoded by an H~V strain
that is not specific for that strain only, but is present
on at least one other HIV strain. Preferably, the
antibody is capable of binding to the surface of HIV
envelope glycoprotein expressing cells; is capable of
recognizing the region of the envelope protein between
amino acid residues 473 and 759, inclusive, according to
the numbering convention of Ratner et al., 1985, Nature
313:277; and is capable of recognizing that portion of
gp120 contained within the 473 through 7S9 amino acid
region, one example of such an antibody is that produced
by cell line A.T.C.C. No. ~B 10321.
In other pre~erred embodiments, the antibody is
covalently linked to a toxin to ~orm a conjugate, and the
conjugate is capable o~ killing HIV-in~ected cells in the
presence o~ human HIV~ serum; killing may occur via
internalization of the antibody-toxin conjugate by HIV-
infected cells. The conjugate may be made at the protein
level by chemically linking the antibody and the toxin
molecule or at the DNA level, by cloning the DNA sequence
'
... :~.
: .
8UE~STITUTE SHEET
.- . . : . . . , ` :. `.
W091~10742 207'~`~G ~ PCT/USgl/003t9
-- 4 --
encoding the antibody and ligating it to a toxin-encoding
DNA sequence, as described in Huston, s~pra.
As employed herein, the term "toxin" is used to
include the commonly designated toxins such as poisonous
lectins, ricin, abrin, modeccin, diphtheria toxin,
pseudomonas exotoxin or preferably the toxic A chain
portions thereof, as well as other toxic agents such as
radioisotopes, cytotoxic and carcinostatic drugs.
"Toxin" may also refer to combinations of the various
toxins, which can be coupled to one antibody molecule
thereby accommodating variable cytotoxicity.
In another preferred embodiment of the inventiQn,
the HIV-specific antibody is linked to a second antibody
that is specific for an effector cell to form an antibody
heteroconjugate (also known in the art as
heteroaggregates or heteroantibodies). The anti-HIV
antibody of the heteroconjugate ~inds to an HIV-infected
cell, i.e., the target cell to be killed, while the anti-
effector antibody of the heteroconjugate binds to an
effector cell such as those found within the peripheral
blood lymphocyte (PBL) population, e.g., cytotoxic T
lymphocytes (also known as T cells), monocytes (in
particular, macrophages), granulocytes, or large granular
lymphocytes which inclùde cells with natural killer
acti~ity or antibody-dependent cellular cytotoxic
activity, with the result that the antibody components of
the heteroconjugate bridge the effector and target cells
and thus promote killing of the target cell by the
cytotoxic e~fector cell.
HIV-$nfected patients can be treated by
administering an amount of the antibody-toxin conjugate
or an antibody heteroconjugate of the invention
sufficient to kill HIV-infected cells. During active
production of ViXU5, the viral envelope protein is
expressed on the surface of infected cells. By
SUBSTITUTE SHEEI-
WO91/10742 2 ~ 7 ~ a ~ ~ PCT/US91/00319
selectively killing the cells in which the virus is
replicating using an antibody conjugate that reacts with
virus-specific cell-surface antigens, the infectious
cycle of the virus may be interrupted.
one advantage of an antibody or antibody-toxin
conjugate sr an antibody heteroconjugate of the invention
is the non-immunodominance of the HIV envelope
glycoprotein epitope that the antibody is specific for.
A consequence of non-immunodominance, or failure of the
human immune system to mount a detectable immune response
to an epitope, is that there are few, if any, circulating
antibodies to that epitope; i.e., to a gpl60 non-
immunodominant epitope of the envelope protein in an non-
immunodominant epitope of the envelope protein in an HIV-
lS infected patient. Therefore, when an antibody-toxin
conjugate or antibody heteroconjugate of the invention is
used to treat an HI~-infected patient, it can selectively
target HIV-infected cells without competing with
circulating antibodies for the target non-immunodominant
epitope.
Another advantage of certain antibodies, antibody-
toxin conjugates, and antibody heteroconjugates of the
invention are their ability to recognize a group common
determinant of the ~IV envelope glycoprotein. Group
common determinants are portions of the envelope
polypeptide that are essentially invariant among
di~ferent HIV strains. Therefore, an antibody capable of
recognizing a group com~on determinant can recognize
gpl60 from any strain o~ HIV. Treatment of an HIV-
infe¢ted patient according to the in~antion will thus notbe limited to any one strain of HIV, but will inc}ude all
strains to which the target determinant is common.
Other features and advantages of the invention
will be apparent from the following description of the `
preferred embodiments thereof, and from the claims.
, .
~'
S~lBSTlTlJl-E S~
WO91/10742 ~ 0 7 ~ Jvi PCT/US91/00319
- 6 -
pesçription of the Preferred Embodiment~ -
we first briefly describe the drawings.
Drawinqs
Fig. l is an schematic illustration of the gpl60
protein, showing the gpl20, gp41, pl21, and pENV9
regions.
Fig. 2 is a graph showing results of an ELISA in
which the binding specificity of the lCl antibody to a
target antigen was tested.
Figs. 3(a) - 3(d) are graphs showing results of
ELISAs in which the binding speci~icity was determined
~or the lCl antibody or a control antibody in the
presence of HIV positive serum.
Figs. 4(a) - 4(d) are graphs showing results of
FACS analyses using the lCl antibody.
We now describe the preparation and use of
antibodies of the invention.
Immunoqens
Two immunogens were used to generate antibodies of
the invention: gpl60 (Repligen Corp., Cambridge, MA) and
the envelope protein fragment extending from amino acid
residue 473 through residue 759, denoted pENV9 (Ivanoff
et a}., U.S. Patent No. 4,861,707).
gp 160 was prepared for immunization by
emulsification in complete Freund's adjuvant (CFA)
according to standard techniques (Difco Labs, Grand
Island, NY).
Production Q~ Monoclonal Antibodies
Balb/cJ female mice (Jackson Labs., Bar Harbor,
ME) were immunized intraperitoneally with 70~g per mouse
o~ gpl60/CFA. ThQ mice were given a booster immunization
of gpl60 in an emulsification in incomplete Freund's
adjuvant three weeks later. Mice were ~led and the sera
assayed for the presence o~ antibodies reactive with the
immunogen. Mice showing a strong serological response
Sl)BSTITUTE SHEET
.i. ~ .. ........ ~, - ., ~ . . . ..... . . .. . .
W09lt1074~ 2 ~ 7 ~ P~T/US9~/00319
were given a final booster immunization of response were
given a final booster immunization of pENV9 in soluble
form five weeks ~ollowing the first booster immunization
and, 3 days later, spleen cells from these mice were
fused at a ration of 5:1 with SP2/0 (A.T.C.C. No.
CRL8287, A.~.C.C. No. CRL8006) myeloma cells incapable of
secreting both heavy and light immunoglobulin chains
(Kearney et al., J. Immunol., 1979, 123:1548), ~y
standard procedures based on the method of Kohler and -
Milstein, Nature (1975) 256:495. ~ `!
Supernatants from hybridomas which appeared 10-21
days after fusion were screened for production of
antibodies reactive with the pENV9 protein fragment.
Each well of a 96-well Costar flat-bottom
microtiter plate was coated with pENV9 by placing a fifty
microliter aliquot of a P~S solution containing the
protein fragment at a final concentration of 2-10 ug/ml
in each well The pENV9 solution was aspirated, the
wells washed and replaced with PBS + 0.5~ BSA and
incubated for 2 hours. Followi~g incubation, the wells
were aspirated, washed, and 50 ul o~ hybridoma
supernatant was added and incubated for 2 hours.
Following incubation, the wells were washed 3 times with
PBS, and then incubated for 1 hr. with 50 ul of an
appropriat~ dilution of goat anti-mouse immunoglobulin
conjugated with horseradish peroxidase (HRP, Boehringer
Mannheim, West Germany). The wells were wash~d again 3
times with PBS and 100 ul of 1 mM ABTS (2,2 azino-bis (3-
ethyl benzth, azoline 6-sulfonic acid) in O.lM Na-
Citrate, p~ 4.2, to which a 1:1000 d~lution of 30% H2O2had been added), the substrate for HRP, was added to
detect bound antibody. After 30 minutes, absorbance was
measured at OD410 on a Dynatech spectrophotometric
aukoreader ~Virginia).
.,
SUE~STITUTE SHEET
W09l/10742 2 ~ r~ PCT/VS91/00319
Four hybridomas (lCl, lH9, 2D7, and 2H8) that
tested positive for binding to the first immunizing
antigen, gpl60, were tested for their ability to bind to
the second immunizing antigen, pENV9, and to gpl60 from
another different HIV strain. One hybridoma clone,
designated lCl, which demonstrated reactivity with the
pENV9 fragment by ELISA, produced antibody which was also
capable of recognizing an envelope protein determinant ~ ,
that is present in more than one strain of HIV, i.e., a
group common determinant, as described below.
The above four antibodies were tested in an ELISA
for bi~ding to either gpl60 from the ~IV-IIIB isolate or
gpl60 from the HIV-RF isolate. The results, presented in
Table I below, show that the pENV9-specific lCl antibody
binds to gpl60 of both the IIIB and RF isolates, as well
as to pENV9, whereas the remaining three antibodies do
not recognize all three proteins.
Table I
clone gpl60~ gpl60RF pENV9 BSA
(control)
lCl + ~ +
lH9 + _
2D7 + +
2H8 ~ + O - -
The isotype of the lCl clone was determined by the
ELISA method to be IgG2~ using goat-anti-mouse HRP (Zymed
Labs, San Francisco, CA) preparations which correspond to
each of the major immunoglobulin isotypes. ~he lCl clone
was subcloned and rescreened ~or the ability to bind to
the Antigens described above. The lCl subclone was
expanded by intraperitoneal in~ection into pristane
primed Balb/c mice. Ascites fluid was recovered from the
mice and the antibody was purified by Protein A affinity
chromatography, as described below.
Ampli~ication and Purification of Monoclonal Antibodies
5UBSTIT~JTE SHEFT'
; ~ ' ' " ' '" "' ' ' ' '' . ' ,'; ' ,
. "" '` " ' ', . ' '
, ''.. ~ ,, ,`', ' ', "" ', " ` ' ,, ,, :, ,, '
WO 91/10742 2 ~ 7 3 ~3 ~ ~ Pcr/usg1/0031g
g
Purified lC1 antibody was prepared by injecting a
hybridoma subclone that repeatedly tested positive by
ELISA intraperitoneally into pristane-primed syngeneic
mice. The ascites which developed were recovered two to
three weeks after injection, and the antibody was
purified as follows, and then dialyzed against PBS.
Ascites fluid containing IgG2a lC1 antibody was
diluted five-fold in 0.1 M Tris/3 M NaCl pH 8.9, bound to
a Protein-A-Sepharose affinity column equilibrated with
the same buffer, and then eluted from the column with
Q.15 M NaCl, 0.1 M acetic acid, Ph 3Ø Following
elution, the antibody was immediately neutralized by the
addition of 1 M Na2HC03.
Bindinq SPecificit~ of the lC1 AntibodY
The lC1 antibody was tested in the ELISA ~or
binding with pENV9, gp120, and pl21, in order to map the
epitope to which lC1 binds. pl21 ~Chang et al~ U.S.
Patent No. 4,774,175) is an 83 amino acid protein
fragment spanning amino acids from approximately 566-648
within the gp41 portion o~ qpl60, and is completely
contained within the pENV9 se~uence. (These regions of
gpl60 are schematically illustrated in Fig. 1.) pl21
includes a major immunodominant epitope of the gp41
protein ~Chang et al., U.S. Patent No. 4,724,175 and Wang
et al., 1986, ~E~. Nat. Aca. Sci. 83:6159). Fig. 2
shows the results of the ELISA assay. These results
demonstrate lC1 binds specifically to both pENV9 and
gpl20, but does not bind pl21. Therefore, the lCl
antibody binds to a region of pENV9 that is also present
30 in gpl20, but that is not contained within the pl21 `
portion. In a control experiment, an antibody specific
for gp41 (Epitope, Inc., ~eaverton, OR) was tested ~or
binding to either pENV9, pl21, or gp120, showing that the
anti-gp41 antibody bound to pENV9 and pl21, but did not
bind to gpl20, as expected.
~U~STITlJTE SHEET
WO91/10742 PCT/US91/~319
2~7~
-- 10 --
Bindinq of the lCl Antibodv in the ;:
Presence of Human Serum
A therapeutically useful exogenous antibody
specific for the HIV envelope protein must be able to
bind HIV, or HIV-infected cells expressing the envelope
protein, in the presence of competing circulating
antibodies.
The lC1 antibody was tested for its ability to
bind to target antigen in the presence of serum from an ~
lo HIV-infected patient, and compared with a control ~-
antibody known to bind to an immunodominant region of ;
gp41. Figs. 3(a) - 3(d3 show the results of ELISAs in
which microtiter wells were coated with the capture ~ -
antigen. The lCl or control antibodies were then added
in 50 ul of (1) undiluted HIV-negative serum, (2)
undiluted HIV-positive serum, or (3) 0.5% BSA. After 2
hrs., the wells were washed and secondary antibody (sheep
anti-mouse-H~P) which did not cross-react with human Ig
was added. After 1 hr., the secondary antibody was
removed, the wells were washed, ABTS was added, and the
OD410 was measured after 30 min.
In ~ig. 3(a), the lC1 antibody was assayed for
binding to the capture antigen, pENV9. ~he results
demonstrate that lC1 bound with almost egual efficiency
to pENV9 in the presence of either HIV-positive serum or
HIV-negative serum (Fig. 3a). Although the lC1 antibody
bo~nd with the highest efficiency to pENV9 in the
presence o~ HIV negative serum at antibody concentrations
above 0.01 ~g/ml, the binding o~ this antibody to pENV9
in the presQnce of HIV positive serum was about 88~ as
efficient as binding to pENV9 in the presence of HIV
negative serum at an antibody concentration of 1 ~g~ml,
and over 95% as efficient at 10 ~g/ml. Similar results
were obtained when lCl binding was tested in the presence
o~ HIV positive sera from four other patients. These
SU8STITUTE SHE~ET
WO 91tlO742 2 0 7 3 ~ 5 ~ PCT/US~1/G0319
results demonstrate that, if there are pENV9-specific
antibodies present in HIV positive serum, they are
present in low enough titers or have a weak binding
affinity or react with different pENV9 epitope so that
they do not significantly interfere with the binding of
the lC1 antibody to pENV9. Thus, lC1 is potentially
useful as a therapeutic agent.
In Fig. 3(b), khe ability of the lCl antibody to
bind the pl21 protein was tested. The lC1 antibody did
not bind pl21 at all. (~he minimal reactivity observed
with pl21 at lC~ concentrations above O.l~tml in 0.5~ BSA
was probably due to nonspecific binding.)
Two additional control experiments were performed
using a control antibody, anti-gp41 (Epitope, Inc.) and
tow target antigens, pl21 and pENV9. Results present in
Figs. 3~c) and 3~d) show that anti-gp41 binds to both
target antigens, but binds more ef~iciently to pl21 than
to pENV9, at antibody concentration~ above 0.1 ~g/ml.
The results also show that, at these same concentrations
of antibody, the binding of anti-gp41 antibody is
partially blocked by HIV positive serum. These results
indicate that there are antibodies specific for pl21 and
p~NV9 present in ~IV positive serum that are capable of
partially blocking the binding o~ the anti-gp41 antibody.
Bindina of lCl An~ibody_to Cells
Whether the lC1 monoclonal antibody binds to the
surface of cells expressing the HIV envelope glycoprotein
was determined by indirect immunofluorescence and
analysis by FACs (Fluorescence Activated Cell Sorter,
~ethods in Enzymology, 1984, Parks et al., 108:197), as
~ollows.
The lCl antibody was bound to either CV1 cells
~A.T.C.C. No. CCL70) infected with a Vaccinia Virus
reaombinant containing the HIV env gene, which express
both gpl20 and gp41 on their surface (CV1-Env), or CV1
SUBSTITUTE SHEET
: ;~
WO91~10742 PCT/US91/00319
2 ~ 7 ~
- 12 -
cells infected with a Vaccinia Virus recombinant that
does not contain the HIV env gene (CVl-Lac), as a
negative control. Construction of a recombinant capable
of expressing the full-length HIV envelope gene is
described in EPO 243 029, hereby incorporated by
reference. Figs. 4~a) - 4(d) show these results.
In Fig. 4ta), the lCl antibody was bound to CVl-
Env cells. If Fig. 4(a) is superimposed on Fig. 4(b),
which shows a FACS profile for CVl-Lac cells incubated
with the lCl antibody, there is a rightward ~hift (i.e.,
an increase) in fluorescence intensity in the CVl-Env
cells compared to the CVl-lac cells, indicating that the
lCl antibody binds HIV envelope glycoprotein expressing
cells significantly better than the cells not expressing
the enYelope protein. This result is significant because
it demonstrates that lCl binds to the target antigen in
its native state, and suggests that cells expressing HIV
envelope glycoprotein may be specific targets for an
immunotoxin conjugate composed of lCl linked to a toxin.
Figs. 4(c) and 4(d) are controls in which the lCl
antibody was bound to uninfected CVl cells and in which
buffer alone was bound to CVl-Env Cells, respectively.
(The background fluorescence that is apparent in the FACS
analyses of CVl-Env and CVl-Lac cells is probably due to
alterations in the cell membrane resulting from
expression of foreign viral proteins.)
Pre~aration of ~ntibod~ Coniu~ates o~ Heterocon~u~ates
$he antibodies may be conjugated to cytotoxic
agents and used as immunotoxins (as described in, e.g.,
Vitetta et al., 1987, Science 238: 1098), or
incorporated onto the surface of liposomes containing
anti-HIV drugs or toxins to specifically target such
drugs or toxins to HIV-infected cells. As employed
herein, the term immunotoxin refers to a conjugate of an
antibody with one or more toxins. Where co~binations of
. . .
S~JB8T~ E 9HE~Ef .
.. . .. .. . .. . .. .
WO~1/10742 ~ 0 7 3 ~ ~ ~ PCT/USgl/00319
- 13 -
various toxins are coupled to one antibody molecule,
coupling may occur by different chemical mechanisms; for
example, covalent binding, affinity binding,
intercalation, coordinate binding and complexation. The ~`
preferred coupling of the antibody to the toxin is,
however, covalent binding either by chemical or genetic
fusions.
In a pre~erred embodiment, the immunotoxin
comprises an antibody reactive with a non-immunodominant,
group common epitope of the HIY envelope protein linked
to the exotoxin form Pseudomonas aeruqinosa. Pseudomonas
exotoxin (PE) is particularly preferably to other toxins
because large amounts are easily prepared, because humans
do not usually have neutralizing antibodies against it,
and because it does not need to be separated into
subunits be~ore being conjugated. PE is an extremely
active monomeric protein (~olecular weight 66kD),
secreted by P. aeru~inosa which inhi~its protein
synthesis in eukaryotic cells. A preferred form of`PE is
the truncated molecule, designated PE40, from which the
cellular binding domain has been removed (Pastan et al.
EP Publication No. 0 261 671). PE~0 can be linked to an
antibody o~ the invention by chemical coupling, for
exa~ple, using the heterobifunctional cross-linker SPDP `
(N-succinimidyl-3-~2-pyridyldithiol~ propionate) Sigma,
St. Louis, M0) ~Pastan et al, 1986, Cell 47:641), or by
genetic fusion ~Cbaudhary et al.,) 1989, Nature 339:394).
Where an antibody heteroconjugate is preferred, any
suitable method of conjugation of the antibodies may be
used; for example, preferred method involves cross-
linking the antibodies using the cross-linker SPDP
according to the method of Karpovsky et al. (1984, J. ~`
Exp. Med. 160:1686). Following cross-linking, the
heteroconjugates are separated from fee antibody by size
35 exclusion chromatography. `;;
,:
SURSTITUTE SHEET
wo 91~ln742 ~ o 7 ~ ~ ~J~ PCT/~S91/00319
An antibody-toxin conjugate or heteroconjugate of
the invention can be tested for target specificity and
efficiency by killing by incubating the conjugate with
HIV chronically-in~ected cells that express HIV envelope
glycoprotein or with uninfected cells, and pulsing the
cells with 3H-Thymidine of 14C-Leucine. Toxicity can be
measured by a decrease in cell division or protein
synthesis in the infected cells relative to the
uninfected control cells. The efficiency of cell killing
can be calculated using a clonogenic assay, in which
infected cells are incubated with a conjugate of the
invention, plated by limiting dilution (Tarwell, 1981, J.
Immunol. 126.1614), and the number of surviving cells is
compared to identically treated uninfected cells.
Use
In a typical treatment employing antibodies of the
invention as immunotoxins, the antibody (which binds to a
protein that is expressed only in HIV-infected cells) is
conjugated to a toxin (e.g. pseudomonas exotoxin~ that i5
toxic to the HIV-infected cells ~and to non-infected
cells as well). By coupling the cytotoxic agent to the
antibody, a high level of toxic efficacy can be achieved
specifically against the targe~ cell with a ~arkedly low
level of non-specific toxicity. The use of the toxic
agent is possible because the antibody to which the agent
is coupled will carry the agent specifically to the
target (in this case, HIV-in~ected cells), thereby
sparing non-infected cells from the toxin. Techniques
that may be employed to con~ugate antibodies to cytotoxic
agents are described in detail in ~itetta et al, su~ra,
and in European Patent Applica~ion No. 279,668, published
August 24, 1988.
The antibodies of the invention can be
incorporated into conventional pharmaceutical
formulations ~or use in treating individuals that are
Sl.3BSTIT~JTE SHEET
` . . . . ., -
: .. , :
WO91/10742 2 ~ ~ 3 ~ ~ Q PCT/US9l/00319
-- 15 --
infected with HIV. In addition, such formulations may
comprise pharmaceutically-acceptable 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
material may be used as diluent , carriers or solvents in
preparing the pharmaceutical formulations comprlsing
antibodies of the invention.
~he phaxmaceutical compositions may be in a
variety of dosage forms which include solid, semi-solid
and liquid forms as powders, pills, tablets, liquid
solutions or su~pension, suppositories, polymeric
microcapsule, liposomes or injectable or infusible
substances. The pharmaceutical formulations may be
administered using conventional methods which include,
but are not limited to, intravenous, oral, subcutaneous,
intraperitoneal or intralymphatic. In addition, the
antibody, i~munotoxin or heterocon~ugates of the
invention may be administered in conjunction with other
treatments to augment the e~fectiveness of the treatment.
Other E~bodiments
Other embodiments are within the following claims.
For example, since, for the most part, monoclonal
antibodies are produced in species other than humans,
they are often i D unogenic to humans. In order to
successfully use these monoclonal antibodies in the
treatment of humans, it may be necessary to create a
chimeric antibody molecule wherein the portion o~ the
polypeptide involved with ligand binding ~the variable
region) is derived ~rom one specie~ and the portion
involved with providing structural stability and other
biological functions (the constant region) is derived ;~
from a human antibody. Methods for producing chimeric `
antibodies in which the variable domain is derived from
one host and the constant domain is derived from a second
SUE~STITUTE SHEET
- . :, ......... ... . ~` : . . . :
~ ;: ; ? :::: ~ ` :
.... . . . ..
wo 91~10742 ~ 0 7 ~ ~ J~ PCT/US91/~319
- 16 -
host are disclosed by ~euberger et al. (Wo Publication
No. 86/01533) and Morrison et al. (EP Publication No. o
173 494), hereby incorporated by reference.
An alternative method, in which an antibody is
produced by replacing only the complementarity
determining regions ~CDRs) of the variable region with
the CDRs ~rom an immunoglobulin of the desired antigenic
specificity, is described by Winter (GB Publication No.)
2 188 638). For example, the CDRs of a pENV9-specific,
murine monoclonal antibody which recognizes a group
common determinant and a nonimmunodominant domain can be
grafted onto the framework of a human antibody by
recombinant DNA techniques. This arrangement is ;~
particularly beneficial for use in the therapeutic
applications of monoclonal antibodies.
DePosit
Cell line lC1-lH5 was deposited in the American
Type Culture Collection on January 10, 1990, and assigned
Accession Number HB 10321.
Applicants' assignee, Repligen Corporation,
represents that the A.T.C.C. is a depository affording
permanence of the deposit and ready accessibility thereto
by the public i~ a patent is granted. All restrictions
on the availability to the public of the material so
deposited will be irrevocably removed upon and granting
of a patent. ~he material will be available during the
pendency of the patent application to one determined by
the Commissioner to be entitled thereto under 37 C.~.R.
1.15 and 35 U.S.C. 122. The deposited material will be
maintained with all the care necessary to k~ap it viable
and uncontaminated for a period of at least five years
after the most recent request for the furnishing of a
sample of the deposited microorganism, and in any case,
~or a period of at least thirty ~30) years after the date
of deposit or for the enforc-able life of the patent,
. i . " : .
,
W~91/10742 PCT/US91/0~319
2~7~Q6~
- 17 -
whichever period is longer. Applicants' assignee
acknowledges its duty to replace the deposit should the
depository by unable to furnish a sample when requested
due to the condition of the deposit. A copy of the
5 A . T ~C.C. Budapest Treaty deposit receipt will be
furnished upon request.
BS~lT~ S~EE~ ~
.. .. ..... . , . .................. ... . .; .. ~ .. . .... ~ . ~ .
::: .
