Note: Descriptions are shown in the official language in which they were submitted.
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Anti-HIV Monoclonal Antibody
The present invention relates to an immunological
technique which provides a novel substance useful for
prevention, treatment, diagnosis of viral infection, and for
study of biochemistry and histology. More particularly, it
relates to a monoclonal antibody having a broad neutralization
spectrum against human immunodeficiency virus (HIV), a
causative virus of acquired immunodeficiency syndrome (AIDS),
a hybridoma secreting said antibody and a process for
preparing the same.
The present invention further relates to a humanized
recombinant monoclonal antibody for clinical application.
Human immunodeficiency virus (HIV) is a human
retrovirus which causes a series of diseases such as acquired
immunodeficiency syndrome (AIDS) and AIDS related complexes
(ARC). Today, these diseases have become a serious problem
in the world, but no vaccines or established therapies
effective for these diseases have been provided.
As an anti-viral agent against HIV, reverse
transcriptase inhibitors of nucleic acid analogues such as 3'
azido-2',3'-dideoxythymidin(AZT)or2',3'-dideoxyinosin (ddI)
have been used, and thereby therapeutic efficacy such as
inhibition of viral growth, increase in the number of CD4
positive cells and prolongation of life span has been
_;:
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observed. However, in most cases, therapeutic efficacy of
these drugs to AIDS is partial or temporal, and in addition,
these drugs exhibit toxicity or growth inhibition to hemato-
poietic cells, and thereby inhibit reconstruction of an immune
system which has become deficient . From these points of view,
development of more effective anti-HIV agents has been
desired.
An antibody is an important protein which plays a
role in an immune reaction in mammals including humans and has
a function to neutralize and remove foreign substances invaded
from outside or substances recognized as foreign substances
by the living body. In this respect, an antibody is expected
to be useful for treatment of infectious diseases.
Karpas et al. observed remission of clinical
symptoms after administration of anti-HIV antibodies derived
from healthy patients infected with HIV to AIDS patients
(Proc. Natl. Acad. Sci. USA, 85, p.9234 (1989), Proc. Natl.
Acad. Sci. USA, 87, p. 7613 (1990)). Jackson et al. also
obtained similar results (Lancet, 2, p. 647 (1988)). These
results show usefulness of an antibody therapy in AIDS.
Apart from such a passive immunotherapy, an active
immunization of patients with a component vaccine of HIV has
also been attempted in order to enhance immune capacity (AIDS
Res. Hum. Retroviruses, 8, P1051 (1992)). This treatment was
found to be effective to patients who have not yet developed
symptoms, but did not show significant effect in patients who
CA 02157874 2004-03-12
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developed AIDS with a decreased number of CD4-positive cells
since they are deficient in active immune response.
Accordingly, in the case of those patients whose disease has
progressed, one cannot but rely on passive immunotherapy, and
hence, a neutralizing antibody has a great significance.
Epitopes recognized by an antibody neutralizing HIV
are located in a glycoprotein antigen having a molecular
weight of about 1. 2 x 105 daltons ( gp120 ) present on a coating
membrane of HIV, a transmembrane glycoprotein antigen having
a molecular weight of about 4 . 1 x 104 daltons ( gp41 ) and a
nuclear protein antigen having a molecular weight of about 1.7
x 104 daltons (p17). Among these epitopes, that located in
the third variable region (V3) of gp120 (amino acid number
303-338), which is also referred to as Principal Neutraliza-
tion Domain (PND), can induce a potent neutralizing
antibody, and hence, is a major target in developing medica-
ments or vaccines.
- Although a correct role of PND region in viral
infection still remains unknown, it is assumed to help
invasion of viruses after binding between gp120 and CD4. PND
region also plays an important role in formation of multinu-
clear giant cells by CD4-positive cells. Accordingly, if an
antibody which binds to this region and inhibits infection and
growth of viruses is prepared, this can possibly be an
effective anti-HIV agent.
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However, since PND region shows a high variability
in amino acid sequence as compared to other epitopes in gp120,
most of the monoclonal antibodies which recognize this region are
viral strain-specific neutralizing antibody which recognizes
only a specific HIV strain. If such strain-specific
monoclonal antibody is used for treatment or prevention, its
efficacy is restricted to those patients who are infected with
HIV strain that can be neutralized with that antibody.
Furthermore, in an individual HIV-infected patient, HIV is
never present as a single HIV strain but usually as
quasispecies of many HIV variants whose amino acid sequence
show several percent variation.
Therefore, the possibility of a monoclonal antibody as
a medicament is closely related to what extent of the many HIV
variants present in patients or within a single patient said
antibody can bind and neutralize, i.e. the range of neutral-
ization spectrum of antibody. In order to obtain a clinically
useful HIV medicament, a monoclonal antibody having as broad a
neutralization spectrum as possible is preferably established.
An object of the present invention is to provide a
monoclonal antibody which recognizes the above PND region of
HIV and has a broad neutralization spectrum and a hybridoma
which is capable of producing said antibody, and further to
provide a chimeric or humanized antibody which is prepared
from said monoclonal antibody for administration to humans and
=~a process for preparing the same. The present inventors have
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studied PND region of viruses obtained from many HIV-infected
individuals, and as a result, have revealed that the so-called
PND-Tip region, said region including Gly-Pro-Gly-Arg sequence
present in the central area of PND region, is relatively
conserved although some regions show high variability in amino
acid sequence (AIDS Res. Human Retroviruses, 7 p.825 (1991)).
Therefore, if there can be prepared an antibody which
recognizes this conserved region, it is expected to be a
clinically effective monoclonal antibody capable of neutraliz-
ing many kinds of viral strains.
However, it is foreseeable that such antibody
recognizing this region can only be prepared with low
efficiency. Boudet et al. (Int. Immunol., 4, p.283 (1992))
suggested, within in PND region, high immunogenicity is shown
by a basic amino acid residing outside the above-mentioned
Gly-Pro-Gly-Arg (GPGR; hereinafter the same) but the
immunogenicity of GPGR sequence is low. The fact that most
antibodies recognizing PND region are strain-specific also
suggests that antibody-producing cells which produce an
antibody having a broad neutralization spectrum as mentioned
above are scarce in HIV-infected patients or animals (mouse
etc.) immunized with HIV antigens. Accordingly, it is
required to increase the number of cells which produce said
antibody in any way.
Under the circumstances, the present inventors have
provided a novel method for immunization as will be discussed
~ereinbelow and thereby succeeded in positive derivation of
P s. .
of
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anti-HIV antibodies which recognize PND-Tip region. That is,
a test animal is firstly immunized with one strain of HIV
which includes GPGR sequence. Then, the second and the
following immunizations are conducted sequentially with
another HIV strains which also contain GPGR sequence of PND
region in common but have different amino acid sequences at
the side of the N and C termini from those of the HIV strain
used for the first immunization. An antigen for immunization
includes a viral particle, an infected cell, a purified gp120,
gp120 with a suitable treatment (e. g., with enzymatic treat-
ment), or a peptide synthesized based on the amino acid
sequence of PND region and a derivative thereof etc. Among
these, a synthetic peptide comprising an amino acid sequence
of PND region or a conjugate thereof with a serum albumin or
keyhole limpet hemocyanin (KLH) etc. are preferable for
inducing immunization which is limited within PND region. A
mouse used for immunization includes BAI,B/c, C57BL/6, C3H/HeN,
and_F1 mouse therefrom. Immunization is conducted using 20
to 200 ~g of an antigen per one mouse (4 to 8 weeks old,
weighing 20 to 30 g) four to seven times with an interval of
1 to 2 weeks.
With such immunization procedure, as a result of a
booster effect of the second and the following immunizations,
antibody-producing cells which produce an antibody that
recognizes an overlapping region of the antigens used for the
first, the second and the following immunizations, i.e. PND-
Tip region, should increase in a population of antibody-
2157874
producing cells which are induced within the living body of
the immunized animal. Spleen cells of this immunized mouse
can be used as a material for cell fusion in order to
efficiently produce the desired antibody-producing cells.
Without using a cell fusion technique, monoclonal
antibody-producing cells may be produced by another method
which comprises transforming B cells from HIV-infected
patients with Epstein-Barr virus (EBV), and converting said
antibody-producing cells into monoclonal antibody-producible
one. However, this method is not suitable for the method of
the present invention since the source of antibody-producing
cells is peripheral blood in the case of the method of the present
invention, and hence, the materials are hardly available. In
addition, this method is not an efficient method since most
of the neutralizing antibodiesfound in HIV-infected individuals
are strain-specific antibodies whereas antibodies recognizing
the above-mentioned PND-Tip region are quite rare. On the
contrary; a hybridoma method is advantageous in that it
employs an experimental animal such as a mouse to allow for an
immunogen being readily available and for devising the above-
mentioned immunization procedure. Accordingly, for positively
inducing the desired monoclonal antibody-producing cells
(recognizing PND-Tip region), a hybridoma method can effi-
ciently be used in the present invention.
In the drawings:
Fig. 1 shows reactivity between anti-serum
obtained from mouse sequentially immunized with multiple PND
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peptides (SP-1, SP-17, SP-11, SP-12, SP-14, SP-30) and each
of V3 peptides.
Fig. 2 shows reactivity between anti-serum
obtained from mouse sequentially immunized with a single PND
peptide (SP-1) and each of V3 peptides.
Fig. 3 shows activity of the monoclonal antibody
C25 of the present invention and of the strain-specific
neutralizing antibodies u5.5, oc64 to inhibit infection of
various HIV variants ( infection by cell-free viruses and cell-
to-cell infection).
Fig. 4 shows reactivity of C25 antibody with
various PND peptides.
Fig. 5 shows reactivity of X5.5 antibody with
various PND peptides.
Fig. 6 shows reactivity of C25 antibody with each of
ten nonapeptides which are obtained by deleting each of one amino
acid from decapeptides IHIGPGRAFY derived from MN, IRVGPGRAIY
derived from NI54-2 and IRVGPGRTLY derived from NI53.
Fig. 7 shows reactivity of C25 antibody with
overlapped peptide groups comprising 3 to 10 amino acids (a
series of peptides having an amino acid sequence shifted one
by one from the N terminus) which are prepared based on the
MN-derived peptide IHIGPGRAFY.
Fig. 8 shows reactivity of C25 antibody with
decapeptides which are obtained by substituting the 1st amino
acid ( I ) in the MN-derived peptide IHIGPGRAFY with other amino
'°,~cids .
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_ 9 _
Fig. 9 shows reactivity of C25 antibody with
decapeptides which are obtained by substituting the 2nd amino
acid ( H ) in the MN-derived peptide IHIGPGRAFY with other amino
acids.
Fig. 10 shows reactivity of C25 antibody with
decapeptides which are obtained by substituting the 3rd amino
acid ( I ) in the MN-derived peptide IHIGPGRAFY with other amino
acids.
Fig. 11 shows reactivity of C25 antibody with
decapeptides which are obtained by substituting the 4th amino
acid ( G ) in the MN-derived peptide IHIGPGRAFY with other amino
acids.
Fig. 12 shows reactivity of C25 antibody with
decapeptides which are obtained by substituting the 5th amino
acid ( P ) in the MN-derived peptide IHIGPGRAFY with other amino
acids.
Fig. 13 shows reactivity of C25 antibody with
decapeptides which are obtained by substituting the 6th amino
acid (G) in the MN-derived peptide IHIGPGRAFY with other amino
acids.
Fig. 14 shows reactivity of C25 antibody with
decapeptides which are obtained by substituting the 7th amino
acid ( R ) in the MN-derived peptide IHIGPGRAFY with other amino
acids.
Fig. 15 shows reactivity of C25 antibody with
_''.~decapeptides which are obtained by substituting the 8th amino
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acid (A) in the MN-derived peptide IHIGPGRAFY with other amino
acids.
Fig. 16 shows reactivity of C25 antibody with
decapeptides which are obtained by substituting the 9th amino
acid ( F ) in the MN-derived peptide IHIGPGRAFY with other amino
acids.
Fig. 17 shows reactivity of C25 antibody with
decapeptides which are obtained by substituting the 10th amino
acid ( Y ) in the MN-derived peptide IHIGPGRAFY with other amino
acids.
Fig. 18 shows reactivity between consensus PND
peptides derived from HIV-infected individuals in Japan and
C25 antibody in comparison with those of X5.5 and cc64.
Fig. 19 shows nucleic acid and amino acid sequences
of H chain variable region of C25 antibody.
Fig. 20 shows nucleic acid and amino acid sequences
of L chain variable region of C25 antibody.
Fig. 21 shows a nucleic acid sequence at the 5'
terminus of a gene of H chain variable region of humanized C25
antibody (RC25) and an amino acid sequence at the N terminus
thereof.
Fig. 22 shows a nucleic acid sequence at the 3'
terminus of a gene of H chain variable region of humanized C25
antibody (RC25) and an amino acid sequence at the C terminus
thereof.
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Fig. 23 shows a nucleic acid and amino acid
sequence of L chain variable region of humanized C25 antibody
(RC25).
Fig. 24 shows antibody dependent complement-mediated
cytotoxicity (ACC) of humanized C25 antibody (RC25) against
HIV-infected cells in comparison with those of C25 antibody
and normal human immunoglobulin.
Fig. 25 shows antibody dependent cell-mediated
cytotoxicity (ADCC) of humanized C25 antibody (RC25) against
HIV-infected cells in comparison with. those of C25 antibody
and normal human immunoglobulin.
Fig. 26 shows a neutralizing activity of humanized
C25 antibody (RC25 ) against virus derived from plasma obtained
from HIV-infected patient (YHI).
Fig. 27 shows a neutralizing activity of humanized
C25 antibody (RC25) against virus derived from mononuclear
cells in peripheral blood obtained from HIV-infected patient
(ASA).
Fig. 28 shows a neutralizing activity of humanized
C25 antibody (RC25) against virus derived from mononuclear
cells in peripheral blood obtained from HIV-infected patient
(HHA).
Fig. 29 shows a neutralizing activity of humanized
C25 antibody (RC25) against virus derived from mononuclear
cells in peripheral blood obtained from HIV-infected patient
(MNI).
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Fig. 30 shows a neutralizing activity of humanized
C25 antibody (RC25) against virus derived from mononuclear
cells in peripheral blood obtained from HIV-infected patient
(MOK).
Fig . 31 shows decrease in an effective concentration
of humanized C25 antibody (RC25) against virus derived from
mononuclear cells in peripheral blood obtained from HIV-
infected patient (KMO) after reconstitution of CD8.
Fig. 1 shows reactivity, of a serum of mouse
immunized in accordance with the above-mentioned procedure
with various PND region peptides. That is, anti-serum
obtained from mouse immunized sequentially with multiple PND
peptides, each having GPGR sequence in common but a different
remaining amino acid sequence, reacted all the PND peptides
used for immunization. More surprisingly, said anti-serum
reacted not only with the peptides used for immunization but
also with other PND peptides comprising GPGR sequence in
common . It also reacted strongly with the peptide ( SP-30 )
comprising 5 repeats of the amino acid sequence GPGR which is
common in the immunogenic PND peptides. It was further
confirmed that this anti-serum could neutralize many HIV
strains.
On the contrary, as shown in Fig. 2, anti-serum
obtained from mouse immunized 5 to 6 times with a single
peptide (SP-1) reacted with the immunogenic PND peptide but
~''
tshowed a low reactivity with other PND peptides. Reactivity
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with the synthetic GPGR peptide was also studied but it hardly
reacted. That is, such a mouse immunized with a single peptide
showed merely a response of a strain-specific antibody even
with hyperimmunization.
This reveals that such a mouse immunized sequentially
with several different PND peptides of HIV induced anti-PND-
Tip antibody having a broad neutralizing spectrum against
various HIV strains. Therefore, it was expected that cell
fusion with spleen cells from this mouse could efficiently
prepare cells which produce monoclonal.antibody having a broad
neutralization spectrum.
A hybridoma is prepared in accordance with the
procedures by Kohler and Milstein (Nature 256, p.495 (1975)).
A myeloma cell preferably includes MOPC-21NS/1 (Nature 256,
p.495 (1975)), SP2/0-Agl4 (Nature 276, p.269 (1979)),
p3X63Ag8-U1 (Eur. J. Immunol. 6, p.511 (1976)), p3X63-Ag8
(Nature, 256, p.495 (1975)), p3X63-Ag8.653 (J. Immunol. 123,
p. 1548 { 1979 ) ) , etc . Spleen cells and myeloma cells are mixed
together at a ratio of 1:1 to 10:1. Fusion is conducted in
a phosphate buffer (pH 7.2 to 7.4) containing NaCl (about
0.850 and polyethylene glycol having a molecular weight of
1, 000 to 6, 000 . Fusion is conducted by incubating the mixture
of both cells at 35 to 37°C for 1 to 5 minutes. Selection of
fused cells (hybridomas) is made by selecting growing cells
using a basal medium containing hypoxanthine (1.3 to 1.4
mg/dl), aminopterin (18 to 20 ul/dl), thymidine (375 to 4,000
[,_~ul/dl), streptomycin (50 to 100 ~g/ml), penicillin (50 to 100
2157874
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Units/ml ) , glutamine ( 3 . 5 to 4 . 0 g/1 ) and fetal calf serum ( 10
to 20$). The basal medium includes those which are generally
used for culture of animal cells, such as RPMI1640 medium,
Eagle MEM medium, etc. Cloning of fused cells is conducted
at least twice by a limiting dilution method.
Another important aspect to be considered in
preparing a monoclonal antibody is what kind of antibodies
produced by a hybridoma should be selected. That is, cell
fusion provides many hybridomas, but among these, hybridomas
producing a desired antibody must be,cloned. Selection is
usually made by using a reactivity with HIV strains employed
in laboratories such as HIV-MN or a neutralizing epitope, PND
peptide, derived therefrom, as an index. However, such
strains are those subcultured in vitro, and hence, do not
always reflect HIV strains actually occurring within the
living body of patients. Since an object of the present
invention is to drive away HIV strains which are present
within the body of patients and actually epidemic, selection
of a hybridoma producing a neutralizing antibody for this
purpose is preferably conducted by using HIV strains derived
from infected patients . Therefore, a gene coding for an amino
acid sequence of PND region is directly isolated from HIV-
infected individuals and expressed in E. coli. Using the
reactivity with this recombinant PND peptide as an index,
selection of a desired hybridoma is conducted to select a
hybridoma producing a monoclonal antibody which binds to HIV
.. '.ky present within the body of patients. Furthermore, in order
?'e'
2157874
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to establish an antibody having a broad neutralization spec-
trum, recombinant PND peptides are prepared from as many HIV-
infected individuals as possible, and an antibody capable of
reacting with most of these peptides is selected.
Employing the above-mentioned method, the present
inventors have established a monoclonal antibody C25 which
broadly neutralizes various HIV variants. C25 antibody
strongly inhibited infection by cell-free viruses and cell-to-
cell infection in vitro. C25 antibody reacted with most
peptides used as an immunogen and neutralized many additional
HIV strains, suggesting that an epitope recognized by this
antibody is a region conserved among strains, i.e. GPGR and
surroundings thereof.
As a result of detailed analysis of epitopes using
synthetic peptides, C25 antibody of the present invention was
found to react with a series of peptides comprising the
following amino acid sequence at around PND-Tip in PND region:
Xal-Gly-Pro-Xa2-Arg-Xa3
wherein Xal is Ala, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val
or Tyr
Xa2is Gly or Ala
Xa3is Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn,
Gln, Arg, Ser, Thr, Val, Trp or Tyr.
Furthermore, C25 antibody of the present invention
was found to react with the peptides:
Xaa-Gly-Pro-Gly-Arg-Ala
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wherein Xaa is Ala, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr,
Val, Tyr;
Ile-Gly-Pro-Gly-Arg-Xaa
wherein Xaa is Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr;
Val-Gly-Pro-Gly-Arg-Thr;
Val-Gly-Pro-Gly-Arg-Ser; or
Ile-Gly-Pro-Ala-Arg-Ala.
That is, C25 antibody of the present invention
recognized an epitope formed by six amino acids comprising
GPGR as a core and each one amino acid adjacent thereto at the
N and C termini.
Based on the above observation, it was confirmed
that C25 antibody has a broad neutralization spectrum since
it recognized a highly conserved amino acid sequence within
PND region of HIV and inhibited many HIV infections.
Then, the present inventors have examined usefulness
of .C25 antibody in actual clinical usage by studying a
neutralization spectrum of C25 antibody against HIVs derived
from actual HIV-infected individuals.
Clinical usefulness means, first of all, what range
of actually epidemic HIV variants C25 antibody can bind to and
neutralize. In this respect, PND region of HIV variants
present within the living body of each infected individual is
first studied to determine an amino acid sequence of HIV
having the highest frequency as a consensus sequence, and
._
!reactivity between PND peptides having said sequence and C25
2157874
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antibody was studied. As a result, C25 antibody reacted with
about 80$ of the consensus peptides of HIV variants derived
from each patient. Viewing that the strain-specific neutral-
izing monoclonal antibody X5.5 previously established by the
present inventors showed a binding rate of 30~, C25 antibody
was found to have quite a broad neutralization spectrum.
A second aspect of clinical usefulness is to what
range of variants it is effective among quasispecies of HIV
variants occurring in a single patient. When an amino acid
sequence of viral variants occurring in a respective HIV-
infected individual is determined, they do not have a
completely identical amino acid sequence, but quasispecies of
viral variants having a slightly diverse amino acid sequence
infect a patient. Accordingly, for driving away all the
infected viruses from the living body by administering an
antibody as a medicament, the antibody must react with most
HIV variants occurring in a patient. In this respect,
reactivity between C25 antibody and PND region peptides
isolated from a single patient was determined. When the
strain-specific antibody u5.5 was used, there remained in the
living body several HIV variants which did not reacted with
the antibody. On the contrary, C25 antibody could react with all
or most HIV variants occurring in an infected individual.
As mentioned above, it was revealed that C25
antibody of the present invention is a monoclonal antibody
which recognizes a highly conserved PND-Tip region of PND
;,'region and shows a strong neutralizing activity, and hence,
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can sufficiently cope with the diversity of HIV. In fact, C25
antibody reacted with a majority of HIV variants occurring in
the living body of infected individuals, and hence, has much
clinical usefulness, suggesting clinical applicability of said
antibody.
A representative hybridoma producing the monoclonal
antibody C25 of the present invention has been deposited at
National Institute of Bioscience and Human-Technology, Agency
of Industrial Science and Technology with the accession number
of FERM BP-4561 in accordance with the Budapest Treaty on
February 10, 1994.
Although C25 antibody of the present invention has
a broad neutralization spectrum and is suggested to be
clinically useful as mentioned above, since it is a mouse-
derived antibody, its administration to humans is actually
impossible from the viewpoint of safety (induction of antigenicity)
or effectiveness(shortening of a half-life). Therefore, it is
necessary to modify C25 antibody to a molecule having an amino
acid sequence of a human antibody without altering an antigen-
binding capacity by using a genetic engineering technique.
In order to prepare a so-called chimeric or
humanized antibody wherein the antigen-binding site of C25
antibody is linked to a human antibody constant region, a
variable (V) region gene of C25 antibody was firstly cloned
and a base sequence and an amino acid sequence coded thereby
were determined.
~:
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A V region gene can be isolated by a usual gene
manipulation technique. For example, it can be isolated by
cloning a V region gene from a chromosomal DNA of a cell in
accordance with the conventional method (for example, see T.
Maniatis, "Molecular Cloning" Cold Spring Harbor Lab. (1982))
or by synthesizing cDNA from mRNA materials of a cell in
accordance with the conventional method (for example,
D.M.Glover ed. "DNA cloning Vol. I" IRL press (1985)) and
cloning a V region gene. In either procedure, as a probe for
cloning a V region gene, DNA probes synthesized with reference
to the nucleic acid base sequence of a mouse immunoglobulin
gene which has already been reported (for example, Sakano et
al., Nature, 286, p.676 (1980); E.E.Max et al., J.Biol.Chem.,
256, p5116, (1981)) can be utilized. Cloning using PCR
(polymerase chain reaction) can also be conducted (R.Orlandi
et al., Proc. Natl. Acad. Sci. USA, 86, 3833 (1989); W.D.Huse
et al., Science, 246, 1275 (1989)).
Using the above procedures; a variable region gene
of C25 antibody was isolated and the base and amino acid
sequences were analyzed, and as a result, it was found that
the variable region of C25 antibody has quite a novel sequence
different from those of antibodies which hitherto have been
reported . CDRl to CDR3 regions in Figs . 19 and 20 are regions
which actually bind to an antigen, and a sequence thereof is
assumed to be closely related to a broad neutralization
spectrum of C25 antibody.
n~
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In this respect, there were prepared a chimeric
antibody gene by linking a gene fragment coding for an amino
acid sequence of the regions to the upstream of a gene
fragment of a human antibody constant region, or a humanized
C25 antibody gene by transplanting the above-mentioned CDR
regions alone at CDRs of a human antibody variable region.
These genes were expressed and expression products were
analyzed for their properties, and as a result, it was found
that the chimeric and humanized C25 antibodies had a neutral-
ization spectrum equivalent to that of mouse C25 antibody.
Among other things, the fact that the humanized C25 antibody,
wherein CDR regions alone were transplanted, reacted with PND
means that, among V region of.an antibody, CDRs are just the
most important amino acid for binding. In addition, said
antibody reacted only with an anti-human IgG but not with
anti-mouse IgG, revealing that said antibody shows an
antigenicity as a human antibody. Accordingly, it was
suggested that, when administered to humans, the chimeric and
humanized C25 antibodies do not provoke severe antigenicity.
Another advantage of the chimeric and humanized
antibodies is that they have an effector activity due to a
constant region of a human antibody such as an antibody
dependent complement-mediated cytotoxicity (ACC) and an
antibody dependent cell-mediated cytotoxicity (ADCC). As
mentioned hereinabove, C25 antibody inhibits infection by
cell-free HIV viruses or cell-to-cell infection. However, in
addition to this, whether it can destroy infected cells is
~:~ ~rz
~a r
2157874
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another important factor for a medicament. Generally, an
antibody alone cannot kill and destroy infected cells but can
destroy infected cells via complement or effector cells having
FcR. It is a constant region where an antibody binds to the
complement or FcR (Mol.Immunol, 22, p161 (1985)).
In this respect, ACC and ADCC activities of the
chimeric and humanized C25 antibodies of the present invention
based on the antibody constant region were examined. As a
result, the chimeric and humanized C25 antibodies significant-
ly destroyed cells with continuous ,HIV infection in the
presence of complement or effector cells. This suggests that
the chimeric or humanized C25 antibody of the present
invention can not only inhibit infection by free viral
particles or a viral antigen on the surface of infected cells
but also destroy infected cells.
As mentioned hereinabove, HIV variants actually
occurring in HIV-infected patients do not have a completely
identical amino acid sequence but they are quasispecies of
many variants having different amino acid sequence. Accord-
ingly, for driving away infected viruses from the living body
by administering an antibody for treating purpose, the
antibody should neutralize most of the quasispecies of
variants. In this respect, said humanized C25 antibody was
tested for a neutralizing activity against viruses derived
from plasma and peripheral blood mononuclear cells from
patients and as a result, apparently showed a neutralizing
,"activity against many viruses derived from the patient. This
'.
2157874
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revealed that said humanized C25 antibody is clinically useful
against actual patients.
As mentioned above, the present inventors have
prepared the monoclonal antibody C25 which is capable of
broadly neutralizing many kinds of HIV strains from infected
individuals. Furthermore, humanization of said antibody could
decrease antigenicity in humans and confer an effector
activity such as destruction of infected cells. Thus, the
present invention provides an antibody useful for prevention,
treatment and diagnosis of HIV infection.
Example 1: Preparation of monoclonal antibody
1-1) Preparation of antigen (synthetic PND peptide)
Synthetic PND peptides corresponding to the amino
acid Nos. 303-322 of gp120 as shown in Table 1 were used as
an antigen for immunization and an antigen for assay.
Table 1
Synthetic PND Isolated HIV Amino acid sequence
peptides strains
S P -1 MN YNKRICRIH IGPGRAFYTTFQQ-C
SP-6 NI16-1 NNTRKGIRIGPGRAVYATGK-C
S P - 9 N I 2 3 NNTRIGS IP IGPGRAFYTTGE-C
SP-10 NI63-1 NN'IRKRVTMGPGRVYYTTGE-C
SP-11 NI54-2 NNTRKGIRVGPGRAIYATEK-C
SP-12 NI53 NNTKKAIRVGPGRTLYATRR-C
SP-14 RF NNTRKSITKGPGRVIYATGQ-C
S P-17 N I 18 NNTRE~tITIGPGRVYYTTGE-C
SP-20 NI63-2 NN'IRRGIRIGPGRAFYATDK-C
SP-30 - GPGRGPGRGPGRGPGRGPGR-C
For chemical synthesis of the above peptides,
ABI430A peptide synthesizer (Applied Biosystem) was used. As
s.',
2157814
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a result, a crude product was obtained. The peptide was
removed from the resin by the TFMSA method and then purified
by a reverse high performance liquid chromatography (HPLC).
Purification by a reverse HPLC was repeated three times and
the obtained fractions were collected.
Then, each of the obtained synthetic peptides was
lyophilized and bound with KLH to prepare a peptide-KLH
conjugate. First, each of the above peptides (10 mg) was
dissolved in 10 mM PBS (pH 7.0; 2 ml) and thereto was added
a solution of dimethylformamide (MBS type crosslinking agent)
( 40 mg/100 ~1 ) and the mixture was stirred at room temperature
for 30 minutes. The reaction solution was then washed three
times with dichloromethane (2 ml) and the obtained aqueous
layer was separated (Solution A) . Separately, KLH (20 mg) was
dissolved in 0.2 M Tris-HC1 (pH 8.6, 8M urea) (5 ml) and
thereto was added dithiothreitol (DTT) and the mixture was
stirred at room temperature for 1 hour. To the reaction
solution was added 10~ trichloroacetic acid (3 ml). The
precipitate was filtered by suction, washed with distilled
water ( 2 ml ) , and then dissolved in 20 mM NaPB (pH 7 . 0 6M
urea) (5 ml) (Solution B). Solutions A and B were mixed
together and the mixture was stirred at room temperature for
3 hours. The reaction product was dialyzed and then lyophi-
lized.
As mentioned above, PND peptide and PND peptide-KLH
conjugate were prepared and used as an antigen for immuniza-
'A''~,~ tion and an antigen for assay.
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1-2) Immunization of mouse
By way of example, immunization with the synthetic
peptides prepared as mentioned above is shown hereinbelow but
the order of the peptides for immunization can be varied.
BALB/c and C3H/HeN mice of 4 to 8 weeks old were
used. Immunization was conducted by five intraperitoneal
inoculations and one following intravenous inoculation.
That is, there were made intraperitoneal (i.p.) administration
of SP-1-KLH in the presence of Freund's complete adjuvant on
Day 0; i.p. administration of SP-17-ICHL in the presence of
Freund's incomplete adjuvant on Day 7; i.p. administration of
SP-11-KHL in the presence of Freund's incomplete adjuvant on
Day 14; i.p. administration of SP-12-KHL in the presence of
Freund's incomplete adjuvant on Day 21; i.p. administration
of SP-14-KHL in the presence of Freund's incomplete adjuvant
on Day 28; and intravenous administration of SP-30-KLH in the
absence of adjuvant on Day 35.
1-3.) Measurement of antibody titer in anti-serum of immunized
mice
This was conducted by EIA method. The synthetic
peptide antigen (2 ug/ml) prepared as mentioned above was
added to a 96-well microtiter plate at 100 ul/well and the
plate was incubated at 4°C overnight to immobilize the
antigen. Thereto was further added 1~ BSA (bovine serum
albumin) solution (150 ul) and the plate was incubated in the
same way for masking. To the thus prepared antigen-immobi-
~lized plate were added hybridomas obtained by a cell fusion
2157874
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method or a culture supernatant of hybridomas after cloning.
After incubating the plate at 4°C for 1.5 hours, it is washed
three times with 0.1~ TweenTM20/PBS and then a solution of
peroxidase-labelled anti-mouse immunoglobulin antibody
(manufactured by Kappel; 5,000 times dilution) was added
thereto at 100 ul/well. After incubating the plate at 4°C for
1 hour, it was washed five times with 0.1$ Tween20/PBS. Then,
a solution of TMBZ substrate was added to develop a color
reaction by a conventional procedure and an absorbance at
a wave length of 450 nm was measured.
As is clear from Fig. 1, the anti-serum obtained
after final immunization reacted all the peptides SP-1, SP-17,
SP-11, SP-12, SP-14 and SP-30. Surprisingly, the anti-serum
reacted not only with the peptides used for immunization but
also with PND peptides SP-6, SP-9 and SP-20, derived from
other HIV strains. In contrast, when a mouse is immunized
with a single peptide SP-1 five to six times, the obtained
anti-serum was highly reactive with the immunogenic peptide
( SP-1 ) but showed a lower reactivity with other peptides ( Fig .
2). As to the reactivity with SP-30, the anti-serum of the
mouse immunized with a single peptide hardly reacted with this
peptide (Fig. 2) whereas the anti-serum of the mouse immunized
with different PND peptides showed strong reactivity ( Fig . 1 ) .
A neutralization test was also conducted for anti-sera, and
as a result, it was found that the anti-serum of the mouse
immunized with SP-1 alone could neutralize only HIV-MN strain
,a
~S~hereas the anti-serum of the mouse immunized with many kinds
2157874
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of PND peptides could neutralize many HIV strains. This
revealed that the immunized mouse induced an anti-GPGR
antibody having a broad neutralization spectrum against
various HIV strains. The anti-serum was also confirmed to
neutralize various HIV strains. Therefore, a cell fusion was
conducted using spleen cells of this mouse.
1-4) Cell fusion and culture of hybridomas
Three days after the final immunization, spleen
cells were collected from the mouse by the conventional
procedure.
The spleen cells were mixed with myeloma cells
p3X63Ag8-U1 at a ratio of cell number, 1:5, and the mixture
was subjected to centrifugation (1,200 r.p.m. for 5 minutes)
to remove supernatant. After loosening the precipitated cell
lump sufficiently, a polyethylene glycol solution (polyethyl-
ene glycol-4000 (2 g), RPIM1640 (2 ml)) (1 ml) was added
thereto while stirring. The mixture was incubated at 37°C for
minutes, and then RPMI1640 was added slowly to the mixture
to give a total volume of 50 ml. After centrifugation (900
r . p . m . for 5 minutes ) , the supernatant was removed and the
cells were loosened mildly. Thereto was added a normal medium
(RPMI-1640 medium supplemented with 10~ fetal calf serum) (100
ml) and the cells were mildly suspended using a measuring
pipet.
The suspension was poured into each well of a 24
well culture plate (at 1 ml/well), and culture was conducted
q in an incubator containing 5~ carbonic acid gas at 37°C for
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24 hours. Then, 1 ml/well of an HAT medium (a normal medium
supplemented with hypoxanthine (1.3 to 1.4 mg/dl), thymidine
(345 to 4,000 ~1/dl) and aminopterin (18 ~1/dl)) was added to
the plate and culture was further continued for 24 hours.
Thereafter, the culture supernatant (1 ml) was exchanged with
the same volume of the HAT medium at an interval of 24 hours
for 2 days and culture was conducted for 10 to 14 days in the
same manner.
For each well where fused cells (about 300
cells) were observed to grow in the, shape of colony, the
culture supernatant (1 ml) was exchanged with the same volume
of an HT medium (the above HAT medium devoid of aminopterin)
and thereafter the same exchange was conducted at an interval
of 24 hours for 2 days. After culture on the HT medium for
3 to 4 days, a portion of the culture supernatant was taken
and a desired hybridoma was selected by the screening method
as mentioned hereinbelow.
1-5.) Screening of hybridoma
Selection of a desired hybridoma was made by a
combination of the following EIA method and the Western
blotting method. The thus selected clone was measured for its
neutralizing activity.
(1) EIA method
To a 96-well microtest plate were added the
synthetic PND peptide antigens prepared as mentioned above or
PND peptides expressed in E.coli as described in Examples 2
'~:(2-5) (protein concentration: 1 to 10 ~g/ml) at 100 ~.1/well,
,.
2157874
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and the plate was incubated at 4 °C overnight to immobilize the
peptide. Thereto was further added 1$ BSA (bovine serum
albumin) solution (150 ~1) and the plate was incubated in the
same manner for masking. To the thus prepared antigen-
immobilized plate were added the hybridomas or the culture
supernatant of the hybridomas after cloning. After incubating
the plate at 4°C for 1.5 hours, the plate was washed three
times with 0.1~ Tween20/PBS and a solution of peroxidase-
labelled anti-mouse immunoglobulin antibody (manufactured by
Kappel; 5,000 times dilution) (100 ~1/well) was added to the
plate. After incubating the plate at 4°C for 1 hour, the
plate was washed five times with 0.1~ Tween20/PBS. Then, a
solution of TMBZ substrate was added to develop a color
reaction by the conventional procedure and an absorbance at
a wave length of 450 nm was measured. Hybridomas which commonly
react with a group of peptides whose GPGR sequence in PND
region is conserved are selected and cloned. Hybridoma clones
after cloning were also selected in the same manner.
(2) Western blotting method
This method was conducted in accordance with Towbin
et al. (Proc.Natl.Acad.Sci.U.S.A., 76, p.4350 (1979)).
Viral particles of laboratory strains such as HIV-
MN, HIV-LAV or HIV-RF or of clinically isolated strains such
as NI61, NI23, NI54-2, NI53, NI18 or NI63, or PND peptides
expressed in E.coli as described in Examples 2 (2-5), were
prepared. The prepared viral particles or peptides were
subjected to electrophoresis using 10$ SDS-PAGE, the gel was
2157874
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transferred to a nitrocellulose membrane to transfer the
viruses to said membrane, and the membrane was cut with 0.4
to 0.5 cm width. Each strip was immersed into the culture
supernatant of the hybridomas and incubated overnight.
Thereafter, the strips were washed three times with PBS and
incubated in 1:750 dilution of a biotin-labelled anti-mouse
IgG (manufactured by TAGO) for 2 hours. After washing the
strips three times with PBS, they were immersed into an avidin
coupled with horseradish peroxidase (manufactured by Sigma)
(1:1000 dilution) and incubated for 1 hour. After washing the
strips three times with PBS, a color reaction was developed
with a coloring agent using 4-chloro-1-naphthol (manufactured
by Bio-Rad) and the hybridomas were selected and cloned which
commonly reacted with bands of the viral gp120 and the PND
peptides expressed in E . coli whose GPGR sequence in PND region
is conserved. Hybridoma clones after cloning were also
selected in the same manner.
(3)_Measurement of neutralizing activity
For measurement of neutralizing activity, various
laboratory viruses having respective PND amino acid sequence
and various viruses isolated from patients and a culture
supernatant of the viruses after cloning were used as a viral
source (104'5 to 105 TCIDSO) . Cloning of the viruses was made
by a limiting dilution method, or a plaque method in CD4-HeLa
cells.
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First, the viral solution adjusted to 10 TCIDso/50
~1 and a culture supernatant of the hybridoma clone or
purified ascites (50 ~1) (with various dilutions) were
inoculated onto a 96-well flat.bottom plate and the plate was
incubated at 37°C for 1 hour. Then, 104 cells/100 ~1/well of
MT4 cells (suspended in RPMI1640 medium containing 10~ FCS,
L-glutamine 3.5 to 4.0 g/1, penicillin 50 U/ml and streptomy-
cin 50 ul/ml) were added and cultured for 5 days.
Neutralizing activity was evaluated based on
whether an antibody inhibits syncytium formation occurring
upon infection or not. A neutralization titer was expressed
as a minimum effective concentration of an antibody which
inhibits the syncytium formation by 100.
A hybridoma producing the monoclonal antibody C25
was obtained by the above-mentioned selection method.
1-6) Preparation of C25 monoclonal antibody
To pristan-treated SPF female mouse (BALB/c,
C3H~HeN, and F1 mouse thereof) of 8 weeks old, 5 x 106
cells/mouse of the hybridoma C25 antibody strain obtained in
the above 1-5 was administered intraperitoneally. After 10
to 21 days, ascitic cancer was induced. Ascites was taken out
of mice and, after removing solid components by centrifugation
at 3000 r.p.m. for 5 minutes, purified by affinity chromatog-
raphy using AffigelTM Protein A MAPS-II kit (manufactured by
Bio-Rad).
Example 2: Analysis of properties of C25 monoclonal antibody
~..ys,~:
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2-1) Activity of C25 antibody to neutralize virus
A neutralizing activity of C25 antibody against HIV
was analyzed using various HIV strains. Fig. 3 shows viral
strains used and an amino acid sequence of PND region of the
viruses. A neutralization test was conducted as shown in
Example 1. As a control, monoclonal antibodies X5.5 which
neutralizes HIV-MN strain in a strain-specific manner and cc64
which neutralizes NI53 strain in a strain-specific manner were
used and compared with C25 antibody.
The left side of Fig. 3 shows a minimum effective
concentration (~g/ml) of antibodies which inhibit infection
of cell-free viruses by 100. The neutralizing activity of
C25 antibody is almost the same as those of X5.5 or cx64, and
hence, is quite strong. As to specificity to HIV strains, C25
antibody neutralized much more HIV strains as compared to the
strain-specific antibodies such as u5.5 or oc64, revealing a
broad neutralization spectrum of C25 antibody.
The right side of Fig. 3 shows a minimum effective
concentration of antibodies which inhibit a cell-to-cell
infection of infected cells by more than 80~. An inhibitory
activity of C25 antibody against cell-to-cell infection is
also almost the same as those of u5.5 or oc64, and hence, is
quite strong. C25 antibody inhibited cell-to-cell infection
of much more HIV strains as compared to the strain-specific
antibodies such as X5.5 or c~c64, revealing a broad neutraliza-
tion spectrum of C25 antibody.
r v~'~-i''~eo
,,'~'=2) Reactivity with various synthetic peptides
2157814
- 32 -
The neutralization test revealed that C25 antibody
had a broad neutralization spectrum. Then, how C25 antibody
reacts with various PND peptides (comprising 20 amino acids)
shown in Example 1 (1-1) was examined by EIA method.
As is clear from Fig. 4, C25 antibody reacted with
SP-1, SP-6, SP-9, SP-11, SP-12, SP-20, and SP-30. This
suggested that C25 antibody recognized PND-Tip region, a
consensus sequence of the peptides. On the contrary, the
strain-specific antibody X5.5 bound only with the antigen used
for immunization, SP-1, but did not react with other peptides
(Fig. 5).
2-3) Analysis of epitope recognized by C25 antibody
Since it was suggested that the epitope recognized
by C25 antibody is located in Tip sequence around GPGR in PND
region, the recognized epitope was identified in accordance
with the following method.
(1) Binding test with PND peptides which is devoid of a
specific amino acid
The above experiment 2-2 revealed that C25 antibody
reacts with the peptides SP-1 (YNKRKRIHIGPGRAFYTTKN-C) derived
from HIV-MN strain, SP-12 (NNTKKAIRVGPGRTLYATRR-C) derived
from NI53, and SP-11 (NNTRKGIRVGPGRAIYATEK-C) derived from
NI54-2. By synthesizing peptides devoid of either one of
amino acids of these peptides and examining the reactivity of
the resulting peptides with C25 antibody, specific amino acids
which contribute to the binding will be clarified. According-
~.;ly, each ten nonapeptide which is devoid of either one of
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amino acid from the decapeptides, IHIGPGRAFY derived from MN
strain and IRVGPGRTLY derived from NI53 strain, both
decapeptides being known to bind to C25 antibody, were
synthesized on a polyethylene rod and were tested for
reactivity with C25 antibody (Fig. 6).
In the case of IHIGPGRAFY derived from HIV-MN strain,
the peptides devoid of the 3rd to 8th amino acids, I, G, P,
G, R and A, hardly react with C25 antibody. In case of
IRVGPGRTLY derived from NI53 strain, the peptides devoid of
the 3rd to 8th amino acids, V, G, P, G., R and T, do not react
with C25 antibody. Furthermore, in case of IRVGPGRAIY derived
from NI54-2 strain, the peptides devoid of the 3rd to 8th
amino acids, V, G, P, G, R and A, did not react with C25
antibody.
These results revealed that the sequence IGPGRA is
indispensable for binding of C25 antibody with HIV-MN strain,
the sequence VGPGRT for binding with NI63 strain, and the
sequence VGPGRA for binding with NI54-2 strain, suggesting
that six amino acids comprising a GPGR core and each one amino
acid adjacent to both sides of said core is an epitope
recognized by C25 antibody.
(2) Binding test with overlapped hexapeptides
Then, based on the peptide IHIGPGRAFY derived from
HIV-MN strain with which C25 antibody reacts, a group of
overlapped peptides comprising 3 to 10 amino acids (a series
of peptides which have an amino acid sequence shifted one by
''~~~ne from the N terminus) were synthesized on a solid phase as
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in (1). The reactivity between these peptide groups and C25
antibody was examined by EIA, and a possible portion as an
epitope was assessed (Fig. 7).
As a result, C25 antibody had a low reactivity with
shorter peptides of less than a pentapeptide but showed a
sufficient reactivity with longer peptides of more than a
hexapeptide. Among these overlapped hexapeptides, the peptide
which showed the strongest reaction was IGPGRA and the peptide
whose reactivity comes second was GPGRAF. However, HIGPGR and
PGRAFY showed an extremely decreased .reactivity, suggesting
that the epitope recognized by C25 antibody is the six amino
acid sequence, IGPGRA, like the result shown in (1).
2-4) Amino acid substitution analysis
In order to investigate neutralization broadness
(neutralization spectrum) of C25 antibody to HIV variants,
the reactivity with PND peptides prepared by serially
replacing each amino acid with either one of the other
19 amino acids was examined (Figs. 8 to 17). By way of
example, the decapeptide IHIGPGRAFY derived from HIV-MN strain
was used herein for substitution.
When G1, P and R in IIHIzGIPGZRAFY were replaced with
other amino acids, C25 antibody hardly bound to these
peptides. C25 antibody reacted strongly with a peptide wherein
the 6th GZ is replaced with A, and hence, it was found that GZ
can be replaced with A. Accordingly, the amino acids which
most contribute to the binding were considered to be GPGR.
r ; 4.~ ';
2157874
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IZ located at the N terminal side of GPGR could be replaced
with A, L, M, N, P, Q, S, T, V and Y, whereas A located at the
C terminal side of GPGR could be replaced with every amino
acid other than P. This proved that the amino acids located
at both sides of GPGR are not crucial but contribute to
the binding to some extent. Even after replacement of I1, H,
F and Y, which are located biased more to the N and C termini,
with other amino acids, the peptides maintained the reactivity
with C25 antibody.
The results of the above experiments 2-3 and 2-4
proved that C25 antibody recognized the epitope formed by six
amino acids comprising GPGR as a binding core and each one
amino acid adjacent to both sides thereof as shown below.
In addition, the results of the amino acid substitu-
tion analysis in 2-4 revealed that C25 antibody could cope
with the following many amino acid variances:
(1) Xal-Gly-Pro-Gly-Arg-Ala
wherein Xal is Ala, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr,
Val or Tyr;
(2) Ile-Gly-Pro-Gly-Arg-Xa2
wherein Xa2 is Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
(3) Val-Gly-Pro-Gly-Arg-Thr;
(4) Val-Gly-Pro-Gly-Arg-Ser;
(5) Ile-Gly-Pro-Ala-Arg-Ala.
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2-5 ) Reactivity of C25 antibody with PND peptides derived from
viruses of infected individuals
Then, the reactivity of C25 antibody with HIV
actually occurring within the living body of HIV-infected
individuals was examined by the following method.
First, peripheral blood lymphocytes (PBL) of HIV-
infected individuals were suspended in 1 x RSB buffer and
thereto were added SDS (final concentration 1~), Proteinase
K ( final concentration 1 mg/ml ) , and the mixture was incubated
at 37°C for 2 hours. Then, phenol extraction and ethanol
precipitation procedures were repeated to give DNAs (genomic
DNAs) having a high molecular weight. Also, HIV particles
were precipitated from serum of infected individuals and cDNAs
were synthesized with a reverse transcriptase. Using these
DNAs having a high molecular weight or cDNAs as a template,
gp120/PND region of HIV in infected patients were amplified
using the following primers A and C.
Primer A; (5')GCCGGATCCACACATGGAATTAGGCCAGTA(3')
Primer B; (3')AGTCCTCCCCTGGGTCTTTAAACTGACGTCTCG(5')
Amplification was carried out using Taq polymerase
for 30 to 35 cycles.
The thus obtained amplified DNA fragments were
cloned into pUCl8 plasmid and the amplified DNA fragments were
sequenced by a dideoxy method. Furthermore, the cloned DNA
fragments were incorporated into pUEX2 expression vector and
E.coli was transfected with this vector and subjected to heat
induction at 42°C for expression. The expressed protein in
.q
2157874
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the form of a fused protein with j3-galactosidase was purified
from the inclusion in E.coli as follows. E.coli cells which
undertook expression were destroyed with glass beads and then
treated with lysozyme (final concentration 0.1 mg/ml) at 4°C
and the precipitates obtained from centrifugation were treated
with TritonTM X-100(final concentration 0.5~). The precipi-
tates obtained from centrifugation were solubilized with 8M
urea and then reacted with C25 antibody. The binding property
was confirmed by the EIA method and the Western blotting
method as described in Example 1 (1-5).
Fig. 18 shows an amino acid sequence of PND region
of HIVs which occur most frequently in patients and the
reactivity of C25 antibody therewith. C25 antibody bound with
the consensus sequences derived from twenty five patients
among thirty HIV-infected individuals examined herein. That
is, the binding spectrum of C25 antibody was as broad as 83~.
On the contrary, the strain-specific monoclonal antibodies,
X5.5 or cz64, had a spectrum of only about 30~.
When viruses in an HIV-infected individual were
examined for their amino acid sequence, they do not show a
completely identical sequence but they infect a patient as
quasispecies of viruses having a somewhat different amino acid
sequence. To drive away viruses from the living body of
infected individuals by administration of an antibody, the
antibody must react with most of HIVs present within the
living body of the patient . Tables 2 and 3 show the reactivity
s ~ .~h:.
2157874
- 38 -
of C25 antibody with PND region peptides derived from HIVs
isolated from a single patient.
Table 2
HIV clone No. of Amino acid sequence C25 u5.5
analysis of PND
HIV-MN - YNKRKRIHIGPGRAFYTTKNIIG
TIW-Ol 7 N-T--S-P----------GE--- + -
TIW-02 4 N-T--S-P----------GEV-- + -
TIW-09 1 N-T--G-P----------GE--- + -
Binding rate (~) 100 0~
(12/12) (0/12)
Table 3 .
HIV clone No. of Amino acid sequence C25 u5.5
analysis of PND
HIV-MN - YNKRKRIHIGPGRAFYTTKNIIG
NI230-1 3 N-T--S------------GE--- + +
NI230-4 1 N-T--S------------GE-M- + +
NI230-8 2 N-T--G-Y------V---ER--- + -
NI230-7 1 N-T--G-Y------V---GR--- + _
NI230-5 1 N-T--G-Y------V---ER--- + -
Binding rate (~) 100 50~
(8/8) (4/8)
In the case of the patient TIW, C25 antibody bound all
the HIVs whereas u5.5 bound none of HIVs (Table 2). In case
the infected individual NI230, C25 antibody could bind all the
HIVs whereas there remained some HIVs with which the antibody
u5.5 could not react (Table 3). Table 4 summarizes results
obtained from such 13 HIV-infected individuals. C25 antibody
showed a reactivity at a high rate in almost all the infected
individuals, wherein 100 reactivity was shown in 8 individu
als, and more than 90~ reactivity was shown in as many as 11
r y
2157874
- 39 -
individuals. On the contrary, the strain-specific u5.5
antibody showed a low reactivity wherein more than 90$
reactivity was shown in only one individual. These proved
that C25 antibody could sufficiently cope with a high
variability of HIVs and hence could actually be clinically
applicable.
Table 4
HIV-infected Bidinct rate of neutralizing antibody
($~
individuals C25 ,-
u5.5-
NI56 100$(11/11) 100$(11/11)
KMO 92$(12/13) ~ 46$(6/13)
TI 93$(14/15) 20$(3/15)
TIW 100$(12/12) 0$(0/12)
YHI 92$(11/12) 0$(0/12)
HHA 100$(35/35) 60$(21/35)
NI229 75$(6/8) 0$(0/8)
NI230 100$(8/8) 50$(4/8)
NI334 100$(8/8) 25$(2/8)
NI373 100$(10/10) 0$(0/10)
NI252 100$(11/11) 0$(0/11)
NI380 82$(9/11) 46$(5/11)
NI382 100$(11/11) 0$(0/11)
No. of applica- 8(11) 1(1)
ble patientsl
Note 1) Number of patients having an antibody binding rate of
100$
The number in the parenthesis shows number of patients having
an antibody binding rate of more than 90$.
Example 3: Preparation of chimeric C25 antibody (CC25)
3-1) Isolation of V region gene of C25 antibody
Isolation of a gene coding for mouse immunoglobulin
variable (V) region was carried out as mentioned hereinbelow.
Whole RNAs were extracted from C25 cells in accordance with
A
2157874
- 40 -
a , conventional procedure [Glober ed. "DNA cloning Vol. 1"
IRL press (1985)] and a single-stranded cDNA was synthesized
using cDNA Synthesis System Plus (Amersham). Using this
single-stranded cDNA as a template, a polymerase chain
reaction (PCR) was carried out using DNA primers synthesized
based on the nucleic acid base sequence of V region and J
region as classified by Kabat et al. (Sequences of Proteins
of Immunological Interest 4th ed. , Public Health Service, NIH,
Washington DC, 1987). HindIII and BamHI sites were included
in the V region primer and J region primer, respectively. PCR
was conducted in accordance with the protocol of CETUS. That
is, each 100 pmol of the primers were used and PCR reagents
were a kit from CETUS. The PCR conditions were 94°C for 1
minute, 55°C for 1 minute and 72°C for 1 minute, and PCR was
conducted for 25 cycles. After PCR, the obtained DNA
fragments were subcloned into HIncII site of pUCl8 (manufac-
tured by Takara Shuzo K.K.; the reagents used in this Example
were those manufactured by Takara Shuzo K.K. or Toyobo K.K.
unless otherwise mentioned).
3-2) Nucleic acid base sequence of mouse V region gene of C25
antibody
Using SequenaseTM Ver. 2 kit manufactured by Toyobo
K.K., the V region gene incorporated in pUCl8 was sequenced.
The thus obtained nucleic acid base sequences of C25 antibody
are shown in Figs. l9 and 20. Amino acid sequences deduced
from the nucleic acid base sequences are also shown in Figs.
19 and 20. The nucleic acid base sequence of C25 antibody
m
2157874
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showed a rearrangement specific to the V region gene and
formed an open reading frame (ORF) which allows for expres-
sion.
3-3) Construction of gene expressing chimeric C25 antibody
(CHC25, CLC25)
In order to confirm that the isolated V region gene
of C25 antibody is actually a gene coding for V region
responsible for anti-HIV activity, a mouse-human chimeric
antibody was prepared. For expression of a chimeric antibody,
expression vectors, AG-x and AG-yl, ,having a 8-actin (AG)
promoter were used. AG-x contains a human x chain constant
region gene and a DHFR gene as a selection marker whereas AG-
yl contains a human yl chain constant region gene and a neo
gene as a selection marker. The V region of C25 antibody as
prepared above was digested with HindIII and BamHI restriction
enzymes and the obtained VH and VL fragments were incorporated
into the HindIII-BamHI site of AG-yl and AG-x, respectively
(CH~25 and CLC25).
3-4) Expression of chimeric C25 antibody (CC25)
The chimeric C25 antibody gene constructed as
mentioned above was tested for its antibody activity in a
transient expressian system using COS7 cell [ATCCCRL 1651].
A mixture of CHC25 and CLC25 plasmid DNAs was introduced into
COS7 cells using an electroporation device manufactured by
Bio-Rad in accordance with the protocol of Bio-Rad, and the
COS7 cells were cultured in DMEM medium containing 10$ fetal
calf serum (GIBCO) . After three days, the culture supernatant
2157874
42
was collected and the activity of antibodies present in the
culture supernatant was measured by ELISA method using anti-
human IgG or PND peptides derived from various HIVs. As a
result, an expression product of the mixture of CHC25 and
CLC25 plasmid DNAs could bind with the anti-human IgG. The
reactivity with various PND peptides was compared with that
of the original C25 antibody and thereby the expression
product showed a reaction spectrum similar to that of the
original C25 antibody. Furthermore, the neutralizing activity
against HIV-MN strain was also tested,, and as a result, the
expression product was found to inhibit viral infection by
100 at a minimum effective concentration of 1 ug/ml like the
mouse C25 antibody. Accordingly, it was proved that the C25
antibody V region gene isolated as mentioned above is exactly
a gene coding for V region of an antibody having a neutraliz-
ing activity.
3-5) Preparation of a cell strain producing chimeric C25
antibody at high rate
For preparing a stable plasma cell line producing
chimeric C25 antibody (CC25), the above-mentioned plasmid
DNAs, CLC25 and CHC 25, were linearized with PvuI, and CHO-
DG44 cells and P3-653 cells were transformed with a mixture
of the linearized DNAs and lipofectin. As in the temporal
expression of the chimeric antibody, a culture supernatant of
Neo-resistant DHFR-resistant cells where the genes are intro-
duced was collected, and the activity of antibodies present
~in the culture supernatant was measured by ELISA method using
2157874
43
an anti-human IgG and various PND peptides. An expression
product by cotranfection with CLC25 and CHC25 plasmid DNAs
bound with various PND peptides, and hence, this transformed
cell was cloned. Furthermore, an amplification procedure for
DHFR gene was repeated by adding MTX at a concentration of 4
to 32 x 10' M. As a result, a stable plasma cell line which
is resistant to MTX and produces CC25 antibody at a level of
50 to 70 ~g/ml.
Example 4: Preparation of humanized C25 antibody (RC25)
4-1) Transplantation of CDRs of C25 antibody V region gene by
PCR mutagenesis
In order to investigate an important region for
antigen binding among VH and VL regions of the cloned C25
antibody, CDR (complementarity determining) regions of C25
antibody were transplanted into a human V region. This was
carried out in accordance with the method for preparing a
humanized antibody (Japanese Patent First Publication No. 4-
141095). CDR region of C25 antibody VH region was transplant-
ed into VH region having a framework (FR) region of human
subgroup II (NEW: donated by Dr. Bendig of U.K. MRC Collabora-
tive Centre) whereas CDR region of C25 antibody VL region was
transplanted into VL region having FR region of human x chain
(REI: W.Palm and N.Hilscmann, Z.Physiol.Chem., 356, 167
(1975)). Specifically, this was conducted by PCR-mutagenesis
wherein mutation is introduced by PCR (Saiki, R.G. et al.,
Science, 239, 487 (1988)) into the humanized antibody u5.5 or
,0.53 which the present inventors have previously prepared.
2157874
- 44 -
Figs: 21, 22 and 23 show the synthetic primers used for
mutagenesis which are annealed to a PCR template, i.e. VH and
VL regions of the humanized antibody.
The condition of PCR was 94°C for 1 minute, 55°C for
1 minute and 72°C for 1 minute and 25 cycles were repeated.
In the case of VH, using VH gene of the humanized
antibody X5.5 (Japanese Patent First Publication No. 4-152893)
as a template and primers #1 and #2, the 5' site of the VH
gene was amplified (Fig. 21) . This was linked via the central
BglII site to a gene fragment (Fig. 22) which is prepared by
amplification with primers #3 and #4 using the VH gene of the
humanized antibody 0.5J3 (Hum.Antibod.Hybridomas, 2, p124
(1991)) as a template. On the other hand, in case of VL, the
5' site of VL was amplified using #5 and M13-M4 primers and
the 3' site of VL was amplified using primer #8 and M13-
reverse primer, and the resulting amplified genes were linked
together to form KpnI site, which site is self-annealed with
synthetic DNAs. #6 and #7 (Fig. 23).
Thus, V regions of the humanized C25 antibody (RHC25
and RLC25, respectively: cf. SEQ ID N0:2 and SEQ ID N0: 4)
were obtained. These humanized V region fragments were
digested with HindTII and BamHI restriction enzymes as in
preparation of the chimeric antibody (cf. Example 3) and the
resulting VH and VL fragments were incorporated into the
HindIII-BamHI site of AG-yl and AG-x, respectively. Thus,
expression vectors for humanized C25 antibody (RHC25 and
RLC25, respectively) were prepared.
rte:
l,~:w,~,
2157874
- 45 -
4-2) Expression of humanized C25 antibody (RC25)
The activity of antibodies obtained by the thus
prepared humanized antibody gene was examined in a transient
expression system of the above-mentioned COS7 cells. As in
the transient expression of the chimeric antibody, the culture
supernatant of cells where the gene was introduced was
collected and the activity of antibodies present in the
culture supernatant was measured by ELISA method using an
anti-human IgG or PND peptides derived from various HIVs. As
a result, expression products of a mixture of RHC25 and RLC25
plasmid DNAs bound various PND peptides. Furthermore, the
expression products were examined for the neutralizing
activity against HIV-MN strain, and as a result, it was found
that they inhibit the viral infection by 100 at a minimum
effective concentration of 1 ~g/ml like C25 mouse antibody and
the chimeric antibody. Accordingly, among the amino acid
sequence of C25 antibody as shown in Figs. 19 and 20, the
transplanted CDR regions are an important, region for exerting
the anti-HIV activity, and hence, the gene coding for these
regions is the most important gene for preparing a recombinant
antibody.
4-3 ) Preparation of cell line producing humanized C25 antibody
at high rate
In order to prepare a stable plasma cell line
producing the humanized C25 antibody (RC25), the above-
mentioned plasmid DNAs RLC25 and RHC25 were linearized with
s~ ~PvuI and the linearized DNAs, as a mixture with lipofectin,
2157874
- 46 -
were used for transformation of CHO-DG44 cells and P3-653
cells. As in the case of the transient expression of the
chimeric antibody, the culture supernatant of neo-resistant
DHFR-resistant cells wherein the gene is introduced was
collected and the activity of antibodies present in the
culture supernatant was measured by ELISA method using an
anti-human IgG and various PND peptides. An expression
product by cotranfection with RLC25 and RHC25 plasmid DNAs
bound with various PND peptides, and hence, this transformed
cell was cloned. Furthermore, an amplification procedure for
DHFR gene was repeated by adding MTX at a concentration of 4
to 32 x 10' M. As a result, a stable plasma cell line which
is resistant to MTX and produces RC25 antibody at a level of
80 to 100 ~g/ml was prepared.
Example 5: Effector activity of chimeric and humanized C25
antibodies
5-1) Antibody dependent complement-mediated cytotoxicity (ACC)
C25 antibody, chimeric C25 antibody (CC25),
humanized C25 antibody (RC25) and normal human IgG (NHG) were
diluted in RPMI1640 containing 5~ FCS to a final concentration
of 0.1 to 50 ug/ml and each 50 ~1 was added to a 96-well
plate. Then, H9 cells with continuous infection of HIV-MN
(2.5 x 105 cells; 100 ~1) and fresh human serum (30 ~1) were
added and the plate was allowed to stand at 37°C for 1 hour.
After 1 hour; the cells were dyed with trypan blue and the
numbers of living cells and of dead cells were counted.
~ r,
2157874
- 47 -
As shown in Fig. 24, 10 ~g/ml of CC25 antibody and
RC25 antibody destroyed about 70~ of the target cells whereas
C25 antibody and NHG showed a low cytotoxicity. This proved
that the chimeric and the humanized C25 antibodies had a
strong ACC activity.
5-2) Antibody dependent cell-mediated cytotoxicity (ADCC)
Cells with continuous infection of HIV-MN were
established using CEM cells resistant to NK cells (CEM-NKR)
and used as a target cell. The infected cells (3 x 106 cells)
were suspended in RPMI1640 containing 10~ FCS (1 ml) and
labelled with SICr for 90 minutes. The cells (104 cells) were
inoculated on a 96-well plate and thereto were added each 0.1
to 10 ug/ml of C25 antibody, CC25 antibody, RC25 antibody and
NHG. Then, normal human peripheral blood lymphocytes (5 x 105
cells ) were added and the plate was incubated for 4 hours .
Percentage of destroyed cells was obtained in the usual
manner.
As shown in Fig. 25, under condition of effector
cells / target cells = 50, 1 ~.g/ml of CC25 antibody and RC25
antibody destroyed about 50~ of the target cells whereas C25
antibody showed as low cytotoxicity as that of NHG. This
revealed that C25 antibody and NHG showed a low cytotoxicity
activity and proved that the chimeric and humanized C25
antibodies had a strong ADCC activity.
Example 6: Effectiveness of humanized C25 antibody (RC25) to
viruses derived from patients
2157874
- 48 -
6-1) Binding property of RC25 antibody with viruses derived
from patients
Since it is known that HIV occurs as quasispecies
of variant viruses in a single patient, in accordance with the
procedures of Example 2 (2-5), a base sequence of PND region
of viral RNA in plasma and of proviral DNA in peripheral blood
mononuclear cells derived from anti-HIV antibody positive
patients was analyzed with multiple clones per one specimen,
and the binding property of RC25 antibody with recombinant PND
proteins prepared based on the obtained sequence was examined .
Table 5 shows the binding property of RC25 antibody with PNDs
of peripheral blood mononuclear cells derived from various
patients in comparison with HIV-IIIB type virus-specific
chimeric antibody (C(31) and MN type virus-specific humanized
antibody (R~5.5). As is clear from Table 5, RC25 antibody
bound with the PND recombinant proteins derived from various
patients at a high rate of 91 to 100.
Table 5
HIV-infected Binding rate of neutralizing antibody
individuals
RC25 Ru5.5 C 1
YHI 91$(11/12) 8(1/12) 0(0/12)
ASA 100(7/7) 0(0/7) 0(0/7)
HHA 100(46/46) 69(32/46) 0(0/46)
MNI 100$(24/24) 91(22/24) 0(0/24)
KMO 92(12/13) 53(7/13) 0(0/13)
MOK 100(24/24) 100(24/24) 0(0/24)
6-2 ) Neutralizing RC25 antibody viruses derived
activity of to
rom patient plasma
2157874
- 49 -
RC25 antibody (2 mg/ml; 5 ul) was reacted with the
patient plasma (50 ~1) used in the above 6-1 at room tempera-
ture for 30 minutes. Culture was started by adding a mixture
of RC25 antibody and plasma to normal human peripheral blood
mononuclear cell system (50 ~l) wherein CD8-positive cells
were removed with anti-CD8 antibody-bound magnetic beads
(manufactured by Dinal) in ten times higher amount than that
of the mononuclear cells in order to enhance production of
viruses and, after activation with 10 ~g/ml of
phytohemagglutinin for three days, the. mononuclear cells were
cultured in a culture medium containing interleukin-2 for 4
days. After four days, the cells were washed with fresh
medium and then culture was continued while collection of
supernatant and culture exchange were conducted at an interval
of 5 to 7 days. A concentration of HIV-1 p24 antigen in the
collected culture supernatant was measured using a kit for
detecting HIV antigen (manufactured by Dinabbott). In all the
tested cases where viral infection from patient plasma to
normal human peripheral blood mononuclear cells occurred, as
shown in Fig. 26, HIV-IIIB type virus-specific chimeric
antibody (CJ~1) and MN type virus-specific humanized antibody
(R~5.5) used as a control antibody were ineffective, but the
group in which R25 antibody was added showed less than
detection limit of p24 antigen production, and thereby an
apparent effect of RC25 antibody to inhibit infection was
confirmed.
v3
2157874
- 50 -
6-3 ) Neutralizing activity of RC25 antibody to viruses derived
from peripheral blood mononuclear cells of patients
Mononuclear cells were prepared from the above-
mentioned patient peripheral blood ( 20 to 40 ml ) , CD8-positive
cells were removed by the above-mentioned procedure in order
to enhance production of viruses, and then the cells were
cultured in the presence of anti-CD3 monoclonal antibody (0.5
~g/ml) for 3 to 5 days to produce viruses. Culture was
further continued in the presence of 60, 120 and 240 ug/ml of
RC25 antibody while collection of supernatant and culture
exchange were conducted at an interval of 5 to 7 days, and a
concentration of HIV-1 p24 antigen in the culture supernatant
was measured as mentioned hereinabove. In all the tested
cases using peripheral blood mononuclear cells of patients
showing more than 90~ binding of RC25 antibody with the PND
proteins prepared based on proviral DNA in peripheral blood
mononuclear cells, RC25 antibody inhibited production of viral
antigen in a concentration dependent manner as shown in Figs.
27 to 30.
6-4 ) Neutralizing activity of RC25 antibody to viruses derived
from peripheral blood mononuclear cells from patients after
reconstitution of CD8
The above test procedures for confirming the
effectiveness of the present invention, which mimic in vivo
style of HIV infection by activating latent proviruses in
infected cells of patients to induce viral infection, were
~~'! ~ conducted under quite severe conditions wherein CD8-positive
a~
2157874
- 51 -
cells were removed and viral activation was artificially
introduced by the anti-CD3 antibody. Accordingly, RC25
antibody which was proven to be extremely effective in the
test procedures is expected, if clinically applied to
patients, to be much more effective than in the test proce-
dures.
Thus, the same test procedures mentioned hereinabove
were conducted with reconstitution of one tenth amount of the
removed CD8-positive cells at the addition of the antibody.
As a result, as shown in Fig. 31, an effective concentration
of RC25 antibody was reduced to as low as 30 ~g/ml, suggesting
that RC25 antibody is quite effective in clinical application.
HIV is a highly variable virus which infects a
single patient as quasispecies of variant viruses having a
different amino acid sequences. In order to exert treating
efficacy even to such quasispecies of variant viruses, it is
essential to identify a conserved region among HIV strains and
establish a neutralizing antibody which recognizes said
conserved region. V3-PND region of HIV is an important site
which induces a strong neutralizing antibody and a monoclonal
antibody to PND-Tip region comprising the conserved sequence
GPGR is believed to have a broad neutralization spectrum.
The present inventors have established a novel
method for immunization which allows for efficient preparation
of the neutralizing monoclonal antibody recognizing the region
,~v
'::'conserved among each strain, and have established C25
2157874
- 52 -
monoclonal antibody which neutralizes many HIV strains
isolated from infected individuals. Furthermore, by humaniz-
ation of said antibody, the antibody was reduced in antigenic-
ity in humans and endowed with the ability to destroy infected
cells in Fc region dependent manner. Furthermore, contrary
to the conventional HIV neutralizing antibodies which showed
effectiveness only to a single virus isolated in a laboratory,
the antibody of the present invention was confirmed to be
apparently effective to quasispecies of many variant viruses
within the body of a patient due to its broad neutralization
spectrum. Accordingly, the antibody of the present invention
can respond to diversity and variability of various HIVs and
can be clinically applicable as a medicament for prevention,
treatment or diagnosis of HIV.
215'~~7~
- 53 -
SEQUENCE LISTING
SEQ ID N0: 1
SEQUENCE LENGTH: 355
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: cDNA to genomic RNA
ORIGINAL SOURCE
ORGANISM: mouse .
SEQUENCE
CAG GTC CAG CTG CAG CAG TCT GGA GCT GAG CTG GTA AGG CCT GGG ACT 48
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr
1 5 10 15
TCA GTG AAG ATG TTC TGC AAG GCT GCT GGA TAC ACC TTC ACT AAC TCC 96
Ser Val Lys Met Phe Cys Lys Ala Ala Gly Tyr Thr Phe Thr Asn Ser
20 25 30
TGG ATA GGT TGG TTT AGG CAG AGG CCT GGA CAT GGC CTT GAG TGG ATT 144
Trp Ile Gly Trp Phe Arg Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45
GGA GAT ATT TAC CCT GGA GGT GGT TAT ACT AAC TAC AAT GAG ATC TTC 192
Gly Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Ile Phe
50 55 60
AAG GGC AAG GCC ACA CTG ACT GCA GAC ACA TCC TCC AGC ACA GCC TAT 240
Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
_ 215'874
- 54 -
ATG CAG CTC AGC AGC CTG ACA TCT GAG GAC TCT GCC ATC TAT TAC TGT 288
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
g5 90 95
TCA AGG GGG ATA CCG GGA TAT GCT ATG GAC TAC TGG GGT CAA GGA ACC 336
Ser Arg Gly Ile Pro Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
TCA GTC ACC GTC TCC TCA G 355
Ser Val Thr Val Ser Ser
115
SEQ ID N0: 2
SEQUENCE LENGTH: 354
SEQUENCE TYPE: nucleic acid
STR.ANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (modified nucleic acid)
ORIGINAL. SOURCE
ORGANISM: mouse and human
SEQUENCE
CAG GTG CAA CTA GTG CAG TCC GGC GCC GAA GTG AAG AAA CCC GGT GCT 48
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
TCC GTG AAG GTG AGC TGT AAA GCT AGC GGT TAT ACC TTC ACT AAC TCC 96
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Ser
20 25 30
_ 2157874
- 55 -
TGG ATA GGT TGG TTT AGA CAG GCC CCA GGC CAA GGG CTC GAG TGG ATT 149
Trp Ile Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
GGC GAT ATT TAC CCT GGA GGT GGC TAT ACA AAC TAT AAC GAG ATC TTT 192
Gly Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Ile Phe
50 55 60
AAG GGC AAG GCT ACA ATG ACC GCA GAC ACC TCT ACA AAC ACC GCC TAC 240
Lys Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr
65 70 75 80
ATG GAA CTG TCC AGC CTG CGC TCC GAG GAC ACT GCA GTC TAC TAC TGC 288
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
TCA AGG GGG ATA CCG GGA TAC GCT ATG GAC TAT TGG GGA CAG GGT ACC 336
Ser Arg Gly Ile Pro Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
CTT GTC ACC GTC AGT TCA 354
Leu Val Thr Val Ser Ser
X15
SEQ ID N0: 3
SEQUENCE LENGTH: 340
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: cDNA to genomic RNA
ORIGINAL SOURCE
ORGANISM: mouse
2157874
- 56 -
SEQUENCE
GAC ATT GTG ATG ACA CAG TCT CCA TCC TCC CTG ACT GTG ACA GCA GGA
48
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 S 10 15
GAG AAG GTC ACT ATG AGC TGC AAG TCC AGT CAG AGT CTG TTA AAC AGT
96
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
GGA GAT CAA AAG AAC TAC TTG ACC TGG TAC CAG CAG AAA CCA GGG CAG 144
Gly Asp Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 . 45
CCT CCT AAA CTG TTG ATC TAT TGG GCA TCC ACT GGG GAA TCT GGG GTC 192
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Gly Glu Ser Gly Val
50 55 60
CCT GAT CGC TTC ACA GGC AGT GGA TCT GAA ACA GAT TTC ACT CTC ACC 240
Pro Asp Arg Phe Thr Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr
65 70 75 80
ATC AGC AGT GTG CAG GCT GAA GAC CTG GCA GTT TAT TAC TGT CAG AAT 288
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
GAT TAT AGT TAT CCG TGG ACG TTC GGT GGA GGC ACC AAA CTG GAA ATC 336
Asp Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
C 340
Lys
SEQ ID N0: 4
SEQUENCE LENGTH: 339
215'~87~
- 57 -
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (modified nucleic acid)
ORIGINAL SOURCE
ORGANISM: mouse
and
human
SEQUENC E
GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC AGC GTG GGT 48
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 , 15
GAC AGA GTG ACC ATG AGC TGT AAG TCC AGC CAA AGT CTG TTA AAC AGT 96
Asp Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
GGA GAT CAA AAG AAC TAC TTG ACC TGG TAC CAG CAG AAG CCA GGT AAG 144
Gly Asp Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45
GCT CCA AAG CTG CTG ATC TAC TGG GCA TCC ACT GGG GAA TCT GGT GTG 192
Ala Pro ~ys Leu Leu Ile Tyr Trp Ala Ser Thr Gly Glu Ser Gly Val
50 55 60
CCA AGC AGA TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTC ACC 240
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
65 70 75 80
ATC AGC AGC CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGT CAG AAT 288
Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn
85 90 95
215'874
- 58 -
GAT TAT AGT TAC CCA TGG ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC 336
Asp Tyr Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110
339
Lys
SEQ ID NO: 5
SEQUENCE LENGTH: 6
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
ORIGINAL SOURCE
ORGANISM: human immunodeficiency virus
SEQUENCE
Xaa Gly Pro Xaa Arg Xaa
SEQ ID N0: 6
SEQUENCE.LENGTH: 6
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
ORIGINAL SOURCE
ORGANISM: human immunodeficiency virus
SEQUENCE
Xaa Gly Pro Gly Arg Ala
SEQ ID N0: 7
2157874
- 59 -
SEQUENCE LENGTH: 6
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
ORIGINAL SOURCE
ORGANISM: human immunodeficiency virus
SEQUENCE
Ile Gly Pro Gly Arg Xaa
SEQ ID N0: 8
SEQUENCE LENGTH: 6
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
ORIGINAL SOURCE
ORGANISM: human immunodeficiency virus
SEQUENCE
Val Gly Pro Gly Arg Thr
SEQ ID N0: 9
SEQUENCE LENGTH: 6
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
ORIGINAL SOURCE
ORGANISM: human immunodeficiency virus
SEQUENCE
_ 2f 57874
- 60 -
Val Gly Pro Gly Arg Ser
SEQ ID N0: 10
SEQUENCE LENGTH: 6
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
ORIGINAL SOURCE
ORGANISM: human immunodeficiency virus
SEQUENCE
Ile Gly Pro Ala Arg Ala
SEQ ID N0: 11
SEQUENCE LENGTH: 5
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
FRAGMENT, intermediate fragment
ORIGINAL SOURCE
ORGANISM: mouse
SEQUENCE
Asn Ser Trp Ile Gly
SEQ ID N0: 12
SEQUENCE LENGTH: 17
SEQUENCE TYPE: amino acid
. 2~.~ X874
- 61 -
TOPOLOGY: linear
MOLECULE TYPE: peptide
FRAGMENT: intermediate fragment
ORIGINAL SOURCE
ORGANISM: mouse
SEQUENCE
Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Ile Phe Lys
50 55 60 65
Gly
SEQ ID N0: 13
SEQUENCE LENGTH: 9
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
FRAGMENT: intermediate fragment
ORIGINAL SOURCE
ORGANISM: mouse
SEQUENCE
Gly Ile Pro Gly Tyr Ala Met Asp Tyr
100 105
SEQ ID N0: 14
SEQUENCE LENGTH: 17
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
215784
- 62 -
FRAGMENT: intermediate fragment
ORIGINAL SOURCE
ORGANISM: mouse
SEQUENCE
L~ys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asp Gln Lys Asn Tyr Leu
25 30 35
Thr
SEQ ID N0: 15
SEQUENCE LENGTH: 7
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
FRAGMENT: intermediate fragment
ORIGINAL SOURCE
ORGANISM: mouse
SEQUENCE-
Trp Ala Ser Thr Gly Glu Ser
SEQ ID N0: 16
SEQUENCE LENGTH: 9
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
FRAGMENT: intermediate fragment
_ 215'874
- 63 -
ORIGINAL SOURCE
ORGANISM: mouse
SEQUENCE
Gln Asn Asp Tyr Ser Tyr Pro Trp Thr
95 100
