Note: Descriptions are shown in the official language in which they were submitted.
W093/20210 ~ PCT/GB93/00725
Y~ t~ h
ANTIBODIES FOR TREATMENT AND PREVENTION OF
RESPIRATORY SYNCYTIAL VIRUS INFECTION
.F_el~ ~ ~
This invention relates generally to the field of
monoclonal, and recombinant, humanized antibodies, and
specifically, to antibodies directed to epitopes on
Respiratory Syncytial Virus.
sackgl~und of the T~ inn
Respiratory syncytial virus ~RSV) is a pneumovirus of
the family Paramyxovirldae and is the major cause of severe
lower respiratory tract infections in children and calves
during the first year of life [Kim et al., Am~r. J.
E~ LL11~r ~:216-225 ~1973); Stott et al., J~ Hyqi~n~,
R5:257-270 ~1~80); McIntosh and Chanock, in B. N. Fields et
al. (eds), V;rology, RavPn Press, New York ~1990)]. Human
and bovine strains of RSV are antigenically distinct, but
closely related, and two subgroups (A and B) of both human
and bovine strains have been identified [Lerch et al., ~_
V; rol . j 63:833-840 ~1989~; Anderson et al ., J . I~f ec~ Dis .,
626-633 ~1985)].
The use of anti-RSV antibodies for treatment of RSV in
murine and bovine species has been suggested. However, the
treatment of non-murine or non-~ovine species is potentially
25 limited by the immune response of these species to the
foreign murine or bovine antibodies. For example, immune
responses in humans against murine antibodies have been
shown to both immunoglobulin constant and variable regions.
There remains a need in the art to identify
specifically the protective epitopes on RSV proteins and the
- immune effector mechanisms that protect against infection,
and to produce and characterize RSV antigens, epitopes and
antibodies thereto for use in safe, effective RSV vaccines.
Summary of the Invention
SUBSrrrVTE SHEET
WO93/20210 ~1 ~ 5~ ~ PCT/GB93/0072S
The present invention provides a variety of anti-RSV
antibodies, functional fragments thereof including CDRs.
These antibodies and fragments are useful in the
construction of fusion proteins, particularly chimeric and
humanized antibodies, which are characterized by the binding
specificity and/or neutralizing activity of an anti-RSV
monoclonal antibody (mAb). Alsc provided is a novel
humanized antibody containing bovine antibody variable
sequences in association with human immunoglobulin framework ;`
l0 and constant regions. Methods for producing these products, ;'
which further include therapeutic and pharmaceutical
compositions for treating RSV are also disclosed.
Other aspects and advantages of the present invention
are described in the following detailed description.
~
Fig. l is a graph illustrating the isolation of
recombinant LFl~1298, which contains the RSV Long strain F
glycoprotein cDNA with a single transversion C to A at
nucleotide 1298, cloned in the polylinker of pGEM4. This
recombinant permits expression of the F protein in selected
host cells.
Fig. 2 is a diagram of the F glycoprotein primary
structure denoting the hydrophobic regions (~), the site of
proteolytic processing (~), the potential sites for N-
glycosylation (~), the cysteine residues (-) and the amino
acid residues which are changed in neutralization escape
mutants (_). The locations of the trypsin fragments
recognized by different mAbs are shown below the diagram.
Figs. 3A and 3B compare partial B4 and Bl3/Bl4 antibody
variable light (VL) chain amino acid sequences [SEQ ID NOS:
l iand 2~. The B4 sequènce is reported above the ~13/Bl4
sequence to more readily illustrate comparison between the
sequences. In the sequences, the symbol "-" represents a
gap in the sequence introduced to improve the alignment
SVBSTITUTE S~tEET
- W093/20210 ~ PCT/GB93/00725
between the sequences. The CDRs are boxed. The underlined
sequences correspond to the sequences of the polymerase
chain reaction (PCR) oligonucleotide primers used in
amplifying these antibody sequences.
Figs. 4A and 4B compare partial B4 and B13/B14 antibody
variable heavy (VH~ chain amino acid sequences [SEQ ID NOS:
3 and 4] with the B4 sequence reported above the B13/B14
sequence. The symbol ~ , CDRs and PCR oligonucleotide
primers sequences are defined and illustrated as in Figs. 3A
and 3B.
Fig. 5 is a bar diagram showing the competitive binding
of 10 anti-F bovine mA~s, labelled with l2sI, to the A2
strain of RSV in the presence of increasing amounts of
unlabelled antibodies. "Neut" represents the ability of the
mAb to neutralize the RSV in a plaque neutralization assay.
"FI" refers to the ability of the antibody to inhibit fusion
of multinucleated giant cells in an assay~ "Protection"
refers to whether the m~b was able to protect mice against
RSV infection in an in vivo assay. Symbols: less than 10~
(-), 11 to 80% (cross-hatched box), or greater than 80% (O)
remaining bound at the highest amount of competing antibody
tested.
Fig. 6 is a bar diagram showing the competitive binding
of anti-F murine mAbs. "Neut", "FI", "Pro~ection" and the
symbols are defined as in Fig. 5.
Fig. 7 is a bar diagram showing the binding of anti-F
mAbs to the RSV A2 strain and antibody-escape mutant RSVs.
The antibodies were tested in an ELISA using the purified
viruses indicated at the top of the figure to coat
microtitre plates. Symbols: less than 20% (H), 20 to 80%
;~cross-hatched box), greater than 80~ (O) of the absorbance
values obtained with the A2 strain.
Fig. 8 is a bar diagram showing the binding of anti-F
mAbs to RSV Long strain and antibody-escape mutant RSVs.
SUBSm~JTE SH EET
,~
WO93/20210 ~ 6 6 2 PCT/GB93/00725 -
The antibodies were tested as described in Fig. 7. Symbols:
less than 25% (open box), 25 to 50% ~cross-hatched box),
greater than 50% (-) of the absorbance values obtained with
the Long strain.
S Fig. 9 is a series of 8 bar diagrams showing the
binding of mAb B4 to synthetic octomeric peptides, bound to ;
polyethylene pins, where each amino acid in the sequence
corresponding to amino acid #266 through 273 of the RSV F
protein ~SEQ ID NO: l9] has been replaced in turn with other
amino acids (indicated on the abscissa). The sequence of
amino acids beneath each bar diagram shows which amino acid
has been replaced (indicated by a box around the letter).
The antibody binding was tested in an ELISA and the black `
bars represent the absorbance values obtained with the
native sequence of the peptide and the grey bars represent
the absorbance obtained with the peptides containing the
substituted amino acids.
__Fig. lO is a predicted humanized V~ region sequence
B4HuVH wherein bovine mAb B4 is the donor antibody [SEQ ID
20 NO: 5]. CDRs are boxed. Underlined residues in the -
framework regions are murine residues which have been
retained.
Fig. ll is a predicted humanized constant heavy region
sequence B4HuVK for use in cons~ructing an altered antibody,
wherein 84 is the donor antibody [SEQ ID NO: 6]. CDRs are
boxed.
Figs. 12A and 12B provide a contiguous predicted ;
humanized VH region sequence Bl3~Bl4HuVH [SEQ ID NO: 7] for
use in constructing an altered antibody, wherein Bl3/Bl4 is
the donor antibody. CDRs are boxed and retained murine
residues are underlined.
Fig. 13 is a predicted humanized constant hea~y region
sequence Bl3/Bl4HuVK [SEQ ID NO: 8~ wherein Bl3/Bl4 is the
donor antibody. CDRs are boxed.
SUBSTITUTE SHEET
'W093/20210 ~J ~ r~62 PCT/GB93/0072~
Figs. 14A and 14B provide a contiguous DNA sequence and
corresponding amlno acid sequence [SEQ ID NOS: 9 and 10] for
the VH region of RSV19. CDRs are boxed. Underlined
sequences correspGnd to the primers used.
Figs. 15A and 15B provide a contiguous DNA sequence and
corresponding amino acid sequence of the RSVl9 VL region
- ~SEQ ID NOS: 11 and 12]. CDRs are boxed. Primers are
underlined.
Fig. 16 shows the plasmid pHuRSV19VH comprising a human
Ig VH region framework an~ CDRs from murine RSV19.
Flg. 17 shows the plasmid pHuRSV19VK comprising a human
Ig VL framework and CDRs derived from RSV19.
Fig.~;18 shows the derived Ig variable region amino acid
sequences encoded by murine RSV19VH [SEQ ID NO: 13].
Fig. 19 shows the derîved Ig variable region amino acid
sequences encoded by pHuRSV19VH [SEQ ID NO: 14].
F~ig. 20 shows~the~derived Ig variable region amino acid
sequences~encoded~by pHuRSV19VHFNS [SEQ ID NO: 15]. ;~
F~i~g.~,21~shows~the~derived~Ig variable region amino acid
O~ s'equenc~es~encoded~by~pHuRSV19VHNIK lSEQ ID NO: 16]. ',
Fig.~22~shows the~derived Ig variable region amino acid
sequences~encoded by~pHuRSV19VK ~SEQ ID NO: 17].
Fig. 23 is~the DNA and amino acid encoding the HuVL
framework ~4,;~5EQ ID~,~NOS: 2~0 and 21]~showing the potential
,",~ 25 ~spl1ce~site~ ,The~underlined bases were changed to provide `~
,the,~-gen,ùine~Jl~gene~sequence~[SEQ ID NO: 22]. ;~
etai/lé~d~ne~ crtDt~;`on~Qf~the In~ent'
Dè~-lnl:tlons`.~
As used~herein,~the~term "first fusion partner" refers '`
to a nucleic~acid sequence encoding an amino acid sequence,
ich~can'be'all~or part of a heavy chai'n variable region, ~'
light~;chain~var~iable~region, CDR, functional fragment or
analog~thereof, having the antigen binding specificity of a `~
selected antibody, prePerably an anti-RSV antibody. "
:,',~f ,''"` ~: ~ ` '`
.': .', : ~ :`~
SU8STITUTE SH EET
WO93/20210 i~ c5 ~ ~ 6 2 PCT/GB93/00725
As used herein the term "second fusion partner" refers
to another nucleotide sequence encoding a protein or peptide
to which the first fusion partner is fused in frame or by
means of an optional conventional linker sequence. ~Such
second fusion partners may be heterologous to the first
fusion partner. A second fusion partner may include a
nucleic acid sequence encoding a second antibody region of
interest, e.g., an appropriate human constant region or
framework region.
The term "fusion molecule" refers to the product of a
first fusion partner operatively linked to a second fusion
partner. "Operative linkage" of the fusion partners is
defined as an association which permits expression of the
antigen specificity of the anti-RSV sequence (the first
fusion partner) from the donor antibody as well as the
desired characteristics of the second fusion partner. For
example, a nucleic acid sequence encoding an amino acid -~
linker may be optionally used, or linkage may be via fusion
in frame to the second fusion partner.
The term "fusion protein" refers to the result of the
expression of a fusion molecule. Such fusion proteins may
be altered antibodies, e.g., chimeric antibodies, humanized -;
antibodies, or any of the antibody regions identified herein
fused to immunoglobulin or non-immunoglobulin proteins and
the like.
As used herein, the term "donor antibody" refers to an
antibody (polyclonal, monoclonal, or recombinant) which
contributes the nucleic acid sequences of its naturally-
occurring or modified variable light and/or heavy chains,
~DRs thereof or other functional fragments or analogs
thereof to à first fusion partner, so as to provide the
fusion molecule and resultinq expressed fusion protein with
the antigenic specificity or neutralizing activity
characteristic of the donor antibody. An example of a donor
SUBSTITUTE SHE~T
WO93/20210 PCT/GB93/00725
antibody suitahle for use in this invention is bovine mAb B4
or B13/14.
As used herein the term "acceptor antibody" refers to
an antibody (polyclonal, monoclonal, or recombinant~
heterologous to the donor antibody, but homologous to the
patient (human or animal) to be treated, which contributes
all or a substantial portiorl of the nucleic acid sequences
encoding its variable heavy and/or light chain framework
regions andlor its heavy and/or light chain constant regions
~o a second fusion partner. Preferably a human antibody is
a-n acceptor antibody.
"CDRs" are defined as the complementarity determining
region amino acid sequences of an antibody which are the
hypervariable regions of immunoglobulin heavy and light
chains which provide the majority of contact residues for
the binding of the antibody to the antigen or epitope. CDRs
interest in;this invention are derived from donor j` antibody variable heavy and light chain sequences, and
include functional fragments and~analogs of the naturally
occurring CDRs, which fragments and analogs also share or
retain the same antigen binding specificity and/or
-~ neutrali~zing ability as the donor antibody from which they
were~derived. See, e.g., the CDRs indicated by boxes in
Figs. 3A, 3B, 4A, 4B, and 10 through 13. By 'sharing the
25 ~antigen~binding~specificity or neutralizing ability' is
meant, for example,;that although mAb B13/B14 may be
characterized by~a~ce~tain level of~antigen affinity, and a
CDR encoded~by a~nucleic acid sequence of B13/B14 in an
appropriate structural environment may have a lower
affinity, it is expected that CDRs of B13/B14 in such
env1ronments will nevertheless recognize the same epitope(s)
as B13/B14.
-A~"functional fragment" is a partial CDR sequence or ;-
partial heavy or light chain variable sequence which retains
`
:: i,`:
SUBSTITUTE SHEET
WO93/20210 ~ PCT/GB93/0072i`
the same antigen binding specificity and/or neutralizing
ability as the antibody from which the fragment was derived.
An "analog" is an amino acid or peptide sequence
modified by replacement of at least one amino acid,
modification or chemical substitution of an amino acid,
which modification permits the amino acid sequence to retain
the biological characteristics, e.g., antigen specificity,
of the unmodified sequence.
An "allelic variation or modification" is an alteration
in the nucleic acid sequence encoding the amino acid or
peptide sequences of the invention. Such variations or
modifications may be due to degeneracies in the genetic code
or may be deliberately engineered to provide desired
characteristics. These variations or modifications may or
may not result in alterations in any encoded amino acid
sequence.
As used herein, an "altered antibody" describes a type
of fusion protein, i.e., a synthetic antibody (e.y., a
chimeric or humanized antibody) in which a portion of the
light and/or heavy chain variable domains of a selected
acceptor antibody are replaced by analogous parts of CDRs
from one or more donor mAbs which have specificity for the
selected epitope. These altered antibodies may also be
characterized by minimal alteration of the nucleic acid -
sequences encoding the acceptor mAb light and/or heavy
variable domain framework regions in order to retain donor
mAb binding specificity. These antibodies can comprise
immunoglobulin (Ig) constant regions and variable framework
regions from the acceptor mAb, a~d one or more CDRs from the
anti-RSV donor antibodies described herein.
! ' A "chimeric antibody" refers to a type of altered
antibody which contains naturally-occurring ~ariable region
light chain and heavy chains (both CDR and framework
regions) derived from a non-human donor antibody in
SUBSTmJTE SHEET
-WO93/20210 PCT/GB93/00725
association with light and heavy chain constant regions
derived from a human acceptor antibody.
A "humanized antibody" refers to an altered antibody
having its CDRs and/or other portions of its light ~nd~or
heavy variable domain framework regions derived from a non-
human donor immunoglobulin, the remaining immunoglobulin-
derived parts of the molecule being derived from one or more
human immunoglobulins. Such antibodies can also include
altered antibodies characterized by a humanized heavy chain
associated with a donor or acceptor unmodified light chain
or a chimeric light chain, or vice versa. -~
The term "effector agents" refers to non-protein
carrier molecules to which the fusion proteins, and/or
natural or synthetic light or heavy chain of the donor -;~
15 antibody or other fragments of the donor antibody may be ;
associated by conventional means. Such non-protein carriers
can include conventional carriers used in the diagnostic -~`
field, e.g., polystyrene or other plastic beads, or other
non-protein substances useful in the medical field and safe -;~
for administration to humans and animals. Other effector
; agents may include a macrocycle, for chelating a heavy metal
atom, or a toxin, such as ricin. Such effector agents are ;`
useful to increase the half-life of the anti-RSV derived
amino acid sequences.
25 II. Anti-RSV Antibodies
For use in constructing the antibodies, fragments and `
fusion proteins of this invention, a non-human species may
be employed to generate a desir~ble immunoglobulin upon
presentment with the respiratory syncytial virus (RSV) F
30 protein or a peptide epitope therefrom. Conventional `~
~ hybridoma techniques are employed to provide a hybridoma
; cell line secreting a non-human mAb to the RSV peptide.
For example, several neutralizing, fusion-inhibiting
(FI) and highly protective bovine and murine anti-RSV i
:~ . .. i-
,
~i
`;
SUBSTITIJTE SHEET
W093~20210 ~'J ~ b ~ PCT/G~93tO0725
monoclonal antibodies (mAbs) are provided by this invention.
The production and characterization of the bovine antibodies
capable of binding to the F protein, B13 and Bl4, and other
suitable bovine mAbs designated herein as B4, B7 through
B10, and murine mAbs, designated herein as 16 through 21,
are described in detail in Examples 1 and 2, and in Figs. 5
and 6.
The resulting B13 and B14 anti-RSV antibodies are
characterized by the ability to neutralize RSV in a plaque
reduction neutralization test. Both B13 and B14 are potent
in fusion inhibition assays and are protective in mice.
Competition studies, together with studies of antibody-
escape mutants, binding to F protein fragments and synthetic
peptides suggest that the epitope recognized by mAbs Bl3 and
B14 may be similar to, but not identical to, the epitope
recognized by mAb RSV19 (also known as mAb 19 or RSMUl9),
the IgG2a murine mAb specific for F protein amino acid 417-
438 of and described in Example 11 below and in PCT patent
application No. PCT/GB91/01554. After sequencing, B13 and
20 B14 have been determined to be substantially identical are ;
referred to as a single mAb called Bl3/B14 in certain
instances. Where the mAbs were tested separately, reference
is made to mAb Bl3 or Bl4.
The inventors have determined that a previously
disclosed anti-RSV mAb, B4, is effective in protecting
calves against infection with bovine RSV, as well as
protecting mice against infection with human RSV. The
ability of bovine mAb B4, administered to calves by the i.t.
route, to protect against lower respiratory tract infection
with RSV and against the development of pneumonic lesions,
indicates that bovine mAbs are potentially effective
prophylactic and therapeutic agents in the control of calf
respiratory disease. B4 is also potent in fusion inhibition
and virus neutralization assays (Examples 16 and 17).
SV8ST~TlJTE SH EET
WO~3/20210 ~ ~ PCT/GB93/00725
'
1 1 !
These three bovine mAbs B4, B13, and Blq have been ~
identified as desirable antibodies which may be altered for ;
pharmaceutical or prophylactic use. However, this invention
is not lirnited to the use of these three mAbs or their
hypervariable sequences. These mAbs illustrate the products
and methods of this invention; wherever in the following
description the donor mAb is identified as B4, B13 or B14,
it should be understood that any other appropriate anti-RSV
neutralizing antibodies and corresponding anti-RSV CDRs may
10 be substituted therefor. `~
It is anticipated that other antibodies, bovine as well -
as other species, which are developed against the RSV F
protein epitope spanning amino acid 266 through 273 as well
as other RSV epitopes of interest described herein, may be -~
useful in compositions of this invention for treating RSV in
mice, cattle and humans. Other anti-RSV antibodies may be -~
developed by screening an antihody li~rary including
hybridoma products or libraries derived from any species ;~-
immunoglobulin repertoires in a conventional competition
20 assay, such as described in the examples below, with one or ~`
more bovine antibodies or RSV epitopes described herein. ;`
- Particularly desirable for screening for additional
antibodies are the neutralizing and protecti~e mAbs, B4 and ~
B13/B14. ;
Thus, the invention may provide an antibody, other than `-~
B4 or Bl3/14, which is capable of binding to the RSV peptide
spanning amino acid #266 through #273, ITNDQKKL, of the F `
protein [SEQ ID NO: 19] or other relevant RSV epitopes. `i
This antibody may be a mAb or an altered an~ibody, an analog `
30 of such antibodies, a Fab fragment thereof, or an F(ab') 2 ``
; ~ragment thereof. Such other mAbs generated against a
desired RSV epitope and produced by conventional techniques,
include without limitation, genes encoding murine mAbs,
human mAbs, and combinatorial antibodies.
SUBSTmJTE SHEET `;
O ~
WO93/2~210 PCT/GB93/0072~*~
Th~se anti-RSV antibodies may be useful in
pharmaceutical and therapeutic compositions for treating RSV
in humans and other animals.
III. Antibody Fragnents ~ -
The anti-RSV antibodies described above may be useful
as donors of desirable functional rragments, including the ;~
antibody light and heavy chain variable sequences and CDR
peptides.
The present invention also includes the use of Fab
fragments or F~ab') 2 fragments derived from mAbs directed
against an epitope of RSV as agents protective in vivo
against RSV infection and disease. A Fab fragment is the
amino terminal half of the heavy chain and one light chain,
and an F(ab') 2 fragment is the fragment formed by two Fab
fragments bound by disulfide bonds. MAb Bl3Jl4 or other
suitable RSV binding antibodies, provide a source of these
fragments, which can be obtained by conventional means,
e.g., cleavage of the mAb with the appropriate proteolytic
enzymes, papain and/or pepsin.
These Fab and F~ab') 2 fragments are also useful
themselves as therapeutic, prophylactic or diagnostic agents
for RSV in humans and other animals, and are also useful as
donors of variable cha1n sequences, CDRs and other
functional fragments useful in this invention.
IV. RSV F Protein Æpitopes of Interest
The above-described mAbs recognize certain protective
epitopes on the fusion (F~ protein of RSV which are
recognized by a natural host of RSV. The nucleotide
sequence of the F mRNA and the predicted protein sequence of
the F ~rotein [SEQ ID NO: l9] have been previously reported
in'Collins et al., ~oc. Na~l Acad. Sci, US~, ~l:7683-7687
(1984). The amino acid numbering referred to herein is
identical to the numbering in this latter reference. The
inventors identified an eight amino acid sequence spanning
SUBSTmJTE SHEET
. W093t20210 PCT/GB93tO0725 `-~
13 -
amino acids 266 through 273 of the F protein [SEQ ID NO~
19], as a suitable target for screening for neutralizing ~:~
antibodies, as an antigen useful in therapeutlc agents
against RSV, and in particular, for producing monoclonal ;:-
antibodies against RSV. Other epitopes of interest include
epitopes at around amino acid #429 which are recognized by
neutralizing antibodies, B13 and B14.
The regions of the F protein [SEQ ID NO: 19] which
react with the neutralizing, fusion-inhibiting, and highly ..
10 protective bovine and murine mAbs of the invention were ~`
mapped by competitive binding assays (Example 6); isolation `.
and sequencing of antibody neutralization escape mutants .`
(Examples 7 and 8); and synthesis of peptides with sequences i.`.
containing the amino acids changed in the esc~pe mutants and i---
the assessment of the reactivity of these peptides with the
mAbs (Examples 9-11).
Sequence analysis of the F protein [SEQ ID NO: 19] of
the antibody-escape mutants permits identification of the
: amino acid residues important in the binding of the highly
protecti~e mAbs. Similarly, information on the binding of
the protective mAbs to synthetic peptides permits the
location of the epitopes that they recognize. `~
Briefly described, most of the bovine mA~s recognized ``
epitopes similar to those recognized by the murine mAbs, and :
one of the protective antigenic areas (site B; site II of
Fig. ~) is recognized both by cattle, which are a natural
host for RSV, and mice. The epitope~s) recognized by the ~.
protective bovine mAbs B13 and B14 do not appear to be ~-
identicaI to any recognized by murine mAbs. Bi3 and B14 .:.
bind to a region of the F protein around amino acid 429.
! ; ' Th~s epitope is similar, but distinct from the epitope .
recognized by murine mAb RSVl9 (PCT patent application No.
PCT/GB91/01554 and Example 11). For example, mAb B13/Bl4
does not recognize the peptides spanning F protein amino
. . ,
SU8STITUT~ SHE~T `
hl~5~)h
WO93/20210 ~ PCT/GB93/0072~
acids #417-438, #417-432, and #422-438 all of SEQ ID NO: 19,
which are recognized by mAb RSV19. A second antigenic site
(area C, Figs 5 and 6; area IV, Fig. 8) on the F protein
identified by neutrall~ing, protective murine mAbs~RSV19 and
20 has been located towards the carboxy end of the F1
subunit and has also been described in the above-referenced
PCT patent application.
The RSV epitope recognized by B4 is reproduced by the
RSV F peptide at the amino acid sequence spanning #255-275
of SEQ ID NO: 19. The inventors have determined using the
Geysen pepscan technique, that B4 recognizes an epitope
spanning amino acid 266 to 273 of the F protein [SEQ ID NO:
lg3. Altered antibodies directed against functional
fragments or analogs of this epitope m~y be designed to
elicit enhanced binding with the same antibody. mAbs which
are directed against this epitope have been shown to protect
mice and/or bovines from in vivo RSV infection. ~-
Replacement of each amino acid in the sequence has
enabled the discovery that enhanced binding of B4 occurs in
mutant epitopes. Changes in amino acids 266, 279 and 273
did not affect binding of mAb B4. Changes in amino acid 267
resulted in reduced binding of mAb B4. Changes in amino
acids 268, 269, and 272 resulted in total loss of binding.
Substitution at amino acid 271 resulted in significantly
25 enhanced binding (See Example 10). -
The epitopes of these antibodies are useful in the
screening and development of additional anti-RSV antibodies
as described above. Knowledge of these epitopes enables one
of skill in the art to define synthetic peptides and
identify naturally-occurring peptides which would be
suitable as vaccines against RSV and to produce mAbs useful
in the treatment, therapeutic and/or prophylactic, of RSV
infection in humans or other animals.
IV. Anti-RSV Nucl eotide Sequences o f Inte~est
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~: WO93/20210 ~ v~ b~ Pcr/GBo3/0072s
1 s
The mAbs B4 and B13J14 or other anti-RSV murine, human ;~
and bovine, antibodies described herein may donate desirable ~ `;
nucleic acid sequences encoding variable he~vy and/or light
chain amino acid sequences and CDRs, functional fra~ments,
and analogs thereof useful in the development of the first
fusion partners, fusion molecules and resulting expressed !,~
fusion proteins according to this invention, including
chimeric and humanized antibodies. `;~
The present invention provides isolated naturally-
occurring or synthetic variable light chain and variable
heavy chain sequences derived from the anti-RSV antibodies, ~`
which are characterized by the antigen binding specificity ~-~
of the donor antibody. Exemplary nucleotide sequences of
interest include the heavy and light chain variable chain
sequences of the mAbs B9, B13 and B14, as described below in
the examples. Based on this ~ariable region sequence data,
B13 and B14 appear to ~e substantially identical. ;~
The naturally occurring ~axiable light chain of B13/14
is characterized by the amino acid sequence of Figs. 3A and `~-~
3B [SEQ ID NO: 2] labelled B13~B14VL. The naturally-
occurring variable hea~y chain of B13~14 is characterized by `~
the amino acid sequence illustrated in Figs. 4A and 4B ~SEQ
ID NO: 4] labelled B13VH. These hea~y and light chains are
described in Example 18.
As described above for B13/B14, the amino acid ;`'
sequences of the B4VL and VH chains are reported in Figs. 4A
and 4B [SEQ ID NO: 4] and 3A and 3B [SEQ ID NO: 2~
respectively, with the putative CDR peptides boxed. In both ~`
VH chains of B13/B14 and B4, the CDR3 peptides are unusually
long, having 25 and 21 amino acids, respectively, in
contrast to the vast majority of human and rodent CDR3s
which have less than 20 amino acids.
The nucleic acid sequences encoding the ~ariable hea~y
and/or light chains, CDRs or functional fragments thereof,
SUBSTmlTE SltEET `
WO93~20210 ~ 6 ~ PCT~GR93/00725
16
are used in unmodified form or are synthesized to introduce
desirable modifications. These sequences may optionally
contain restriction sites to facilitate their insertion or
ligation to a second fusion partner, e.g., a suitable
nucleic acid sequence encoding a suitable antibody framework
region or the second fusion partners defined above.
Taking lnto account the degeneracy of the genetic code,
various coding sequences may be constructed which encode the
VH and VL chain amino acid sequences, and CDR sequences
(e.g., Figs. 3A, 3B, 4A, 4B, and 10 through 13) and
functional fragments and analogs thereof which share the
antigen specificity of the donor antibody.
Thus, these isolated or synthetic nucleic acid
sequences, or fragments thereof are first fusion partners,
15 which, when operatively combined with a second fusion ;
partner, can be used to produce the fusion molecules and the
.
expressed fusion proteins, including altered antibodies of
this~ invention. These nucleotide sequences are also useful
or mutagenic insertion of spec~fic changes within the
nucleic acid sequences encoding the CDRs or framework
regions~, and for incorporation of the resulting modified or `
nucleic acid sequence into a vector for expression.
VI~. Fusion Molecules, Fus i on Prote~ns and Other Proteins of ~ `~
thi s: In ven t i on
A fusion molecule may contain as a first fusion partner
a nucleotide sequence~from an anti-RSV donor mAb, fragment
~or analog which sequence encodes an amino acid sequence for
the naturally occurring or synthetic VH or VL chain
- sequences, a functional fragment or an analog thereof. When
the first fusion partner is operatively linked to a $econd `
- fusion partner, the resulting fusion molecule and expressed
fusion protein is characterized by desirable therapeutic or
prophylactic characteristics.
':
: : :
SUBSTITUTE SHEET `
"
WO93/20210 ~ 3~ PCT/GB93/00725
The fusion molecule, upon expressio~, can produce a
fusion protein which is an altered antibody, a chimeric, `~
humani~ed or partially humanized antibody. Altered
antibodies directed against functional fragments or analogs
of RSV m~y be designed to elicit enhanced binding in
comparison to the donor antibody.
An exemplary fusion molecule may cont~in a synthetic VH
and/or VL chain nucleotide sequence from the donor mAb
encoding a peptide or protein having the antigen specificity `~
of mAb B4 or B13/14. Still another desirable fusion
molecule may contain a nucleotide sequence encoding the
amino acid sequence containing at least one, and preferably `~
all of the CDRs of the VH and/or VL chains of the bovine
mAbs B4 or B13/14 or a functional fragment or analog ;
15 thereof. The second fusion partners with which the anti-RSV `-
sequences first fusion partners are associated in thè fusion --
molecule are defined in detail ~bove. ;
Where the second fusion partner is a nucleic acid
sequPnce encoding a peptide, protein or fragment thereof
heterolo~ous to the nucleic acid sequence having anti-RSV
antigen specificity, the resulting fusion molecule may
express both anti-RSV antigen specificity and the ;~-
characteristic of the second fusion partner. Typical
characteristics of second fusion partners can be, e.g., a
25 functional characteristic such as secretion from a ~
recombinant host, or a therapeutic charac~eristic if the ;~-
fusion partner is itself a therapeutic protein, or
additional antigenic characteristics, if the second fusion
partner has its own antigen specificity.
If the second fusion partner is derived from another
antibody, e.g., any isotype or class of immunoglobulin
framework or constant region (preferably human), or the
like, the resulting fusion molecule of this invention
provides, upon expression, an altered antibody. Thus a
SUBSTlTUTE Stl EET
WO93/20210 `` ~ 4~ b~ PCT/CB93/0072~'
fusion molecule which on expression produces an altered
antibody can comprise a nucleotide sequence encoding a
complete antibody molecule, having full length heavy and
light chains, or any fragment thereof, such as th~ Fab or
F(ab')2 fragment, a light chain or heavy chain dimer, or any
minimal recombinant fragment thereof such as an Fv or a ''
single-chain antibody (SCA) or an~ other molecule with-the ~.. -
same specificity as the donor mAb. ' .
As one example, a fusion molecule which on expression `~.
produces an altered antibody may contain a nucleic acid
se:quence encoding an amino acid sequence having the antigen
:specificity of an anti-RSV antibody directed against the F
protein amino acid sequence spanning amino acid #266 through:'.
#273 of SEQ ID NO: l9, ITNDQKKL and analogs thereof, ~S
operatively linked to a selected second fusion partner.
Analogs of that epitope include those identified in the '
examples,~ such as SEQ ID NO:::56 when amino acid #266 is ::~
replaced with Ala, Cys, Asp, Glu,' Phe, Gly, His, Leu, Pro,
:: Gln~,~ Arg;, S:er, Thr, Val, Trp, and Tyr; or SEQ ID NO: 57 when ~
20 amino aci~d #2'69 is replaced with G1u, Phe, Ile, Leu, Met, `:'
Arg,~Ser,~ Thr,~Val, Trp,~and Tyr; or SEQ ID NO: 58 when
am~ino~acid~#271 is replaced with Asp, Glu, Phe, Ile, Leu,
:Met,: Arg,:Ser, Thr, Val, Trp, Tyr and Gln; or SEQ ID NO: 59::
when~amino~acid #~273 is replaced with Ala, Cys, Asp and Glu.
': 25 ~Desirably the source of the nucleic acid sequences is mAb
B4.- ~ ~
;Another'~fus:ion~molecule which~on expression produces an :.
altered~antibody~may contain a nucleic acid sequence
encoding:~the.variable heavy.chain sequence of Figs. 4A and
'30 :4B, a functional fragment or analog thereof, the variable
' light chain sequènce of Figs. 3A and 3B, a functional
: fragment or analog thereof, or one or more B4 CDR peptides.
Another exemplary fusion molecule may contain a nucleic''-
: acid sequence encoding an amino acid sequence having the .'
..:
~ .
- i:
SUBST1TUTE SHEET
' ~ ..
::
WO93/20210 ;~ 2 PCT/GB93/00725
19
antigen specificity of the anti-RSV antibody B13/~14,
operatively linked to a selected second fusion partner. For
example, the nucleic acid sequence may encode the VH chain
sequence of Figs. 4A and 4B [SEQ ID N0: 4], a functlonal ~;
fragment or analog thereof, the VL chain sequence of Figs.
3A and 3B [SEQ ID NO: 2], a functional fragment or analog
thereof, or one or more B13/B14 CDR pept-ides. ~
When the fusion protein which is obtained upon ~-
expression of the fusion molecule is an altered antibody, ~`
the antibody contains at least fragments of the VH and/or VL
domains of an acceptor mAb which have been replaced by
analogous parts of the variable light and/or heavy chains
from one or more donor monoclonal antibodies. These altere~ i
antibodies can comprise immunoglobulin ~Ig) constant regions ~-
and variable framework regions from one source, e.g.,~ the
acceptor antibody, and one or more CDRs from the donor
ar.tibody, e.g~, the anti-RSV antibodies described herein.
An altered antibody may be further modified by changes
in variable domain amino acids without necessarily affecting
the specificity of the donor antibody. It is anticipated
that heavy and light chain amino acids ~e.g., as many as 25%
thereof) may be substituted by other amino acids either in
the variable domain frameworks or CDRs or both. Such
altered antibodi~s may or may not also include minimal
alteration of the accepto- mAb VH and~or VL domain framework
region in order to retain donor mAb binding specificity.
In addition, these altered antibodies may also be
characterized by minimal alteration, e.g., deletions,
substitutions, or additions, of the acceptor m~b VL and/or
30 VH domain framework region at the nucleic acid or amino acid ~
levels may be made in order to retain donor antibody antigen `
binding specificity.
Such altered antibodies are designed to employ one or
both of the VH or VL chains of a selected anti-RSV mAb
SUBSTmJTE SHEE~
W093/20tl0 ~ PCT/GBg3/0072
(optionally modified as descrlbed) or one or more of the
above identified heavy and/or light chain CDR amino acid and
encoding nucleic acid sequences. As another example, an
altered antibody may be produced by expression of ~ fuslon
molecule containing a synthetic nucleic acid sequence
encoding three CDRs of the VL chain region of the selected
anti-RSV antib~dy or a funct~onal fragment thereof in place
of at least a part of the nucleic acid sequence encoding the
VL region of an acceptor mAb, and a nucleic acid sequence
encoding three CDRs of the VH chain region of a selected
anti-RSV antibody, e.g., the bovine mAb B13/14, ox a
functional fragment thereof in place of at least a part of
the nucleic acid sequence encoding the VH region of an
acceptor mAb, such as a human antibody.
The altered antibodies can be directed against a
specific protein epitope of RSV spanning amino acid #266-273
of SEQ ID NO: 19. It has been demonstrated that monoclonal
antibodies which are directed against this epitope protect
mice and/or bovines from in vivo RSV infection.
A suitable acceptor antibody, for supplying nucleic
acid sequences as second fusion partners, may be a human (or
other animal) antibody selected from a conventional ,~
database, e.g., the Kabat database, Los Alamos database, and
Swiss Protein database, by homology to the nucleotide and -
amino acid sequences of the donor antibody. Desirably the
acceptor antibody is selected from human IgG subtypes, such
as IgG1 or IgG2, although other Ig types may also be
employed, e.g., IgM and IgA. For example, a human antibody
characterized by a homology to the framework regions of the
donor antibody (on an amino acid basis) may be suitable to
p~ovide a heavy chain constant region and/or a heavy chain
variable framework region for the insertion of the donor
- CDRs. A suitable acceptor antibody capable of donating
light chain constant or variable framework regions may be
,.':
SUBSTlTUTE SH EET ~
` ` wo93j20210 ~ h~2 PCT~G~93/00725
21
selected in a similar manner. It should be noted that the .
acceptor antibody heavy and light chains are not required to
originate from the same accep~or antibody.
The acceptor antibody need not contribute only~human
S immunoglobulin nucleotide sequences to the desired fusion
molecule, and resulting expressed fusion protein. For ~`
instance a fusion molecule may be constructed in which a DNA
sequence encoding part of a human immunoglobulln chain is
fused to a DNA sequence encoding the amino acid sequence of ;;-
a polypeptide effector or reporter molecule.
Similarly rather than a human immunoglobin, a bovine or
another species' immunoglobulin may be used, e.g., to create
a 'bovinized' or other species' altered antibody. -
One example of a particularly desirable fusion protein
is a humanized antibody. As used herein, the term
"humanized antibody" refers to a molecule having its CDR
regions and/or other partions of its VL and/or VH domain ~
framework regions derived from an immunoglobulin from a non- ```
human species, the remaining immunoglobulin-derived parts of
the molecule being derived from a human immunoglobulin.
Suitably, in these humanized antibodies one, two or
preferably three CDRs from the anti-~SV antibody VH and/or
VL regions are inserted into the framework regions of a
selected human antibody, replacing the native CDRs of that
latter antibody. Preferably, the variable domains in both
human heavy and light chains have been altered by one or
more CDR replacements. However, it is possible to replace
the CDRs only in the human heavy chain, using as the light
chain an unmodified light chain from the bovine donor
antibody. Alternatively, a compatible light chain may be
selected from a human acceptor antibody as described above.
A chimeric light chain may also be employed. The remainder
of the altered antibody may be derived from any suitable
acceptor human immunoglobulin.
SU8STlTUTE SH EET
WO93/20210 ~ ~c~t~ ~ PCT/GB93/0072S
Such altered antibodies according ~o this invention
include a humanized antibody containing the framewor~
regions of a human IgG subtype into which are inserted one
or more of the CDR regions of a bovine antibody. Such a
humanized antibody can contain the VH CDR peptides of the
bovine mAb inserted into the heavy chain framework region of
a human antibody and in association with the bovine light
chain, or a bovine/human chimeric light chain. Such an
exemplary humanized antibo~y is described in Example 20.
Alternatively, such an altered antibody may be associated
with a desired human light chain. Similarly, a chimeric
antibody can contain the human heavy chain constant regions
(preferably IgG) fused to the anti-RSV antibody, preferably
bovine mAb, Fab regions. An exemplary chimeric antibody is
described in ~xample l9.
The altered antibody preferably has the structure of a
natural antibody or a fragment thereof and possesses the
~ombination of properties required for effective prevention
and treatment of a desired condition in animals or man
depending on the antigenicity supplied by the donor
antibody. The altered humanize~ antibody thus preferably
has the structure of a natural human antibody or a fragment
thereof, and possesses the combination of properties ~;
required for effective therapeutic use. Such "humanized"
antibodies are~effective in the prevention and treatment of
, .,
RSV infection in an appropriate animal model for RSV `
~infect~lon in humans,~and recognize a large variety of human
~- ~ clihical isolates of RSV. Because of their above-denoted `
characteristics, nucleic acids encoding the bovine mAbs B4,
Bl3 and Bl4 provide desirable RSV epitope spçcific donor
i ;sequences~(Ifirst fusion partners) for the construction of a
fusion molecule, which upon expression produces a humanized
antibody according to this invention which can elicit a
minimal immune response in humans. See, for example, the t
`,
;:
' ',.
SUBSTmJTE SHEET
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W093/202l0 ~ ~ $ ~ ~ 6 2 PCT/GB93/00725
23 ;
variable heavy and light chain sequences of Flgs. 4A, 4B,
3A, 3B and 10 through 13.
A fusion protein which is a chimeric antibody, as
defined above, differs from the humanized antibodies by
providing the entire non-human donor antibody heavy chain
and light chain variable regions, including framework -
regions in association with human (or other heterologous
animal, where desired) IgG constant regions for both chains.
It is anticipated that chimeric antibodies which retain
additional non-human sequence in comparison to humanized
antibodies of this invention, may also prove likely to
elicit some desirable immune response in the human.
A preferred altered antibody is one directed against
respiratory syncytial virus (RSV), preferably one specific
for the fusion (F) protein of RSV. A particularly preferred
antibody of this kind has all or a portion of the variable -~
domain amino acid sequences of B4 or B13/~14 reported in
Figs. 3A, 3B, 4A and 4B in its light and heavy chains,
respectively. Figs. lO through 13 illustrate predicted
amino acid regions suitable for use in a ~Ihumanized~l
antibody and are described in the Brief Description of the
Drawings section above. Additionally, an altered antibody
of this invention may be characterized by the presence of
one or more of the CDR peptides identified in the above
figures.
As one example, an altered antibody may contain a the
VL chain region of Fig. ll or a functional fragment thereof
in place of at least a part of the VL region of an acceptor
mAb, and a VH chain region of Fig. lO or a functional
fragment thereof in place of at leas~ a part of the VH
region of an acceptor mAb, such as a human antibody. The
resulting humanized antibody is characterized by the antigen
binding specificity of mAb B4.
SUBSTITUTE SHEET
WO93/202l0 ;~ `~ ~ PCT/GB93/00725
24
Still another preferred altered antibody may contain a
VL chain region of Fig. 13 or a functional fragment thereof
in place of at least a part of the VL region of an acceptor
mAb, and the VH chain region of Figs. 12A and 12B or a
S functional fragment thereof in place of at least a part of
the VH region of the acceptor mAb. The altered antibody is `~
thus characterized by the antigen binding specificity of mAb
B13~B14.
Alternatively, functional fragments of the variable
sequences, such as the ~4 CDR peptides, including:
SYSVS (amino acids 31-35 of SEQ ID N0: 3);
DASNGGIIYYNPALKS (amino acids 50-65 of SEQ ID N0: 3); ; :
CSVGDSGSYACTXaaGXaaRKGEYVDA, wherein Xaa is any ;
or no amino acid (amino acids 100-122 of SEQ ID N0: 3); ~:
SGSS(S or D)NIG(R or I)(W or F)(G or A)V(N or G) (amino :.-
acids 22-34 of SEQ ID N0: 1); .`
YESSRPS ~amino aCids 50-56 of:SEQ ID N0~
~ATGDYNIA (amino acids 89-96 of SEQ ID N0: 1);
ATGDYNIAV (amino acids 89-97 of SEQ ID NO: 1); .~.
20~: or the B13/B14 CDR peptides, including ``~
GNTKRPS: ~amino acids 50-56 of SEQ ID N0: 2); ~`
. VCGESKSATPV (amino acids 89-99 of SEQ ID N0: 2); ..
~: ~DHNVG (amino acids 31-35 of SEQ ID N0: 4~; -
-~ VIYKEGDKDYNPALKS ~amino acids 50-65 of SEQ ID N0: 4);
LGCYPV~GVGYDCTYGLQHTTFXaaDA, wherein Xaa is any amino
; ~ acid (amino acids 98-122 of SEQ ID N0: 4), .`"
may be ~used in place of the larger variable region sequences `;
of the figures. .
Such altered antibodies can be effective in prevention .`
and treatment of respiratory syncytial viruS (RSV) infection
'~ ~ in animals~and man. `:`;; Another species of therapeutic, diagnostic or
pharmaceutical protein of this invention is provided by the
proteins or peptides encoded by the first fusion partner
..,
., -
~,
. .
SUBSTITUTE SHEET `~
WO93/20210 f~ 6 2 PCT/GB93/00725
which are associated with above-described effector agents.
One example of such a protein provides an anti RSV
amino acid sequence of the invention associated with a non-
protein carrier molecule. Another example contains~a
5 desired anti-RSV sequence of the inventlon to which is -
attached an non-protein reporter molecule. Additionally, `;
the entire fusion proteins described above may be associated
with an effector agent
The procedure of recombinant DNA technology may be used
to produce a protein of the invention in which the Fc
fragment or CH3 domain of a complete anti-RSV antibody
molecule has been replaced by an enzyme or toxin molecule.
Another example of a protein of this invention
contains an anti-RSV amino acid sequence of th~ invention
with a macrocycle, for chelating a heavy metal atom, or a
toxin, such as ricin, attached to it by a covalent bridging
structure.
In general, fusion or linkage between the anti-RSV
antibody nucleotide sequences sequences and the second
fusion partner in the fusion molecule or association of the
peptides encoded by the first fusion partner and an effector
agent, may be by way of any suitable conventional means.
Such conventional means can include conventional covalent or
- ionic bonds, protein fusions, or hetero-bifunctional cross-
linkers, e.g., carbodiimide, glutaraldehyde, and the like.For association of the non-proteinaceous effector agents,
conventional chemical linking agents may be used to fuse or
join to the anti-RSV amino acid sequences.
Additionally, conventional inert linker sequences which
simply provide for a desired amount of space between the
first and second fusion partners in the fusion molecule may
also be constructed into the molecule. The design of such
linkers is well known. Such techniques and products are
SUBST~TUTE SHEET
i-,..: s
WO93/20210 PCT~GB93/00725
26
known and readily described in conventional chemistry and
biochemistry texts. -
VII. Production of Fusion Proteins and Altered Antibodies
Preferably the fusion proteins and altered antibodies
5 of the invention will be produced by recombinant DNA ~;
technology using genetic engineering techniques The same ~-
or similar techniques may also be employed to generate other
embodiments of this invention, e.g., to construct the
chimeric or humanized antibodies, the synthetic light and
heavy chains, the CDRs, and the nuc~eic acid sequences
encoding them, as above mentioned.
Briefly described, a hybridoma producing the anti-RSV
antibody, e.g., the bovine mAb B4, is conventionally cloned,
and the cDNA of its heavy and light chain variable regions -
15 obtained by techniques known to one of skill in the art, ~-
e.g., the techniques described in Sambrook e~ al., ~l~s~la~ -
Clonln~ (~ Tahoratory Manual~, 2nd edition, Cold Spring
Harbor LabQratory (1989). The variable regions of the mAb
B4 are o~tained using PCR primers, and the CDRs identified -
20 using a known computer database, e.g, Kabat, for comparison `~
to other antibodies.
Homologous framework regions of a heavy chain variable
region from a human antibody are identified using the same ~;-
databases, e.g., Kabat, and a human (or other desired -
25~ animal) antibody having homology to the anti-RSV donor
antibody is selected as the acceptor antibody. The -`
sequences of synthetic VH regions containing the CDRs within
the human antibody frameworks are defined in writing with
optional nucleotide replacements in the framework regions
for restriction sites. This plotted sequence is then
' synthesized by overlapping oligonucleotides, amplified by `
polymerase chain reaction (PCR), and corrected for errors. `~
A suitable light chain variable framework region may be
designed in a similar manner or selected from the donor or
....
, ~ ~
SUBSTITUTE SH EET
.,'
-WO93~20210 ~ PCT/GB93/00725
acceptor antibodies. As stated above, the source of the
light chain is not a limitin~ factor of this invention.
These synthetic VL and/or VH chain se~uences and the
CDRs of the anti-RSV mAbs and their encoding nucleis acid
sequences, are employed in the construction of fusion
proteins and altered antibodies, preferably humanized
antibodies, of this invention, by the following process. By
conventional techniques, a DNA sequence is obtained which
encodes the non-human donor antibody (e.g., B4, Bl3/Bl4) VH
or Vl chain regions. In such a donor antibody at least the
CDRs and those minimal portions of the acceptor mAb light
and/or heavy variable domain framework region required in
order to retain donor mAb binding specificity as well as the
remaining immunoglobulin-derived parts of the antibody chain
are derived from a human immunoglobulin.
A first conventional expression vector is produced by -
placing these sequences in operative association with -~
conventional regulatory control sequences capable of
controlling the replication and expression thereof in a host
cell. Similarly, a second expression vector is produced
having a DNA sequence which encodes the complementary
antibody light or heavy chain, wherein at least the CDRs
~and those minimal portions of the acceptor monoclonal
antibody light and/or heavy variable domain framework region
required in order to retain donor monoclonal antibody
binding specificity) of the variable domain are derived from
a non-human immunoglobulin. Preferably this second vector
expression vector is identical to the first except in so far
as the coding sequences and selectable markers are concerned
so to ensure as far as possible that each polypeptide chain
! ' is equally expressed. Alternatively, a single vector of the
invention may be used, the vector including the sequence
encoding both light chain and heavy chain-derived
.
SUBSTlTlJTE SHEET
W O 93/20210 ~ L ~ PC~r/G~93/00725
28
polypeptides. The DNA in the coding sequences for the light -~
and heavy chains may comprise cDNA or genomic DNA or both. ~-
A selected host cell is co-~ransfected by conventional
techniques with both the first and second vectors ~o create -
5 the transfected host cell of the invention comprising both -;~
the recombinant or synthetic light and heavy chains. The
transfected cell is then cultured by conventional techniques ;
to produce the altered or humanized antibody of the
invention. The humanized antibody which includes the
association of both the recombinant heavy chain and/or light
chain is screened from culture by appropriate assay, such as `
an ELISA assay. Similar conventional techniques may be `~
employed to construct other fusion molecules of this ;~
invention. ;-~
Thus, the invention also includes a recombinant plasmid ;;
containing a fusion molecule, which upon expression produces
an altered antibody of~the invention. Such a vector is
prepared by conventional techniques and suitably comprises
the above described DNA sequences encoding the altered ~r
20 antibody and a suitable promoter operatively linked thereto.
The invention includes a recombinant plasmid containing the
coding sequence of a mAb generated against the F protein i`~
26~6-273 epitope. ~`
Suitable vectors for the cloning and subcloning steps ~`
25 employed in the methods and construction of the compositions `~
of this invention may be selected by one of skill in the .
art. For example, the conventional pUC series of cloning
vectors commercially a~ailable from supply houses, such as ``-`
Amersham (Buckinghamshire, United Kingdom) or Pharmacia
~Uppsala, Sweden), may be used. Additionally, any vector
which is ca~able of replicating readily, has an abundance of ~
cloning sites and marker genes, and is easily manipulated `
may be used for cloning. Thus, the selection of the cloning
vector is not a limiting factor in this invention. -
SUBSTITUTE SHEET
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29
Similarly, the vectors employed for expression of the
altered antibodies according to thls invention may be
selected by one of skill in the art from any conventional
vector. The expression vectors also contain selected
regulatory sequences which are in operative association with
the DNA coding sequences of the immunoglobulin regions and
capable of directing the repLication and expression of
heterologous DNA sequences in selected host cells, such as
CMV promoters. These vectors contain the above described
DNA sequences which code for the altered antibody or fusion
protein. Alternatively, the vectors may incorporate the
selected immunoglobulin sequences modified by the insertion
of desirable restriction sites for ready manipulation.
The expression vectors may also be characterized by
marker genes suitable for amplifying expression of the
heterologous DNA sequences, e.g., the mammalian
dihydrofolate reductase gene (DHFR) or neomycin resistance
gene (neoR~. Other preferable vector sequences include a
poly A signal sequence, such as from bovine growth hormone
(BGH) and the betaglobin promoter sequence (betaglupro).
The expression vectors useful herein may be synthesized by
techniques well known to those skilled in this art~
The components of such vectors, e.g. replicons,
selection genes, enhancers, promoters, and the like, may be
o~tained from natural sources or synthesized by known
procedures for use in directing the expression of the
recombinant DNA in a selected host. Other appropriate
- expression vectors of which numerous types are known in the
art for mammalian, bacterial, insect, yeast, and fungal
expression may also be selected for this purpose.
Such a vector is transfected into a mammalian cell or
other suitable cell lines via conventional techniques. The
present invention also encompasses a cell-line transfected
with these described recombinant plasmids. The host cell
SU8STITUTE SHEET
W093/20210 PCT/GB93/00725`
`~
used to express the altered antibody or molecule is
preferably a eukaryotic cell, most preferably a mammalian
cell, such as a CHO cell or a myeloid cell. Other primate
cells may be used as host cells, including human c~lls which
enable the molecule to be modified with human glycosylation
patterns. The selection of suitable mammalian host cells
and methods for transformation, culture, amplification,
screening and product production and purification are known :
in the art. See, e.g., 5ambrook et al., cited above. ~`~
Bacterial cells may prove useful as host cells suitable ~-
for the expression of the recombinant mAbs of the present
invention. However, due to the tendency of proteins ~`~
expressed in bacterial cells to be in an unfolded or
improperly folded form or in a non-glycosylated form, any ~
15 recombinant mAb produced in a bacterial cell would have to ~`
be screened for retention of antigen binding ability. For ~}`~
example, various strains of E. coli, 3. subtIlis, Y;.
Streptomyces, other bacilli and the like may also be ~a,`
employed in this method.
- Where desired, strains of yeast cells known to those
skilled in the art are also available as host cells, as well
as insect cells and viral expression systems. See, e.g.
Miller et al., ~eneti~ F~nain~er;nc, ~:277-298, Plenum Press
(1986) and references cited therein. ~
The general methods by which the vectors of the -:;
ihvention may be constructedr transfection methods required `~
to produce the host cells of the invention, and culture
methods necessary to produce the fusion protein or altered -~
antibody of the invention from such host cell are all
conventional. Likewise, once produced, the fusion proteins
or altered antibodies of the invention may be purified from ~`
the cell culture contents according to standard procedures
of the art, including ammonium sulfate precipitation,
affinity columns, column chromatography, gel electrophoresis `~
SV8STITUTE SHEET
~` WO93/20210 ~ 6 6 2 PCT/GB93/00725
31
and the like. Such techniques are within the skill of the
art and do not limit this invention.
Yet another method of expression of the humanized
antibodies may utilize expression in a transgenic animal.
For example, a method of expression of the humanized
antibodies of the invention may be by expression in the milk
of a female transgenic animal, such as described in U.S.
Patent No. 4,873,316, which is incorporated herein by -
reference. For example, a DNA sequence for a selected
humanized antibody of the invention may be operatively
linked in an expression system to a milk-specific protein
promoter, or any promoter sequence specifically activated in
mammary tissue, through a DNA sequence coding for a signal
peptide that permits secretion (and maturation, if
necessary) of the desired protein in the mammary tissue.
Suitable promoters and signal peptides may be readily
selected by one of skill in the art.
The expression system is transgenically introduced into ~-
a host genome using standard transgenic techniques, for
example by microinjection into the pronuclei of fertilized
mammalian eggs. See, e.g. B. Hogan et al, "Manipulating The
Mouse Embryo: A Laboratory Manual" Cold Spring Harbor
Laboratory (1986); R.L. Brinster et al, S~ll, 27:223-231
(1991).] As a result, one or more copies of the construct -
or system are incorporated into the genome of the transgenic
mammal. The presence of the expression system permits the
female of the mammalian species to produce and secrete the
recombinant humanized antibody into its milk. This system
allows for high level production of the humanized antibodies
of the invention.
This latter method of expression may be particularly
suitable for a humanized antibody containing bovine CDRs,
and especially suitable for the oral administration of this
SUBSTlllJTE SHE~T
WO93/20210 ~i.~ PCT/GB93/0072~ ;~
.
3~
antibody to bovines as well as human infants. Other
transgenic systems may also be employed.
Once expressed by the desired method, the altered
antibody is then examined for in vitro activity by~use of an
appropriate assay. Presently, conventional enzyme linked
immunosorbent assay (ELISA) formats are employed to assess
qualitative and quan-titative binding of the altered antibody
to the RSV epitope (see Example 3). Other assays may also
be used to verify efficacy prior to subsequent human
clinical studies performed to evaluate the persistence of
the altered antibody in the body despite the usual clearance`~
mechanisms.
Example 11 below demonstrates the method of
constructing the altered humanized antibodies derived from
the murine monoclonal antibody RSV19, such as HuRSV19VH/VK
and HuRSV19VHFNS/HuRSV19VK which are described in copending
PCT patent applicatlon No. PCTtGB91/01554. Following the
procedures described for humanized antibodies prepared from
the murine RSVl9, one of skill in the art may also construct
humanized antibodies from the bovine antibodies, variable
region sequences and CDR peptides described herein (see
Examples 19 and 20). Altered antibodies can be produced ;,
with variable region frameworks potentially recognized as
"self" by recipients of the altered antibody. Minor
modifications to the variable region frameworks can be
implemented to effect large increases in antigen binding ;
without appreciable increased immunogenicity for the
recipient. Such altered antibodies can effecti~ely prevent
and eradicate infection. Of particular interest for such
humanized antibodies are the antibodies B4, B13 and B14
described herein. Such antibodies are useful in treating,
therapeutically or prophylactically, a human against human
RSV infection. Such antibodies may also be useful as
diagnostic agents.
:,
SUBSmUTE SHEET
WO93/20210 ~ 6 2 PCT/GB93/00725
33
VII. Therapeutic/Prophylactic Uses of the Invention
Thls invention also relates to a method of treating,
therapeutically or prophylactically, human RSV infection in
a human in need thereof which comprises administerrng an
effective, human RSV infection-treating dose of antibodies
including one or more of the mAbs described herein, or
fragments thereof, or an altered antibody as described
herein, or another fusion protein, to such human. This
invention also relates to a method of treating,
therapeutically or prophylactically, bovine or other
species' RSV infection in a bovine or other animal in need
thereof which comprises administering an effective, RSV
infection-treating dose of antibodies or molecules including
one or more of the mAbs described herein, or fragments
15 thereof, or an altered antibody as described herein,~to such -
animal.
The fusion proteins, antibodies, altered antibodies or
fragments thereof of this invention may also be used in
conjunction with other antibodies, particularly human
monoclonal antibodies reactive with other markers (epitopes)
responsible for the disease against which the altered -
antibody of the invention is directed. Similarly monoclonal
antibodies reactive with other markers (epitopes)
- responsible for the disease in a selected animal against
which the antibody of the invention is directed may also be
employed in veterinary compositions.
The fusion proteins or fragments thereof described by
this invention may also be used as separately administered
compositions given in conjunction with chemotherapeutic or
immunosuppressive agents. The appropriate combination of
agents to utilized can readily be determined by one of skill
in the art using conventional techniques. As an example of
one such combination, the altered antibody
HuRSV19VHFNS/HuRSV19VK described in Example 11, or a
`,~
SUBSTlTUTE SH EET -~
WO93/20210 ~ O ~ PCT/GB93/0072
34 -~ -
similarly altered 34, Bl3 or Bl4 antibody, may be given in
conjunction with the antiviral agent ribavirin in order to
facilitate the treatment of RSV infection in a human.
One pharmaceutical composition of the present invention ~
5 comprises the use of the antibodies of the subject invention ~-
in immunotoxins, i.e., molecules which are characterized by
two components anà are parti_ularly useful for killing
selected cells in vitro or in vivo. One component is a
cytotoxic agent which is usually fatal to a cell when ~`
attached or absorbed. The second component, known as the
"delivery vehicle" provides a means for delivering the toxic
agent to a particuiar cell type, such as cells comprising a
carcinoma. The ~wo components are commonly chemically
bonded together by any of a variety of well-known chemical
procedures. For example, when the cytotoxic agent is a
protein and the second component is an intact
immunoglobulin, the linkage may be by way of `
heterobifunctional cross-linkers, e.g., carbodiimide,
glutaraldehyde, and the like. Production of various
immunotoxins is well-known in the art.
A variety of cytotoxic agents are suitable for use in
immunotoxins, and may include, among others, radionuclides, -
chemotherapeutic drugs such as methotrexate, and cytotoxic
proteins such as ribosomal inhibiting proteins (e.g., ;
ricin).
The delivery component of the immunotoxin may include
one or more of the humanized immunoglobulins or bovine
immunoglobulins o`f the present invention. Intact
immunoglobulins or their binding fragments, such as Fab, are~;
preferably used. Typically, the antibodies in the
immunotoxins will be of the human IgM or IgG isotype, but
other mammalian constant regions may be utilized if desired. ;
The mode of admlnistration of the therapeutic agent of
the invention may be any suitable route which delivers the
SUBSTITUTE St~E~T
` `WO93/20210 ;~ b ~ PCT/GB93/00725
agent to the host. The fusion proteins, antibodies, altered
antibodies, and fragments thereof, and pharmaceutical
compositions of the invention are particularly useful for
parenteral administrationr i.e., subcutaneously,
intramuscularly or intravenously. The compositions for
parenteral administration will commonly comprise a solution
of the altered antibody of the invention or a cocktail
thereof dissolved in an acceptable carrier, preferably an
aqueous carrier. A variety of aqueous carriers may be
10 employed, e.g., water, buffered water, 0.4% saline, 0.3% -
glycine, and the like. These solutions are sterile and
generally free of particulate matter. These solutions may
be sterilized by conventional, well known sterilization
techniques. The compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions such as pH adjusting and buffering
agents, etc. The concentration of the antibody of the
invention in such pharmaceutical formulation can vary
widely, i.e., from less than about 0.5%, usually at or at
least about 1% to as much as 15 or 20% by weight and will be
selected primarily based on fluid volumes, viscosities,
etc., according to the particular mode of administration
selected.
Thus, a pharmaceutical composition of the invention for
intramuscular injection could be prepared to contain 1 mL
sterile buffered water, and 50 mg of an altered antibody of
the invention. Similarly, a pharmaceutical composition of
the invention for intravenous infusion could be made up to
contain 250 ml of sterile ~inger's solution, and 150 mg of
30 an altered antibody of the invention. Actual methods for ~`
preparin~ parenterally administrable compositions are well
known or will be apparent to those skilled in the art and
are described in more detail in, for example, B~m~ an~5
SUBSTITUTE SH EET
`.`:.
W093/202l0 ~ 6 ~ PCT/GB93/00725~ `~
!
Pharma~utiC~l sclen~e, 15th ed., Mack Publishing Company,
Easton, Pennsylvania.
To effectively prevent RSV infection in a human or -
other animal, one dose of approximately 1 mg/kg to ~
approximately 20 mg/kg of a molecule or an antibody of this
invention should be administered parenterally, preferably
i.v. (intravenously) or i.m. ~in~ramuscularly); or one dose
of approximately 20 ug/kg to approximately 2 mg/kg of such
antibody should be administered i.n. (intranasally). ;~-
10 Preferably, such dose should be repeated every six (6) weeks `-
starting at the beginning of the~RSV season (October-
NovemDer) until the end of the RSV season (March-April). ~!"~'`
Alternatively, at the beginning of the RSV season, one dose
of approximately 5 mg/kg to approximately 100 mg/kg of an
15 antibody of this invention should be administered i.v. or -
i.m. or one dose of;approximately 0.5 mg/kg to approximately ~;~
10 mg/kg of such antibody should be administered i.n.
To effectively therapeutically treat RSV infection in a -~
human or~other animal, one dose of approximately 2 mg/kg to
~- 20 ~approximate~ly 20 mg/kg of an antibody of this invention
~-should be administered parenterally., preferably i.v. or
i.m.; or approximately 200 ug/kg to approximately 2 mgJkg of
such antibody should be administered~i.n. Such dose may, if
nece~ssary, be repeated at appropriate time intervals until `;
25~ the~RSV infection has ~een eradicated.
For;example, in Example 16, the dose of B4 required to ;~
~protect calves~when administered by the i.t. route was 300
g/kg body weight. This is 300 to 1000-fold less than the
amount of human IgG, containing high titres of RSV-
neutralizing antibody, required to reduce RSV infection in
-~ co~ton-rats 'and owl monkeys, passively immunized by the i.t.
~route ~Hemming and Prince, R~,l~y~_~5~ ect;~ s~ases, ;-`
12:S470-S475 ~1990)]. It has been shown that about 10-fold
less antibody is required to reduce virus shedding when
" ,
' 1`'
~ .
"
SUBSTtTUTE SHEET
WO93/2021~ PCT/GB93/00725
given by the topical route when compared with intra~enous
administration [Prince & Hemming, (1990)]. Therefore, it is
estimated that a dose of approximately 3 mg/kg of mAb B4
given i.v. would be needed to significantly reduce~RSV
shedding in calves. This is similar to the amount of murine
or "humanized" mAb required to protect mice against RSV
infection [Tempest et al., (1991)].
The compositions of the invention may also be
administered by inhalation. By "inhalation" is meant
intranasal and oral inhalation administration. Appropriate
dosage forms for such administration, such as an aerosol -
formulation or a metered dose inhaler, may be prepared by
conventional techniques. For example, to prepare a
composition for administration by inhalation, for an aerosol
container with a capacity of 15-20 ml: Mix 10 mg of an
antibody of this invention with 0.2-0.2~ of a lubricating
agent, such as polysorbate 85 or oleic aci~, and disperse
such mixture in a propellant, such as freon, preferably in a
combination of (1,2 dichlorotetrafluoroethane) and ~-
difluorochloromethane and put into an appropriate aerosol
container adapted for either intranasal or oral inhalation
a~mlnistration. As a further example, for a composition for
-~ administration by inhalation, for an aerosol container with -
- a capacity of 15-20 ml: Dissolve 10 mg of an antibody of
this invention in ethanol (6-8 ml), add 0.1-0.2% of a ;~
lubricating agent, such as polysorbate 8S or oleic acid; and
; disperse such in a propellant, such as freon, preferably a
c~ombination of (1.2 dichlorotetrafluoroethane) and
difluorochloromethane, and put into an appropriate aerosol
container adapted for either intranasal or oral inhalation
administration.
The antibodies, altered antibodies or fragments thereof -
described herein can be lyophilized for storage and ``"
reconstituted in a suitable carrier prior to use. This
. ''.':
, ,',
`,`
SU8STlTUTE SHEET ~`
:. . ` `
W093/202l0 PCT/G~93/0072S`! -
h~ ~f li ~ ~ `
38 -
technique has been shown to be effective with conventional
immune globulin~ and art-known lyophilization and
reconstitution techniques can be employed.
Depending on the intended result, the pharmac~utical -
5 composition of the invention can be administered for -~
prophylactic and/or therapeutic treatments. In therapeutic ;~-~
application, compositions are administered to a patient
already suffering from a disease, in an amount sufficient to
cure or at least partially arrest the disease and its
complications. In prophylactic applications, compositions
containing the present antibodies or a cocktail thereof are
administered to a patient not already in a disease state to
enhance the patient's resistance. -
Single or multiple administrations of the
pharmaceutical compositions can be carried out with dose
levels and pattern being selected by the ~reating physician. ~
In any event, the pharmaceutical composition of the ~-
invention should provide a quantity of the altered
antibodies of the invention sufficient to effectively treat
the patient.
It should also be noted that the fusion proteins,
antibodies, variable sequences, CDR peptides and epitopes of
this invention may be used for the design and synthesis of
either peptide or non-peptide compounds (mime~ics) which
would be useful in the same therapy as the antibody. See,
e.g., Saragovi e t a l ., S C i ~ 7 9 2 - 7 9 5 (1991).
Natural RSV infections have also been reported in
cattle, goats, sheep and chimpanzees. Thus, for example, ;
utilizing the methodology described above, an appropriate
mouse antibody could be "bo~inized", and appropriate
framework region residue alterations could be effected, if
necessary, to restore specific bindiny affinity. Once the
appropriate mouse antibody has been created one of skill in
the art, using conventional dosage determination techniques,
SUBSTI~UTE St~ EET
. . J ~ e ;~
; W093~202i0 PCT/GB93/00725
39
can readily determine the appropriate dose levels and
regimens required to effectively treat, prophylactically or
therapeutically, RSV infection in the selected animal.
The following examples illustrate various aspects of
this invention and are not to be construed as limiting the
scope of this invention. All amino acids are identified ~y
conventional three letter codes, single letter codes or by
full name, unless otherwise indicated. All necessary
restriction enzymes, plasmids, and other reagents and ,
materials were obtained from commercial sources unless
otherwise indicated. All general cloning ligation and other
recombinant DNA methodology were as described in "Molecular
Cloning, A Laboratory Manual." (1982), eds. T. Maniatis et
al., published by Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York, ("Maniatis et al") or the second edition
thereof (1989), eds. Sambrook et al ., by the same publisher
("Sambrook et al.").
The following examples illustrate the construction of
exemplary altered antibodies and expression thereof in
suitable vectors and host cells.
F.xam~le l - Pre~arat;on of Monoclonal Ant;hod;es
Murine monoclonal antîbodies 1 to 14 were described in
Taylor et al., (1984) cited above and incorporated herein by
reference. Several of these antibodies were produced by~ 25 immunizing BALB/c mice with bovine RSV, strain 127. The
bovine;~RSV, strain 127 was isolated at Compton in 1973 from
a calf with respiratory disease. Others of these antibodies
were produced wi~th cells persistently infected with the Long
strain of human RSV [Fernie et al., Proc. Soc. Exp. B;o~.
M~d~c ~ 83-86 (1981)). Murine monoclonal antiboqies 16
to 21 were produced from BALB/c mice inoculated intranasally
(i.n.) on two occasions, three weeks apart, with lX104 pfu
- of the human RSV strain A2, grown in Hep-~ cells. Human
RSV, strain A2, subtype A was isolated from a child in `~
,-,~.
SUBSTITUTE SHEET :
WO93/20210 PCT/GB93~00725
Australia [Lewis et al., ed. J Au.~tr , 48:932-933 (1961)].
After an interval of fcur months, the mice were inoculated
intraperitoneally (i.p.) with 2X107 pfu of the bovine 127
strain. Three days after the booster inoculation, the
immune splenocytes were fused with NS-1 myeloma cells
[American Type Culture Collection, designation TIB18]. The
resulting hybridomas were screened for antibody to RSV by
radioimmunoassay and immunofluorescence, cloned twice on
soft agar and cloned cells inoculated into BALB/c mice to
produce ascitic fluid as described in Taylor et al., cited
above.
Bovine monoclonal antibodies B1 to B6 were produced as
described in Kennedy et al., J. Gen. Virnl., ~:3023-3032
(1988), incorporated herein by reference. At the same time,
bovine mAbs B7 to B10, B13 and B14 were produced from bovine
lymphocytes obtained from the same calf, but the lymphocytes
were stored in liquid nitrogen and fused with NSl cells at
later dates. The resulting heterohybridomas were screened
for bovine antibody to RSV by ELISA and in some cases also
by the fusion inhibition assay [essentially as described in
Kennedy et al. (1988), cited abovel, but adapted to
microtitre plates. Cloned heterohybridoma cells secreting
bovine mAbs to RSV were inoculated into pristane-primed nude
BALB/c mice to produce ascitic fluid or grown in serum-free,
DCCM-1 medium [Biological Industries, Ltd., Glasgow, U.K.].
Antibody was purified from cell culture supernatant using
Protein G Sepharose 4 Fast Flow [Pharmacia LKB]. Bound
antibody was eluted with O.lM glycine, pH2.7, neutralized
with lM Tris-HCl (pH9.0) and dialyzed against phosphate
buffered saline (PBS).
I The antibody AK13A2 raised against the Long F protein
was a generous gift of Dr. P. Coppe, Centre d'Economie
Rurale, Marloie, Belgium. The mAbs lBCll (a negative
control antibody), 47F and 49F have been described by
SUBSTITUTE SHEET
WO93/20210 ~ G ~ PCT/GB93/00725
Garcia-Barreno et al., J. Yirol~ 925-932 (1989). MAb
7C2 is described in Trudel et al., (1987), cited above. The
antibodies, 47F, AK13A2 and 49F, were purified from ascitic ~;`
fluids by protein A-Sepharose chromatography and peroxidase -
5 labelled [Garcia-Barreno et al., (1989), cited above] -~
All of the murine and bovine mAbs and hybridoma cell
- lines producing them descri~ed ~lerein, except mAbs lBC11,
47F, 49F, AK13A2 and 7C2, are available from the ~aboratory
of Dr. Geraldine Taylor, Institute for Animal Health, ~
lO ~Compton Laboratory, Compton, Near Newbury, Berks, RG160NN, ~;
England. ~ ~ ~
Exam~l~e 2 - Characterizat;on of Monoclonal ~nt;bodies `-
T~he ~specifLcitles~of the mAbs for F protein viral ~
polypèptides~were determined by radioimmune precipitation of ;`;
(35S)-methionine or (3H)-glucosamine labelled RSV infected
-cell~lysates~performed as described by Kennedy et al.,
Gen.~V~iro~ 2:~30~23-3032 ~l988). The specificity was
med~by~Western~blots~immunoblotting) of non-reduced ';`
a~nd~redùced~RSV-infected~cell lysates~performed as described
20^~ by~ ~ ta~ét~al.,~El~ectrovhor.,~6~:492-497 ~1985). The ~-
ant ~ ~ns~u-sed~in ~immunoblotting were either Hep-2 cells
infected~with the~human~RSV A2 strain or calf kidney (CK) '~
ce~lls in~fected~with the~bovine RSV strain 127. Uninfected
Hep-2 o;r~CK cèlls~were;u~ed~as;control antigens .
25~ Only~ ~ s~Bl,~B4,~B5~EKennedy e:t al., cited abo~e] and `~
s~R 9,~B13 and~B14-reacted~with F protein denatured by
n~dithiothreitol.~ Whereas mAbs Bl, 84 and B5
recognized~46K~and 22K~fraqments of~denatured Fl protein in `-
Westérn b~l~ott1ng,~mAbs~RSVl9~ B13;and Bl4 only recognized
46K fragments. The properties of mAbs 16 to 18, 20 and 21,
SVl9, Bi to Bio~ B13 and B14, not previously described, for
the~assays described b~el`ow are shown in Table 1 below. The
properties~o$ all~the;o~her mAbs in these assays are
summarizèd~in~Figs.~5 and 6.
SU8STITI.JTE SHEET
WO93/20210 ~ PCT/GB93/0072S `
42
The ability of ~he mAbs to inhibit multinucleated giant
cell formation was assayed in MA104 cells [American Type
Culture Collection, Rockville, MD] 24 hours after infection
with the RSV A2 strain [Kennedy et al., cited above,
incorporated herein by reference~. The results of this
assay are reported in Table 1 under the column "Fusion
Inhibition", and in Figs. 5 and 6 as "FI". A "-" indication
means that the mAb did not inhibit the giant cell formation.
A "~" indication means that the mAb inhibited the formation
of multinucleated giant cells.
Four murine mAbs (11, 13, RSV19 and 20) and four bovine
mAbs ~B4, B5, B13 and Bl4) inhibited the formation of
multinucleated giant cells.
The ability of mAbs to neutralize ~SV was assayed by a
plaque reduction neutralization test performed as described
in Kennedy et al., cited above. The results of this assay
are reported in Table 1 under the column "Neut. titre", and
in Figs. 5 and 6 as "Neut". In the figures, a "-"
indication means no neutralization occurred; a "+" -
indication means that the antibody was neutralizing. Seven
of the murine mAbs and four of the twelve bovine mAbs, i.e.,
B4, B5, B13 and B14, neutralized RSV.
The ability of mAbs to protect against RSV infection
was studied in BALB/c mice as follows. 100 ~l of ascitic
~5 fluid containing the mAbs was injected intra-peritoneally
into groups of five mice. One day later, the mice were
inoculated i.n. with 104 pfu of the A2 RSV strain. On day 5
of the infection, the mice were killed and ~heir lungs
assayed for RSV on secondary CK monolayers, according to the
procedure described in Taylor et al ., Infect- . Immun.,
649-655 (1984).
The results of this assay are also reported in Table 1
under the column "Prot. of Mice", and in Figs. 5 and 6 under
"Protection". In the figures, a "-" indication means that
SU8SlTn~TE SHEET
WO93/20210 ~l'7~ PCT/GB93/00725
43
the mAb did not protect the immunlzed mice against RSV ~`
infection. A 1l+-- or ~+++~ indication means that the mAb did
protect the animals to a lesser or greater degree,
respectively. The eight mAbs that were effective ~n the ~-~
S fusion inhi~ition assay ~i.e., murine mAb~ ll, 13, RSVl9 and
20, and bovine mAbs B4, B5, Bl3, and Bl4) were highly
effective in preventing RSV in~ection in BALB/c mice when
adminis~ered i.p. 24 hours prior to i.n. challenge with the `
A2 strain of RSV.
All antibodies, except murine mAbs 9 and lO`~Taylor et
al., ~1984)] and bovine mAb B8, which were specific for
bovlne RSV, reacted with both the A2 and the human B subtype
(8/60) [Coimmon Cold Unit, Salisbury, En~land~ strains of
human RSV (both grown in Hep-2 cells) and with bovine i`
15 strains of RSV ~Taylor et al., ~l984), cited above; Kennedy ~`
et al., cited above]. These results indicate that the
epitopes recognized by the highly protective, fusion-
inhibiting imAbs were highly conserved among strains of RSV.
Table l
20Properties of m~bs to the F protein of RSV
- F.IIS~ ti~re ~lo~ Neut. Fusion ~C Prot
mAb class A2 8/60 BRSV titrel Inhib. lysis2 of
, ~ Tnice~
:
l6 Gl 6.8 6.6 6.82.0 - 0 0.6 ~i
l7 G2b 6.l 6.3 6.l<l.0 - 59 0.6 `~
18 G2a 7.0 6.8 6.23.4 - 43 1.6
RSVl9 G2a 6.4 6.7 6.73.4 + 2 >3.8
G2a >6.0 8.6 7.54.3 + 76 >3.8
21 G2a 8.9 7.4 6.8<l.0 - 68 l.0
B7 Gl 3.0 4.9 4.9<l.0 - 6 0
B8 Gl ~2.0<2.0 4.0<l.0 - 8 0.2 ~`
B9 Gl 5.1 5.4 4.9<l.0 - 2 0.4
BlO Gl 5.1 5.4 6.0<l.0 - 9 ~ 0.5
Bl3 Gl i6.0 5.l 5.45.8 + 0 2.2 ~-
Bl4 Gl 5.6 5.2 5.65.4 + 0 >2.2
._ .. . _ , .
40 1 50% plaque reduction titre expressed as logl~ ~
SUBSTtTUTE SHEET ` -
WO93/20210 ~ t`~ PCT/GB93/00725
44
2 Percent specific release with 1/100 dilution of mAb and
rabbit complement
3 Log10 reduction in titre of RSV in the lungs of passively
5 immunized mice compared with control animals ~ ~
Exampl~ 3 - Enzyme T.; nked Immunosor~ent as~ay (ETTSA) ~
RSV antigens to be tested in the ELISA were each ~-
prepared from Hep-2 cells, 3 to 4 days after infection.
Cells were scraped into medium, spun at 500 g for 5 minutes,
resuspended in distilled water and treated with 0.5% (w/v)
NP40 detergent to yield a cell lysate. Control antigen was
made in a similar way using uninfected Hep-2 cells.
The ELISA was performed as follows: Microtitre plates
were coated with RSV or control antigen, diluted in
distilled water, overnight 2t 37C, incubated with blocking
buffer consisting of 5~ normal pig serum in PBS and 0.05%
Tween 20 for 1 hour at room temperature and washed Sx with
PBS~Tween. Serial 3-fold dilutions of mAb were added to the
wells and the plates incubated for 1 hour at room
temperature. After washing 5x with PBStTween, HRP-
conjugated rabbit anti-bovine IgG (Sigma) diluted 1:4000 or
HRP-conjugated goat anti-mouse IgG (Kpl, Maryland, USA)
~:; diluted 1:2000, was added to each well. After a final wash,
bound conjugate was detected using the substrate 3,3',5,5'-
tetramethylbenzidine (TMB, ICN, Immunobiologicals,
Illinois).
E~m~l e a - Pl~r; fi ca~ton of the F ~1 yconrote;n and trvDs;n
The F protein was purified by immunoaffinity
chromatography from extracts of Hep-2 cells infected with ;
t~e Long strain [See, Walsh et al ., J. Gen. V;rol., ~h:409-
415 (1985); and Garcia-Barreno et al., ~1989), cited above].
Several aliquots of the purified protein (15 ~g each) were
incubated and digested with either 2~g, 4~g, 8~g or 16~g of
trypsin for 4 hours at 37C. The digestion was terminated ~-
SUBSTmJTE SHEET
``- WO93/20210 h i~ PCT/GR93/00725
~
by the addition of electrophoresis sample buffer [Studier,'`~'
~L~I$~L~ , 79:237-248 (1972)] and boiling of the samples
for 3 minutes. SDS-PAGE separated the samples. The samples ,
were elec~rotransferred to Immobilon membranes.
As an initial step to locate the epitopes recognized by',~
the antibodies, AK13A2, 47F, 7C2, RSV19, 20 and B4, used in ~-
the selection of mutant viruses in Example 7, below, the '
binding of mAbs to the trypsin fragments of purified F -~
10 protein were tested by Western blot ~Towbin et al., ,P,~oc. "~'
~S~ IS~, 76:4350-4354 (1979)]. The protein ~,
. .
fragments were either stained with Coomassie blue or ,
developed with antibodies AK13A2 or 19. ',-
Increasing amounts of trypsin generated smaller
15 fragments of the F1 subu,nit which were stained by Coomassie '
blue. Four F1 frag~ents of 30, 20.5, 19 and 15 K were
recognized by mAb AK13A2. The 20.5 and 19 K fragments had
been mapped previously tLopez et al ., 1~ Gen. V;rol.,
~:927-930 (1990~] at the NH2 terminal end of the Fl
20 subunit. Antibodies B4, ~7F, and 7C2 recognized the same j,,~
set of fragments as AK13A2. Thus, the epitope recognized by
these mAbs can be ascribed to amino acid sequences included``
^.
within the NH2 terminal third of the Fl subunit.
In contrast, RSVl9 reacts with a different set of Fl!^'`
fragments. Only large size fragments (26 and 22 K),
generated with low trypsin amounts, reacted with RSV19 (mAb
20 reacted less efficiently with the same set of fragments).
Thus, epitopes 19 and 20 contain trypsin sensitive amino
acid sequences which were tentatively located within the ,~
carboxy terminal two thirds of the F1 subunit (Fig. 2),
' outside the region covered by the fragments recognized by ,',
antibody B9. The NH2 terminal end of the 26 and 22 K ;
fragments could not be determined by direct protein ;,
sequencing because their low yield after trypsin treatment. '
SUBSTtTUTE SHEET .``
~.; .;, 'i
WO93/20210 PCT/GB93/00725
6 2 ! : ~
46
The diagram of Fig. 2 shows the F glycoprotein primary
structure denoting the hydrophobic regions, the site of
proteolytic processing, the potential sites for N-
glycosylation, the cysteine residues and the amino ~cid
residues which are changed in the neutralization escape
mutants (see Table 3A-3C below). The locations of the
trypsin fragments recognized by different mAbs are shown
below Fig. 2.
The region on the F protein recognized by mAbs Bl3 and
Bl4 were identified by examining their binding to F protein
fragments, expressed in E. coLi. Recombinant C protein (rC,
F377s24) of SEQ ID NO: 19 and recombinant D protein (rD, F3"
550) of SEQ ID NO: l9 were used as antigens in ELISA as
described in Example 3. These peptide sequences of the F
protein were fused to an influenza non-structural protein
fragment containing amino acids l - 81 of the influenza `
nonstructural protein 1 (NS-l) at their amino termini,
ins~erted into an expre~ssion plasmid and expressed in E. ~
coli. The production of these ~usion peptides involved ~-
conventional procedures. MAbs Bl3, Bl4 and RSVl9, but not
B4, bound to these protein fragments. Table 2 below
illustrates the binding of anti-F mAbs to recombinant F -
protein~fragments in ELISA. ;These findings suggest that the !'
region~of the F p~rotein recogni~ed by Bl3 and Bl4 is similar
to that~recognized by RSVl9 and is~within the carboxy
terminal thir~ of the Fl subunit.
Tah1~ 2
mA~; ~ rC ~ RSV
B13 5.7 >3.0 5.6
Bl4 6.2 >3.0 5.6
! ~ ' ` I RSVl 9 1 ' 5 . 3 >3 . 0 7 . 4
B4 <1. S <1. 5 5.9
'~
'
SU8STITUTE SHEET
WO93/20210 ~ i G ~ PCT/GB93/00725
47
Log10 titer by ELISA, rC at 2 ~g/well and rD at 1 ~g~well
used as antigens.
FxamDle 6 - Tdentifica~;on ~f anti~enic areas in t~e F ~
5 protein ~ `
The epitope specificity of the 16 murine and 12 bovine
m~bs to the F protein were analyzed by a competitive binding
assay using purified and labelled mAbs. In summary, these ~;~
competitive binding assays identified twelve antigenic sites ~-
10 on the F protein, many of which overlapped extensively. ~-~
Three epitopic sites were recognized by both the
neutralizing mAbs and the highly protective FI mAbs, e.g.,
B4, B5, B13 and B14. These findings are similar to those of Li~
others who have identified three antigenic sites on the F
protein involved in neutralization using murine mAbsl two of
which are involved in FI activity [Walsh et al., J. Gen. ~-
Virol., ~8:505-513 (19-86) and Beeler et al ., J . Virol., -~
fi~:2941-2950 (1989)]. These findings suggest that virus
neutralization can occur by a mechanism independent of --~
preventing the fusion of the ~irus with the cell membrane,
e.g. steric hindrance of virus attachment.
A. ~ "
The IgG from ascitic fluid containing either
murine or bovine mAbs was purified on either Protein A-
sepharose or Protein G-sepharose Fast-Flow [Pharmacia LKB~
The ascitic fluids were mixed with equal volumes of O.lM
phosphate buffer (pH 8), and passed through a Protein A-
sepharose column with the same buffer. Bound antibodies
were eluted with 0.1 M citrate buffer (pH 6.0 to 3.5). `~
Fractions eluted with low pH buffers were collected in lM
Tris-HCl ~pH 9.0). IgG from tissue culture supernatants was
purified on Protein G-sepharose Fast-Flow and eluted with
O.lM glycine as described above. Purified IgG was dialyzed
SUBSmUTE SHEET
WO93/20210 ~ PCT/GB93/00725
48
against ~BS and labelled with 12sI using chloramine T or
coupled to biotin.
B. GQm~eti~i~e Bin~i2a Assay
A dilution of 12sI-labelled, or biotinylated, mAbs,
determined to give approximately l0,000 cpm at 90% of
maximum binding to RSV antigen in a radioimmunoassay, was
allowed to react with RSV antigen in the presence of
increasing amounts of various unlabelled mAbs to the F -
protein. For mAbs Bl3 an~ Bl4, a dilution of biotinylated
10 mAbs, determined to give 90% of maximum binding to RSV- -
infected cell lysate, was allowed to bind to RSV antigen in
the presence of increasing amounts of unlabelled antibody.
An unlabelled mAb to the nucleoprotein (N) was used as a
control.
The results of this assay are illustrated ln Figs.
5 and 6. Some mAbs inhibited the binding in a dose-
dependant manner; other mAbs, however, did not interfere
with the binding of the test antibody. Unlabelled mAb to
the N protein of RSV did not interfere with the binding of
20 any of the m~bs to the F protein. These studies identified -
groups of mAbs that competed for simultaneous binding to
antigen. Epitopes recognized by competing mAbs were
considered to be operationally within the same antigenic
area of the F protein. The competition profiles of the mAbs
overlapped extensively (Figs. 5 and 6).
Therefore the clustering of epitopes was done on
the basis of partial similarities and was analyzed using the
Leucocyte typing database IV [Gilks, "Leukocyte typing
database IV" Oxford University Press (l990)]. These studies
showed that the l6 murine mAbs recognized 7 antigenic areas
on the F protein [SEQ ID NO: l9] (Fig. 6). mAbs 2 and 5
competed with nearly all of the other murine mAbs. Two
high1y protective mAbs, ll and 13, appeared to recognize the
same epitope ~site B), whereas two other mAbs, RSVl9 and 20,
SUBSTmJTE SHEET
- WO93/20210 ~ PCT/CB93/00725
49
which were also highly protective, were slmilar to each
other but different from mAbs 11 and 13, and mapped to site -
C . , . ~
Most of the 12 bovine mAbs mapped to the ~same ;-
S sites as the murine mAbs (Figs. 5 and 6). Murine mAbs 2 and
5 competed with only 4 of the bovine mAbs (B2, B3, B4 and
B6). A neutralizing murine mAb, 14, which mapped to site G `~
in competition studies with the murine mAbs (Fig. 6), showed
a competition profile that was similar to the bovine mAbs
10 B2, B3 and B6 and was therefore placed in group H (Fig. 5)~ -~
The binding of bovine mAbs B1 and B7 were not inhibited ~y ~`~
any of the murine mAbs and, indeed, B7 appeared to recognize
a distinct epitope. The epitopes recognized by 2 highly
protective bovine mAbs B4 and BSt were similar to each other
and to 2 of the highly protective murine mAbs, 11 and 13.
mAb 18, which is partially protective in mice, and B10,
which is not protective, also map in this area (site B).
The binding of the protective bovine mAbs B13
and B14 was inhibited to various degrees by protective
murine mAbs, RSV19 and 20, and the protective bovine mAbs B4
and B5. However, the competition profiles of mAbs B13 and
B14 were different from those antibodies mapping to sites B
and C, suggesting that they recognize a different site on
the F protein. ~`
Taken together, the murine and bovine mAbs ;`
recognized 12 antigenic areas, most of which overlapped
extensively. The highly protective, fusion-inhibiting (FI),
neutralizing mAbs mapped to 2 or possibly 3 sites (areas B, `;
C and L in Fig. 5) on the F protein. mAbs that neutralized
virus but did not have FI activity mapped to 3 sites (areas
, .
! ' ' B, D and H). However, mAbs which have neither neutralizing -:
- nor FI activity also map to these sites.
:,
SUBSl~UTE SHEET ;
WO93/20210 PCT/GB93/0072S
'1'~ S ~) b ~
5 0 :~
FxamDle 7 - Antihody eSca~e mutants
The pattern of reactivity of antibody-escape mutants
with the mAbs confirmed the mapping of the pratective
epitopes deduced from competitive binding assays. ~In ~`
S summary, two regions of the ~ primary structure were
identified where the epitopes recognized by neutralizing
mAbs were located. The first region mapped within the
trypsin resistant amino terminal third of the large F1 ~-
subunit. This region contained the overlapping epitopes
recognized by mAbs 47F, 49F, 7C2, AK13A2, 11 and B4,
ncluded in antigenic area II (Fig. 8) and area B (Figs. S
and 7). Antigenic areas II and B are identical. Most amino ;
acid changes found in mutants selected with these antibodies
were clustered around amino acids 262-272 of SEQ ID NO: 19.
lS Since these antibodies reacted in Western blots with
proteolytic fragments of the Fl subunit, it was originally
thought~that~they recognized~"linear" epitopes determined by
seque~çes of~consecutive amino acids.
However, it seems that some conformations are needed
20 ~for the ~integrity of certain epitopes, because only some of
I ~ them were reproduced by synthetic peptides and amino acid
~substitutions located at a distant site influenced the
b~nding ~of~some~antibodies. For example, the change at
amino~acid~216 (Asn to~Asp), in the mutant 4/4 that
25 ~ conferre;d~resis~tance to mAb AK13A2, also eliminated the
reactivity wit~h~antibod`ies~7C2 and B4 (resistance to which
are als~o conferred~by selected changes at position 272).
`The~change~at 2~1~6 ~is~dist~antly located from the peptide 255-
Z75,~ which~fa~ithfully~reproduced the epitope B4.
Consequently, some long range effect of amino acid 216 in ;
the structù~e adopted by epitope B4 in the Fl subunit is
likely to occur.
Although the competition profiles in Figs. 5 and 6 of
the mAbs overlapped extensively, protective mAbs 11, 13, B4 ¦~
" ~ ` SlJBSTlTUTE SHEET :``
:
~-W093/20210 ~ 2 PCT/GBg3~00725 ~ ~
. ,................................... ~
!
51
and B5 mapped to the same area (site B in Figs. 5 and 7;
site II in Fig. 8) and mutants resistant to these antibodies
failed to bind only those mAbs recognizing site B. MAbs 7C2
and 47F also mapped to this area~ Although there w~as
inhibition of binding of mAbs RSVl9 and 20 to RSV antigen by
antibodies mapping to site B (site II), and vice versa,
cluster analysis suggested that they recognized a different
site (site C). This was confirmed by the finding that `
mutants selected for resistance to mAbs RSVl9 and 2Q still ,~
reacted with mAbs recognizing site B. Similarly, the
binding of mAbs Bl3 and B14 to RSV was inhibited by mAbs
mapping to sites B (II) and C. However, B13 and B14 ~
appeared to map to a different region (site L) and this was ~:
confirmed by the observation that B13 and Bl4 bound to all
15 the mutants selected with mAbs mapping to sites B (II) and `-
. C.
The neutralization of RSV by the mAbs used to select ;
the escape mutants is theorized to be related to their
capacity to inhibit the membrane fusion of the F
glycoprotein [Garcia-Barreno et al. (1989), cited above;
Taylor et al~, (1989), cited above]. By analogy with other
paramyxoviruses [see, e.g., Morrison, Virus ~es., 10:113-136
(1988)], it is assumed that the fusion activity of RSV
depends upon the proteolytic processing of the F protein
precursor. This modification generates the new NH2-terminal
end of the Fl subunit, proposed to interact with lipid
membranes through a short hydrophobic peptide. The
antigenic areas of the F ~lycoprotein identified herein are
distantly located from the fusion peptide in a linear map;
however, it is possible that other regions of the F protein
influence the activity of the fusion peptide. In this
respect, mutants altered in the fusogenic activity of the
influenza virus hemagglutinin [Daniels et al ., Cell, ~Q:431-
SUBSTlTlJTE S~ EET
WO93/20210 ~ ~ 3 ~ ~ PCT/GB93/0072S
,...
52 -
439 (1985)] have been mapped outside the fusion peptide of ;~
the HA2 subunit.
The escape mutants were developed and evaluated as
follows. The wild type and neutralization escape m~tant
S viruses were grown in Hep-2 cells and purified from culture
supernatants as previously described [Garcia-Barreno et al.,
V1ru.~ Res , ~:307-322 (1988)J. The Long and A2 strains of
human RSV were plaque purified before being used to select
~iruses which escaped neutralization (mAb resistant mutants)
by mAbs 47F, AK13A2, 7C2, 11, B4, B5, 19 or 20, and other
mAbs directed against the F glycoprotein as described
herein. These were selected in two different ways:
A. A2 s-~;ra; n ~;sca~ Muta~
Antibody escape mutant ~iruses of the RSV A2
strain, which are refractory to neutralization by one of the
highly protective mAbs, 11, B4, B5, RSV19 and 20, were
produced using plaque reduction techniques. For mAbs RSV19,
- 20, B4 and B5, confluent monolayers of primary CK cells were
infected with the A2 strain at a multiplicity of infection
(MOI) of 0.2. Starting 24 hours after infection and
continuing for 3 to 5 days, the culture medium was replaced
~dally with fresh medium containing 10% mAb. Virus was
harvested when a cytopathic effect tCPE) was apparent.
Virus prepared in this way was mixed with an equal
volume of the mAb under test for 1 hour at room temperature
and inoculated onto CK monolayers in 35 mm multi-well plates
tNunc]. After 1 hour incubation at 37C, the plates were
overlaid with medium containing 0.25% agarose incorporating
`~ ~ a 1 in 10 dilution of the same mAb. Plates were then
incubated at 37C in 5~ CO2 in air for 7 days before adding
'~ the vital stain, 0.3% 3-(4,5-dimethylthiazolyl-2)-2,5-
diphenyltetrazolium bromide in 0.15M NaCl, to the overlay to
visualize virus plaqoe-.
~,.
SUBSTITUTE SH EET
:.
` WO93/20210 PCT/GB93/00725
Putative mutant viruses were removed from plates
in agar plugs containing single plaques, diluted in medium,
mixed with an equal volume of mAb and inoculated onto CK
monolayers as before. Mutant viruses were plaque plcked ~
5 again and inoculated into tubes containing coverslips of :
calf testes cells or Hep 2 cells. After 4 to 6 days
incubation, the coverslips were removed and stained with the
mAb under test followed by FITC-labelled rabbit anti-mouse :-
IgG [Sigma] or FITC-labelled rabbit anti-bovine IgG [Sigma].
A polyclonal bovine antibody to RSV followed by FITC-
labelled rabbit anti-bovine IgG was used as a positive
control. RSVs that failed to react by immunofluorescence to
the mAb under test were classed as mutants and were used to
produce antigen for the ELISA described in Example 3 above.
Mutant viruses rerractory to mAb ll were selected
essentially as described above, but without prior culture of
the virus in cells containing 10% mAb in the supernatant.
Five mutant viruses were independently isolated from
the A2 strain of RSV after plaquing in the presence of mAb
20 ll. Eight mutants were independ2ntly isolated after culture ~`
in the presence of RSVl9, 3 mutants after culture in the ;~
presence on mAb 20, 6 after culture in the presence of B5
and lO after culture in the presence of B4.
After cloning, each escape mutant was used as
antigen in the ELISA described in Example 3 to test its
reactivity with a panel of anti-F mAbs (Figs. 7 and 8).
Mutant viruses selected for resistance to mAb ll
lost the capacity to bind not only mAb ll but also mAbs 13, --~
B4, and B5, and had reduced binding to mAb 7C2, when
30. compared with the parent A2 strain of RSV (Fig. 7). All
mutant viruses selected for resistance to either B4 or B5
lost the capacity to bind not only B4 or B5 but also ll and
13. However, some mutants selected with B4 ~e.g. C4947/5)
SUBSTlTtJTE SHEET
WO93/202l0 ~ 5~ PCT/GB93/00725
54
still bound to B5 but at a greatly reduced level when
compared with the A2 strain.
As seen for mutants selected for resistance to mAb
11, some B4 and B5 mutants showed reduced binding to 7C2,
however others failed to react with 7C2 (e.g. C4947/5). In
contrast to mutants selected with mAb 11, some mutants
selected with B4 or B5 still reacted wi~h mAb 18 (e.g.
C4947/5, 61:19, 61:16, 63:27 and C5014/7). B4 and B5
mutants showed either the same, reduced or no binding to mAb
B10 when compared with the parent A2 strain.
All mutant viruses selected with mAbs RSV19 or 20
failed to reac~ only with mAbs RSV19 and 20 (Fig. 7). The
binding of mAbs B13 and B14 (Fig. 7) and all other mAbs,
described in Fig. 5, to all the mutants was the same as to
the parent A2 strain of RSV, i.e., the mutant viruses
retained the binding of mAbs from other antigenic areas.
B. Ton~ ~X~in~s5~s
Escape mutants of the Long strain were isolated as
previously described tGarcia-Barreno et al. (1989), cited
above]. Briefly, virus stocks were enriched in mutant
. .
viruses by 4-5 consecutive passages in the presence of the
selecting antibody, 47F, 7C2 or AK13A2.
Then, the viruses were plaque purified in antibody
containing agar plates. Several viral plaques were
isolated, and their resis~ance to antibody neutralization
was confirmed. A single plaque originated from each aliquot ;
of the virus stock was chosen for further analysis. ~-
The epitopes recognized by the mAbs 84, 7C2 and ;
AK13A2 were included in antigenic area II previously
described by Garcia-Barreno et al. l1989), cited above,
based solely on their reactivity with antibody-escape
mutan~s. Similarly the epitopes recognized by mAbs RSVl9
and 20 were included in antigenic area IV by the same
criteria (Fig. 8).
'''
SUBSTITUTE SH E~T
WO93/20210 ~ 6 ~ PCT/GB93/00725
The mutations selected in the escape viruses -
affected only epitopes from the antigenic area which
included the selective antibody. For instance, mutant 4/4
did not react with any of the antibodies grouped in~area II,
whereas other mutants selected with the same antibody (11/3,
4, 5 and 7) reacted with mAbs 7C2 and ~4 but not with 47F,
49~ or AK13A2. Similarly, the mu~ants selected with mAbs 19
or 20 did not bind the antibodies grouped in the antigenic
area IV, except mAb 52F. However, in all cases the mutant
viruses retained the bin~ing of mAbs from other antigenic
areas.
The different reactivities of the antibodies from ;~
antigenic area II with the escape mutants lndicated that
their epitopes might overlap on the F molecule but were not
identical. To further differentiate these epitopes, it was
determined whether or not the corresponding mAbs would
compete for simultaneous binding to the virus using a
peroxidase labelled antibody in the ELISA of Example 3 mixed
with increasing, non-saturating amounts of each unlabeled
antibody previously titrated against the Long strain.
The capacity of an anti-idiotype rabbit antiserum
raised against mAb 47F to inhibit the binding of mAbs to RSV
was also tested by ELISA [Palomo et al ., J~ Virol., 64:4199-
4206 (1990)].
The results obtained indicated extensive
competition between these antibodies for virus binding;
howe~er, antibody AX13A2 inhibited the binding of mAbs 47F
and 49F in a non-reciprocal manner. In addition, the anti-
idiotype antiserum inhibited only the virus binding of mAbs
47F and 49F but not AK13A2, 7C2 and B4.
Thus, the epitopes included in antigenic area II
could be distinguished by at least one of the following
criteria: i) the reactivity of mAbs with escape mutants,
ii) the competition of mAbs for virus binding and iii) the
SUBST1TUTE SH EET
W O 93/20210 ~ PC-r/GB93/00725 `~
56
inhibition of virus binding by an antl-Id antiserum. Only
the epitopes 47F and 49F could not be distinguished by the
above criteria, but they differ in both neutralizing
capacity and susceptibility to denaturing agents.
Exam~l~ 8 - T~ocation of amino ac-~ change.s selected ln
neutral-7at;on esca~e mutants ;~
In order t~ identify-the amino acid changes selected in ~-
the escape mutants, the F protein mRNAs obtained from cells
infected with the different viruses were sequenced as
10 follows. Hep-2 cells were infected with the different ~
viruses and harvested 30-40 hours post-infection, when -
cytopathic effect was e~ident by the formation of syncytia.
Total RNA was isolated by the isothiocyanate-CsC1 method
[Chirgwin et al., ~ h~m~ 5294-5299 (1979)] and poly A+
RNA was selected by oligo dT-cellulose chromatography.
These mRNA preparations were used for sequencing by the
dideoxy method [Sanger et al ., Proc~ Nat'l. Aca~ci.~ a,
74:5463-5467 (1977)3 using reverse transcriptase and 5'_32p_
labelled oligonucleotides followed by a chase with terminal
deoxynucleotidyl transferase ~DeBorde et al., ~nal.
275-282 (I986)]. The primers used for
se~quenc~ing were synthesized according to the reported ~
sequence of the Long F protein gene [Lopez et al ., V; rus ` .
Res~ 0.249-262 (1988j].
25~ The~oligonucleotide primers used for sequencing mutants
selected with mAbs RSVl9 and 20 were, in anti-RNA sense:
SEQ ID NO: 23 F1216: 5'-ATCTGTTTTTGAAGTCAT ~``
SEQ ID~NQ: 24 F1300: 5'-ACGATTTTATTGGATGC
SEQ ID NO: 25 F1339: 5'-TGCATAATCACACCCGT
SEQ ID NO: 26 F1478: 5'-CAAATCATCAGAGGGG ! ` ''
SEQ ID NO: ~7 F1548: 5'-AATTCATCGGATTTACGA `~
SEQ ID NO: 28 F1707: 5'-CTCAGTTGATCCTTGCTTAG.
The F mRNA of viruses selected with mAbs AK13A2,
A~13A2, 7C2 and B4 were sequenced between nucleotides 420
.~
: ..
,:
SU8STtTUTE SHEET ` ~:"
. `.
-~W093/20210 h~ 2 PCT/GB93/00725
57
and 920, which encode the trypsin resistant fragments
recognized by those antibodies (Fig. 2). The F mRNA of ;~
viruses selected with mAb 11 were sequenced only between
nucleotides 893 and 906. Similarly, the F mRNAs of viruses
selected with ~Abs 19 and 20 were sequenced between
nucleotides liOO and 1680, which encode the region of the
tentatively located 26 kDa trypsin resistant fragmen~
recognized by those antibodies.
Table 3 illustrates sequence changes selected in -~
different neutralization escape mutants, including two
previously reported mutants selected with mAb 47F [Lopez et
al., (1990), cited above~. Only nucleotide (mRNA sense) and
amino acid changes at the indicated positions, as compared
to the Long and A2 strain sequences, are shown. ND means ;
not done.
This table, parts 3A, 3B, and 3C should be read across
for each antibody. For example, for antibody 47F, virus 4,
note that a nucleotide change from A to U at position 797
~Table 3A) results in an amino acid change from Asn to Tyr
at position 262 (Table 3B), and a loss of antibody binding
at 47F, 49F and AK13A2 (Table 3C).
SU8STITUTE SH EET
W093/20210 ~ 6~ 4~ ~ 6 ~ PCT/GB93tO072S
58
TABL~ 3A ~;
Ab used
for Nucleotide at positi.on
SelectiQn ~i~se.~ ~ 5~3 6~9 786 797 ~1~ 827 ~28 l.298
- Long and A2 C A U A A A A C
11 U ~ '~
47F 4 U -
7 U
AK13A2 4/4 G U
11~3 U -~:
4 U
U
7 G
4' `.
: -
.~;,
7C2 1 G .`~
4 A C ~
11 ' C ,.-
12 C ~.
B4 61:16/7 C --;
. ~
61:16/8 C .
l9 C484f A
;C4909/5 A ~-
C490~/6 : A
~- 30
C4902Wa A
C4902Wb A
C4902Wc A ~
~;.
~ ~.
~ .. ~j,
~.;
SUBSTI~UTE SHEET
WO93/20210 ~ ~ PCT/GB93/00725
59
TABL~
Ab used
for Amino Acid at position
Select;on V;rus~s~ 190 ?16 258 ~2 ?6~ 272 429
- Long and A2 Ser Asn Leu Asn Asn Lys Arg
11 Ile
47F 4 Tyr
7 Ile
AK13A2 4/4 Asp Tyr
11/3 Tyr
4 Tyr
1S 5 Tyr -
7 G1u
4'
7C2 1 G1u
4 Arg Ser
11 Thr
12 Thr
B4 61:16/7 Thr
61:16/8 Thr
19 C484f Ser
C4309/5 Ser
C4909/6 Ser ~.
C4902Wa Ser
C4902Wb Ser
C4902Wc
Ser
SUBSTlTUTE SHE~T
W093/20210 ~ PCI/GB9-/00725~;
~ .
TABLE 3
Ab used . ~
for Loss of binding with ::
SelectiQn Vi ruse~ ArL~ibod; es _ ~.
5 - Long and A2 - ~ :
.', '
11 Not determined .~
''~';~
47F 4 47F, 49F, AK13A2
7 47F, 49F, AK13A2, 7C2, B4 `~:
' -' -;
AK13A2 4/4 47F, 49F, AX13A2, 7C2, B4
11/3 47F, 49F, AK13A2
4 47F, 49F, AK13A2
47F, 49F, AK13A2
7 47F, 49F, AK13A2 ~ `~
41 47F, 49F, AK13A2, 7C2, B4
7C2 1 47F, 49F, AK13A2, 7C2, B4 ~-
4 7C2 ``
11 47F, 49F, AK13A2, 7C2, B4 :~
12 47F, 49F, AK13A2, 7C2, B4 ~-
~ .
`~
B4 61:16/7 47F, 49F, AK13A2, 7C2, B4 ~-`
61:16/8 47F, 49F, AK13A2, 7C2, B4 ~`
19 C484f 56F, 57F, 19, 20 ;~
C4909/5 56F, 57F, 19, 20 .`
C4909/6 56F, 57F, 19, 20
C4902Wa 56F, 57F, 19, 20
C4902Wb 56F, 57F, 19, 20 j:
C4902Wc S6F, 57F, 19, 20
5U8STITUTE SH EET
- W093/20210 ~ V G ~ PCT/GB93/00725
61
MAb 11 selected mutants which had a single transversion
(A to U) at nucleotide 816, which changed Asn-262 to Ile.
This change also led to the loss of the epitopes re~ognized
by mAbs 13, B4 and B5, which are included in antigenic area
B in Fig. 5 and is identical to that found in mutant 7
selected with mAb 47F which led to the loss of all the
epitopes included in antigenic area II of Fig. 8.
Four viruses selected with mAb AK13A2 (11/3, 4, 5 and
7) has a single transversion (A to U) at nucleotide 797
which changed Asn-262 to Tyr. This change ellminated the
binding sites for antibodies 47F, 49F and AK13A2 (see also
Fig. 8) and it is identical to the change observed in mutant
4 selected with mAb 47F. A fifth virus selected with mAb
AK13A2 (4/4) had, in addition, a transition ~A to G) at
nucleotide 659 which Asn-216 to Asp. This second amino acid
change led to the loss of all the epitopes from antigenic
area II (Fig. 8). The last mutant selected with mAb AK13A2
(4') had a single transition A to G at nucleotide 827,
leading to the replacement of Lys-272 by Glu and the loss of
all the epitopes from area II.
All mutants selected with mAb 7C2, except mutant 4,
contained single nucleotide changes (A to G or A to C) at
positions 827 or 828 which changed Lys-272 to Glu or Thr, -
respectively. These changes eliminated the reactivity with
all the m~bs from antigenic area II. Mutant 4 had two
nucleotide substitutions at position 583 tC to A) and 786 (U
to C) which changed amino acids 190 (Ser to Arg) and 258
(Leu to Ser). The last mutant had only lost the binding
site for mAb 7C2 but retained its reactivity with all the
otlher anti-F antibodies (Fig. 8).
The two mutants selected with mAb B4 had a single
- nucleotide transversion at position 828 (A to C) which
changed Lys-272 to Thr. Thus, all the amino acid changes
SUBS rlTU~E SHEET
WO93~20210 ~ PCT/GB93/00725
62
selected with mAbs from antlgenic area II were clustered ln
a small segment of the F protein, between amino acids 262
and 272, except the changes at amino acids 258, 216 and 190
which were detected only in viruses with two amino~acid
S substitutions. -~
All mutants selected with antibodies RSV19 or 20
contained a single C to A tlansversion at nucleotide 1298
which changed Arg-429 to Ser. This amino acid change,
located towards the carboxy terminal end of the cysteine
10 rich region of the F1 subunit (Fig. 2), eliminated the
reactivity of all the mAbs grouped in the antigenic area IV, -;
except antibody 52F. Amino acid 429 (Ser) is therefore ~`~
important for the binding of mAbs RSV19 and 20 to the F
protein. The synthetic peptides 417-432 and 422-438 of the - ~
15 F protein [SEQ ID NO: 19] reproduce at least part of the ~-
epitope recognized by mAb RSV19. The sequence results
confirm the findings shown in Figs. 7 and 8 that antigenic v
areas II and IV do not overlap.
, ~ -.. -,
~.~
SUBSTI~UTE SHEET ` `
WO93t20210 ~` L ~ PCT/GB93/0072
63
Exam~le 9 ~ tivitY of an~ihQ~l~a-with synth~is~
Since the antibodies used to select the escape~mutants
reacted in Western blot with trypsin fragments of the Fl
subunit, whether or not synthetic peptides could reproduce
the epitopes recognized by these antibodies was determined.
In summary, the results obtained with the synthetic
peptides were also indicative of conformational constraints
in the epitopes of antigenic area II. Epitope B4 was
reproduced by the peptide 255-275 of SEQ ID N0: 55; however,
other closely related peptides failed to react with that
antibody. In addition, none of the peptides tested
reproduced other epitopes of antigenic area II (B). The
region of the Fl subunit containing these epitopes is
resistant to high doses of trypsin, indicative of a
particular three-dimensional conformation which might be
preserved in Western blots but not in synthetic peptides.
The peptides shown in Table 4 were synthesized in an
Applied Biosystem 430 instrument, using the solid phase
technology and t-Boc chemistry [Merrifield, Scie~ce,
23?:341-347 (1986)]. The peptides were cleaved off the
resin with trifluoromethyl sulfonic acid and purified from
protecting groups and scavengers by Sephadex G-25
chromatography. The amino acid sequence of each peptide was
confirmed by automated Edman degradation in an Applied
Biosystem 477 protein sequencer.
Three peptides were synthesized with sequences
corresponding to amino acids 250-273, 255-275 or 258-271 of
the Fl subunit [SEQ ID NO: 55~, which surrounded the
! 'pdsitions changed in the mutants selected with mAbs from
antigenic area II.
The binding of mAbs to synthetic peptides was tested by
ELIS~ of Example 3 in polyvinylchloride microtitre plates
SU8STITUTE Sl{EET
WO93/20210 i~ PCT/GB93/00725'`
64
coated overnight with 1-2 ~g of peptide. PBS containing 5
pig serum was used as blocking reagent to eliminate spurious
cross-reactions. The results are reported in Table 9 below. ~`
Only antibody B4 and another bovine antibody, B5, ~-~
reacted with the peptide 255-275 of SEQ ID N0: 55. The B4
titre with this peptide was similar to that obtained against
purified virus. However, this ~ntibody did not react with
peptides 250-273 nor 258-271 of SEQ ID NO: 55, which ~;`
contained almost the entire amino acid seq~ence included in
peptide 255-275 of SEQ ID NO: 55. All other antibodies from
area II failed to react with any of the peptides.
Three other peptides, corresponding to the sequences
417-432, 422-438 and 435-450 of the F1 subunit [SEQ ID NO~
55] which surrounded the position 429 changed in the escape
1$ mutants selected with mAbs RSV19 or 20, were also tested by
ELISA (Table 4). Only antibodies RSV19, B13 and B14 reacted
wi~th~the first two peptides ~417-432 and 422-438 of SEQ ID ~-
NO: $~5)~
Thus~, two antigenlc~sltes recognized by neutralizing, `~
protective mAbs directed against~the F protein have been
identified. The first site contains several overlapping ~`j
epitopes located within the trypsin resistant amino terminal `-`
t~hird~of~the F1 subunit, clustered around amino acids 262-
272 of~SEQ I~D~N0: 55. Only one of these epitopes, that
, ~
25~ recognized~by B4,~was~faithfully reproduced by a short ``~
synt~het~lc~peptide corresponding to amino acids 255-27S of
the F proteln~[SEQ ID NO: 19]. The second antigenic site
was Iocated within the carboxy terminal third of the F1
subunit and the epitope recognized by mAb RSV19 and that
30-~ recognized by B13 and B14 was reproduced by synthetiç
! peptides co~rresponding to amino acids 417 to 432 and 422 ~o
438 of SEQ ID NO: 55. However, the epitopes recognized by ``:
,.:
,',':
: :.
: ' ,:
SU8STITUTE SHEET
: `
` .
--' WO93~20210 ~ ~ ~ G ~ 2 PCT/GB93~0~725
mAbs RSVl9, Bl3, and Bl4 do not appear to be identlcal since
mAbs Bl3 and Bl4 react with antibody-escape mutants selected
with mAb RSVl9 which have a substitution at amino acid 429-
Arg (Fig. 7)j indicating that amino acid 429-Arg is~not
S essential for the binding of mAbs Bl3 and Bl4 to the F
protein. The peptide fragments of the following Table 4 are
taken from SEQ ID NO: 55, the Fl subunit. - ~
:
~:
.
5UBSTITIJTE SHEET :
W093/202l0 ~ 2 PCT/GB93/00725
66 "~
Table 4 .:
Reactivity of monoclonal antibodies with synthetic peptides
- -
MonoclonaL antibody
Peptide 7C2 47F AK13A2 11 B4 RSVl9 20 B13 B14
10 . ''; ``
- ~
~50-273 <2.0 <2.0<2.0<2.0 <2.0 <2.0 ND ND
255-275<2.0<2.0<~.0 <2.0 6.7 <2.0 <2.0 <2.0 <2.0 `.:
258-271<2.0<2.0<2.0 <2.0 <2.0 <2.0 <2.Q ND ND
417-432<2.0<2.0<2.0 <2.0 <2.0 2.8 <2.0 4.5 3.2
422-438<2.0<2.0<~.0 <2.0 <2.0 6.0 <2.0 5.0 4.3
435-450<2.0<2.0<2.0 <2.0 <2.C 2.3 <2.0 ND ND
RSV 8.4 6.1 4.9 6.4 5.3 6.4 6.3 5.6 5~6 `~
strain A2
- . ~
Log10 titre of antibody binding to synthetic peptides dried ``
onto wells or RSV antigen tested in an ELISA.
30 xa~ple lQ - Pe~a~ Analys;s Qf F~i~Q~e ~e~Q nlz~ hy m~h ~.
Overlapping peptides corresponding to amino acids 255
to 275 of the F protein [SEQ ID NO: 19] were synthesized in `-
duplicate as a series of octamers overlapping by seven amino
acids and offset by one amino acid, bound to polyethylene
pins using F-Moc chemistry following the method of Geyson et .
al, J Im=~aL__M~h~, lQ2:259-274 (1987). The software
: package, polyethylene pins and amino acids used to produce :
the peptides were obtained from Cambridge Research ~-
Biochemicals,.Cheshire, England.
SUBSTI~lJTE SH E~T :
-` WO 93~20210 r~ 6 6 ~ PCT/GB93/0072~
67 :
The pins to which the peptides are bound were incubated
with bloc~ing buffer in 96 well microtitre plates (PBS
containing 0.05% Tween 20 and 2% Marvel) on a rotary shaker.
After one hour incubation at room temperature, the Pins were
incubated with mAb B4, ~nd diluted 1:600 in blocking buffer
at 4C with shaking. After being washed 10 times for 5
minutes in PBS containing 0.05~ Tw~en 20 (PBS/Tw), the pins
were incubated with horseradish peroxidase (HRP)-rabbit
anti-bovine IgG [Sigma], diluted 1:4000 in blocking buffer.
After one hour and 4S minutes, the pins were washed ten
times for 5 minutes and incubated, in the dark with
agitation, in microtiter plates containing 150~1/well of 50
mg of azino-di-3-ethyl-benzthiazodisulpho-nate [Sigma]
dissolved in 100 ml of substrate buffer (O.lM disodium
hydrogen orthophosphate; 0.08M citric acid) containing 0.3
l/ml of 120 volume hydrogen peroxide. When sufficient color
had developed, the O.D. was read at 405 nm on a Titertek
MuLtiscan MCC 340 plate reader. MAb B4 recognized a single
peptide extending from amino acid #266-273 of SEQ ID NO: 19
and having the sequence I T N D Q K K L bound to the pins~
The binding of B4 to this octomer was studied further
using peptides, bound to pins, which represented the above
sequence, but where every amino acid in this sequence was
replaced in turn with each of the 20 naturally occurring
amino acids. Duplicate peptides were synthesized as
described above and the binding of mAb B4 to the peptides
was determined by ELISA and is shown in Fig. 9. B4 bound to
all peptides where amino acid 266-Ile was replaced in turn
wlth all other amino acids, indicating that amino acid 266-
Ile was not essential for the binding of B4 to the peptide
! ' 266-273 of SEQ ID NO: 19. Similarly, replacement of amino
acids 270-Glu and 273-Lys did not affect the binding of B4
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;; J ~ `~ J ~ ::
WO 93/20210 PCr/GB93/00725
68
to a significant extent. In contrast, substitution of amino
acids ~68-Asn, 269-Asp and 272-Lys resulted in the to~al
loss of binding of B4, indicating that these amino acids are
essential for the binding of B4 to peptide 266-273~of SEQ ID
S NO: 19. These studies confirm the findings from the
sequence analysis of antibody escape mutants (Example 8) ;~
which also showed that amino acids 268-Asn and 272-Lys were
critical for the binding of B4 to the F protein.
Substitution of amino acid 267-Thr resulted in reduced -~
binding of B4 and replacement of amino acid 271-Lys resulted
in significantly enhanced binding to the peptide. Maximum
binding to the peptide 266-273 of SEQ ID NO: 19 was detected ;~
when amino acid 271-Lys was replaced by Ile.
EX~ - A Hurnanized P~nti-R.SV Ant;~
The following example describes the preparation of an ;
exemplary altered antibody utilizing the murine IgG2a mAb
called RSVl9 or RSMU19, described in co-pending PCT
application No. PCT/GB91/01554 as the source of the donor
variable chain sequences and CDRs. Similar procedures may
be followed for the development of altered antibodies, using ;-~
other anti-RSV antibodies described herein.
RSVl9 is specific for the fusion (F) protein of RSV.
The RSV19 hybridoma cell line was obtained from Dr.
Geraldine Taylor. Methodology for the isolation of
hybridoma cell lines secreting monoclonal antibodies
specific for RSV is described by Taylor et al. ~ Tmn~ Qg
52:137-142 (1984).
As described in the preceding example, cytoplasmic RNA
was prepared by the method of Favaloro et al., (1980) cited
30 above from the RSV19 hybridoma cell line, and cDNA was
synthesized~ using Ig variable region primers as follows.
For the Ig heavy chain variable region, RSV19VH ~see Figs.
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69
15A, 15B and 19), the primer [SEQ ID NO: ~3] VHlFOR
5'TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG3' was used, and
for the Ig light chain variable region, RSV19VK (see Figs.
16A and 16B)~ the primer [SEQ ID NO: 34] ~.~lFOR ~ -
5'GTTAGATCTCCAGCTTGGTCCC3' was used.
cDNA synthesis reactions consisted of 20~g RNA, 0.4~M
VH1-FOR or VKlFOR, 750~M each of dATP, dCTP, dGTP and dTTP,
50mM Tris-HCl pH 7.5, 75mM KCl, 10mM DTT, 3mM MgCl2 and 27
units RNase inhibitor in a total volume o_ 50~1. Samples
were heated at 70C for 10 minutes and slowly cooled to 42C
over a period of 30 minutes. Then, 100~ ~LV reverse
transcriptase was added and incubation at 42C continued for
1 hour.
VH and VK cDNAs were then a~plified using PCR. For
PCR, the primers used were: VHlFOR; VKlFOR; VHlBACK
(described in Example 18), and
; ~ ~[~SEQ ID NO: 35] VKlBACK S'GACATTCAGCTGACCCAGTCTCCA 3'.
Primers VH1FOR, VKlFOR, VHlBA~K and VKlBACK, and their
use for~PCR-amplification of mouse Ig ~NA, are described by
Orlandi~et al., ~1989), cited above.
For PCR amplification of VH, DNA/primer mixtures
consisted;~of 5~1 RNA/CDNA hybrid, and 0.5~M VHlFOR and
VHlBACK~primers. For PCR amplifications of VK, DNA/primer
mixtures~consisted of 5~1 RNA/cDNA hybrid, and 0.5~M VKlFOR
; 25~and VKlBACK primers. To~these mixtures was added 200 ~M
each of~dATP, dCTP, dGTP and dTTP, 10mM Tris-HCl pH 8.3, ~;
~50mM KC1, ~1.5mM~MgCl2, 0.01% ~w/v) gelatin, 0.01~ (v~v)
Tween 20~ 0~.~0~1~% (v~v~ Nonidet P40 and 2 units Taq DNA
polymerase [United States Biochemicals-Cleveland, Ohio,
USA]. ~Samples were subjected to 2S thermal cycles of PCR at
1 940C, 1 minute; 60C, 1 minute; 72C, 2 minutes; ending with
~ 5~ minutes at 72C. For cloning and sequencing, amplified VH
,~ ,
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WO93/202l0 ~J ~ PCT/GB93/0072~;`
DNA was purifled on a low melting polnt agarose gel and by
Elutip-d column chromatography and cloned into phage Ml3.
The general cloning and ligation methodology was as
described in Maniatis et al., cited a~ove. ~ -~
VH DNA was either directly ligated into the SmaI site
or Ml3 mp 18/l9 or, following digestlon with PstI, into the
PstI site of Ml3t~131 [Amersham Internatlonal-Little
Chalfont, UK]. Amplified VK was similarly gel purified and
cloned by the following alternatives: (l) PvuII digest
into Ml3mpl9 ~SmaI site); ~2~ PvuII and BglII digest
into Ml3mpl8/l9 (SmaI-BamHI site)i 13) PvuII and BglII
digest into Ml3tgl31 (EcoRV-BglII site)i (4) BglII cligest
into Ml3tgl31 (SmaI-BglII site). The resultant collections
of ove~lapping clones were sequenced by the dideoxy method
[Sanger et al., cited above] using Sequenase [Unitecl States
Biochemicals-Cleveland, Ohio, USA].
From the sequence of RSVl9 VH and VX domains, as shown
in Figs. 14A and 14B, and 15A, and l5B, respectively, the `
CDR sequen~es were elucidated in accordance with the
methodology of Kabat et al., in "Sequences of Proteins of
Immunological In~erest", US Dept of Health and Human
Services, US Government Printing Office, (1987) utilizing
computer assisted alignment with other VH and VK sequences.
The murine RSVl9 CDRs were transferred to human
frameworks by site directed mutagenesis. The primers used
were:
[SEQ ID NO: 36~ VHCDRl 5'CTGTCTCACCCAGTGCATATAGTAGTCG
CTGAAGGTGAAGCCAGACACGGT 3'
[SEQ ID NO: 37] VHCDR2 5' CATTGTCACTCTGCCCTGGAACTTCGGGG
CATATGGAACATCATCATTCTCAGGATCAATCCA 3'
[SEQ ID NO: 38] VHCDR3 5' CCCTTGGCCCCAGTGGTCAAAGTCACTCCC
CCATCTTGCACAATA 3'
~
SU8STITVTE SHEET
`!; WO 93/202~0 r'~ ~ t~J~ PCT/~B93/00725
[SEQ ID NO: 39] V~CDR1 5' CTGCTGGTACCATTCTAAATAGGTGTTTCCA
TCAGTATGTACAAGGGTCTGACTAGATCTACAGGTGATGGTCA 3'
[SEQ ID NO: 40] VKCDR2 5' GCTTGG~ACACCAGAAAATCGGTTGGAAACTC
TGTAGATCAGCAG 3'
[SEQ ID NO: 41] VKCDR3 5' CCCTTGGCCGAACGTCCGAGGAAGATGT
GAACCTTG~GCAGTAGTAGGT 3'
The DNA templates for mutagenesis comprised human
framework regions derived from the crystallographically
solved proteins, NEW [Saul, et al., J. B;ol..ChemL, 53:585-
597 (1978)] with a substitution of amino acid 27 from serineto phenylalanine [See, Riechmann et al., l~c,i~1 and REI
[Epp et al., ur J. Biochem. 45:513-524 (1974~ for VH and
VK domains, respectively. M13 based ~emplates comprising
human frameworks with irrelevant CDRs were prepared as -~
described by Riechmann et al., Na~ , 332 (1988).
Oligonucleotide site directed mutagenesis of the human ~-
VH and VK genes was based on the method of Nakamaye et al., :.
Nucl. ~cid ~ , 14:9679-9698 (1986). To 5~g of VH or VK
single-stranded DNA in M13 was added a two-fold molar excess
of each of the three VH or VK phosphorylated
oligonucleotides encoding the three mouse CDR
(complementarity determining region) sequences. Primers
were annealed to the template by heating to 70C and slowly `
cooled to 37C. To the annealed DNA was added 6 units T4DNA :~
ligase [Life Technologies, Paisley, UK]; 0.5 mM of each of
the following nucleoside triphosphates (dATP, dGl'P, dTTP and
2'-deoxycytidine 5'-0~ thiotriphosphate) (thiodCTP); 60mM
Tris-HCl (pH 8.0~; 6mM MgCl2i 5mM DTT [Sigma, Poole, UK]; ~.
and lOmM ATP in a reaction volume of 50~1. This mixture was
30 incubated at 16C for 15 hours. The DNA was then ethanol .-
precipitated and digested with 5 units NciI [Life `
Technologies, Paisley, UK] which nicks the parental strand .
SUBSTITUTE SHE~T
W093~20210 PCTiGB93/0072~
but leaves the newly synthesized strand containing thiodCTP
intact. The parental strand was then removed by digestlng
for 30 minutes with lOO units exonuclease III [Pharmacia,
Milton Keynes, United Kingdom] in 50 ~l of 60mM ~r s-HCl (pH
8.0), 0.66mM MgCl2, and lmM DTT. The DNA was then repaired
through addition of 3 units of DNA polymerase I [Life
Technologies, Paisley, UK], 2 units T4 DNA ligase in 50 ~
of 60 mM Tris-HCl (pH 8.0), 6mM MgCl2, 5mM DTT, lOmM ATP and
O.S mM each of dATP, dCTP, dGTP and dTTP. The DNA was
transformed into competent E. coli TGl cells [Amersham
International, Little Chalfont, UK] by the method of
Maniatis et al., cited above.
Single-stranded DNA was prepared from inàividual
plaques and sequenced by the method of Messing, Me~hQ~ in
Fln~mology, lOl:20-78 ~1983). If only single or double
mutants were obtained, then these were subjected to further
rounds of mutagenesis (utilizing the methodology described
above) by using the appropriate oligonucleotides until the
triple CDR mutants were obtained.
The CDR replaced VH and VK genes were cloned in
expression vectors (by the method of Maniatis et al . ) to
yield the plasmids pHuRSVl9VH and pHuRSVl9VK. The plasmids
are shown in Figs. l6 and 17, respectively. For pHuRSVl9VH,
the CDR replaced VH gene together with the Ig heavy chain
promoter, appropriate splice sites and signal peptide
sequences were excised from Ml3 by diqestion with HindIII
and BamHI, and cloned into an expression vector containing
the murine Ig heavy chain enhancer, the SV40 promoter, the
gpt gene for selection in mammalian cells and genes for
replication and selection in E. coli. The variable region
amino acid sequence is shown in Fig. l9. A human IgGl
constant region was then added as a BamHI fragment.
Sl.'BSTITUTE SH EET
O g3/20210 ~J ~ 6 ~ PCT/GB93/00725
The construction of the pHuRSV19VK plasmid was
essentially the same except that the gpt gene was replaced
by the hygromycin resistance gene and a human kappa chain '~
constant region was added (see Figs. 17 and 22)....... ~ ;,
lO~g of pHuRSV19VH and 20~g of pHuRSV19VK were digested ''~.'
with PvuI utilizing conventional techni~ues. The DNAs were ~-
mixed together, ethanol precipitated and dissolved in 25~1
. water. Approximately 107 YB2fO cells [American Type Culture ~:
- Collection, Rockville, Mary:land,~USA] were grown to semi~
10 confluency, harvest~ed by centrifugation and resuspended in ..
0.5ml DMEM [Gibco, Paisley, UK] together with the digested
: DNA:~in ~a cuve~e.~ A~fter 5 m1nutes. on ice,~ the cells were ;~.~ giveA; a single~p~u~lse of 170V at 9~60uF (Gene-Pulser, Bio-Rad- i~
: : :Richmond, Califo:rnia, USA) and left in ice for a further 20.
15 minute. The cells were then put into 20 ml DMEM plus 10% ;,
foetal ca:lf serum and allowed to recover for 48 hours. ''
Aft ~ ~this-~t~ime~,~the:~cel~ls~were~distributed into a 24-well ..'
:plate~:and~s.e~lective~medium~applied~(DMEM,~ 10% foetal calf '~'~,.'
serum,~ 0~.~8~g/ml mycophenolic: acld,~ and 250~g/ml xanthine).
20~ A:fter'~:~3~-~4~days,~:the;medium~and dead cells,were removed and . ~.','.
re'placed wit-h,~fresh''selective~medium. Transfected clones
~?'~ w ~ -vi. ~ ~le~:w~ith~:the~:naked~eye 10:-12 days later. i,
he.~prés:ence::~of~human~ant1body in the medium of wells '',`.
.. ,"~ contain.ing~;,trans~fected,~c:lones~was~measured by conventional
2-:5~ ÉLISA:te ~ ni ~ ~s~ 'Micro-tit:re plates were coated overnight ';
at~4C~wit~goat~;anti-~hùman::IgG~(~gamma chaln specific) `
~ antibodie5~ t~5e~F,a-Lab-Ltd~ Crawley Down, UK] at 1 ~g per ''.. '
,"""~ ~ell~ ~ er~:washing;~with:PBST (phosphate buffered saline
cont~aining'~0~.~02~%~Tween~20x (pH7~5)), 100~1 of culturè medium
30: from the well~s containing transfec,tants was!added to eac~ ,,,"
- m'icrotitre'we~il for 1 hour at 37C. The wells were ehen l,,
empt~ied, washed with PBST and ei:ther peroxidase-conjugated .'.,.
. ~ . - , -.
` SUBSTITUTE St~ EET ``i~``
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WO93/20210 PCT/GB93/00725
74
goat anti-human IgG or peroxidase-conjugated goat anti-human
kappa constant region antibodies [both obtained fro~ Sera-
Lab Ltd., Crawley Down, UK] were added at 100 ng per well.
Plates were then incubated at 37C for 1 hour. Th~ wells
5 were then emptied and washed with PBST. 340 ~g/ml q- ~
phenylenediamine in 50mM sodium citrate, 50mM sodium `;
phosphate (pH 5.0) and 0.003% (v/v~ H2 2 were added at 200
l per well. Reactions were stopped after 1 to 5 minutes by
the addition of 12.5~ sulphuric acid at 50~1 per well. The
absorbance at 492 nm was then measured
spectrophotometrically. ~;
The resulting humanized antibody HuRSV19VH/VK (also
called RSH200), secreted from cell lines co~ransfected with
pHuRSV19VH and pHuRSV19VK, was purified on Protein-A agarose
columns ~Boehringer Mannheim, Lewes, UK] and tested for
binding to RSV virus in an ELISA assay. Antigen consisted
of calf kidney (CK) cells infected with RSV A2 strain [Lewis
et a~., Med. ~ u~tra~ 48:932-933 (1961)] and treated
with 0.5% ~v/v~ NP40 detergent to yield a cell lysate. A
20 control cell lysate was similarly prepared using uninfected ~;
CK cells. Microtitre plate wells were coated with either
infected or control cell lysate. Antigen coated plates were
blocked with PBST for 1 hour at 37C, washed with PBST, and
thereafter humanized antibody was applied (i.e., `
HuRSV19VH~VK). After 1 hour at 37C, the wells were
emptied, washed with PBST and 200 ng goat anti-human IgG
antibodies [Sera Lab-Ltd., Crawley Down, UK] added per well.
After 1 hour at 37C, the wells were emptied, washed with ~;
PBST and 200~1 of a 1:1000 dilution of HRP-conjugated rabbit ~`
anti-goat IgG antibodies [Sigma-Poole, UK] were added.
After 1 hour at 37C, the wells were emptied and washed
with PBST. To each well was added 200~1 substrate buffer
SVBSTlTUTE SHEET
fJ ~ V~
'WO93~20210 PCT/GB93/00725
(340~g/ml q-phenylenediamine in 50mM sodium citrate, 50mM
sodium phosphate (pH 5.0) and 0.003% (v/v) H202). Reactions
were stopped by the addition of 50~l 12.5% sulphuric acid.
The absorbance at 492 nm was then measured. ~ ,
This humanized antibody HuRSVl9VH/VK (RSHZ00),
generated by the straight replacement of the RSVl9 heavy and ,"
light chain CDRs into the human heavy chain framework "
regions (variable and constant regions REI and kappa, '-
respectively) bound to whole RSV preparations, although with '
lO an affinity less than the donor murine RSVl9 antibody. ';~
Exam~le ~2 - Product;on of H~h Affinity ~nti-RSV ~ntihod;es ''~
High affinity antibodies specific for RSV were ',
developed by a method designed to achieve minimal variable
region framework modifications giving rise to high affinity ,'-
15 binding. The method involves the following order of;steps ~'
of alteration and testing~
l. Individual framework amino acid residues which are
known to be critical for interaction with CDRs are compared ','`
in the ,primary~antibody and the altered CDR-replacement ' -
antibody.~ For example, heavy chain amino acid residue 94
Kabat numbering-see Kabat et al., cited above) is compared ~,~
in the primary ~donor) and altered antibodies. An Arg ~,'
residue at this position is thought to interact with the '~,',
invariant heavy chain C~R Asp residue at position lOl. '~;
If amino acid 94 comprises Arg in the framework of ','
the primary~ antibody but not in the framework of the altered ''',~
antibody, then an alternative heavy chain gene comprising '~`~
~ Arg 94 in th:e altered antibody is produced. In the reverse ~,"
- situation whereby the altered antibody framework comprises `~''
30 an Arg residue at position 94 but the primary antibody does ,~
not, then an alternative heavy chain gene comprising the ~`-
original amino acid at position 94 is produced. Prior to
~ . ~
i . ~ .
~ .
~"~
SU8STmJTE SHEET
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WO93/20210 '~ PCT/GB93/0072~-
76 '
any further analysis, alternative plasmids produced on this
basis are tested for production of high affinity altered ',
antibodies.
2. Framework amino acids within 4 residues o~ the
CDRs as defined according to Kabat ~see Kabat et al., cited
above3 are compared in the primary antibody and altered CDR-
replacement antibody. Where differences are present, then
for each region (e.g., upstream of VHCDRl) the specific
amino acids of that region are substituted for those in the ,'
corresponding region of the altered antibody to provide a
small number of altered genes. Alternative plasmids
produced on this basis are then tested for production of ,
high affinity antibodies.
3. Framework residues in the primary and altered CDR- ~
15 replacement ant,ibodies are compared and residues with major '
differences in charge, size or hydrophobicity are
highlighted. Alternative plasmids are produced on this
basis with the individual highlighted amino acids
represented by the corresponding amino acids of the primary '
antibody and such alternative plasmids are tested for
production of high affinity antibodies.
The method is exemplified by the production of a
high affinity altered antibody derivative of HuRSVl9VH/VK
specific for RSV. Comparison of VH gene sequences between '
RSVl9VH and pHuRSVl9VH tFigs. 18-22) indicates that 3 out of
4 amino acid differences occur between amino acids 9l to 94
of the F protein of SEQ ID N0: l9, which defines a framework
sequence adjacent to heavy chain C~R3.
Thus, plasmid pHuRSVl9VHFNS (Fig. 20) was produced by
inserting the RSVl9 heavy chain CDRs and the four ami,no acid
framework sequence amino acids 9l to 94 into the human
framework described in the preceding example. Using
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:~ .
77
oligonucleotide site directed mutagenesis, the following
oligonucleotide was used for mutagenesis of the HuRSV19VH ;~
gene in M13:
~SEQ ID N0: 42] HuRSV19VHFNS - 5'CTCCCCCATGAATTACAGAAATAG
ACCG 3'.
The cell line cotransfected with pHuRSV19VHFNS and
pHuRSVl9VK ~Fig. 22) produced a second humanized antibody,
HuRSV19VHFNS/HuRSV19VK (abbreviated hereafter as RSHZ19).
This antibody was tested in an ELISA assay for analysis of
binding to RSV antigen prepared from detergent-extracted,
virus-infected cells. The substitution of VH residues 91 to
94 in HuRSV19VH/VK with VH residues from mouse RSV19VH
partially restored antigen binding levels. Additional ;
analysis of HuFNS binding properties was performed using an
15 ELISA assay in which intact Type A RSV (Long strain) was ~-~
used as the antigen. The data from such additional analysis -~
show that there is little if any difference between the `;`
ability of the murine mAb RSVl9 and the humanized RSHZl9
~- antibodies to bind to intact, non-denatured RSV. This
20 additional analysis also showed detectable binding of ~-
~HuRSV19VH/VX to intact virus, although of a much lower ,`~
magnitude than was seen with either RSV19 or RSHZl9.
Thus, the data from this additional analysis suggests
that the affinity for the native antigen was restored in the
25 RSHZ19 mAb. Specificity of RSHZl9 for RSV F protein was !"``":
shown by~conventional Western blot analysis using a
truncated soluble F protein construct expressed in CH0 ;^~
cells.
~ le 13 - Tm~unofl~nresce~ce ana1ysis of ~umanlzed ~""
~t;h~d;es
In order to ascertain the potential clinical usefulness
of a humanized antibody specific for RSV, an immuno-
SUBSmtJTE SHEET
WO93/20210 ~ 6 ~ 2 PCT/GB93/00725
78fluorescence analysis of bindlng to 24 RSV clinical isolates
was undertaken. The lsolates were obtained from children
during the winter of 1983-84 by the Bristol Public Health
Laboratory (Bristol, England) and represented both~of the
major subgroups of RSV. Thirteen isolates were serotyped as
subgroup A and ll isolates as subgroup B. HeLa or MA 104
cells infected with RSV isolates were grown in tissue
culture. When the cells showed evidence of cytopathic
effect, 20 ml of 0.02% (w/v) disodium ethylenediaminetetra-
acetic acid (EDTA) [BDH Chemicals Ltd., Poole, UK] in PBS
and 3ml of 0.25% ~w/v) trypsin in PBS were added and the
cell suspension spotted into wells of PTFE-coated slides `~
(polytetrafluoroethylene coated slides) [Hendley, Essex,
UK]. After 3 hours at 37C, the slides were dried and fixed
in 80% acetone. Cells were overlaid with monoclonal
antibody ti.e., either humanized antibody, RSHZl9 or the
murine antibody RSVl9) for l hour at room temperature.
After extensi~e washing, either fluorescein-conjugated
rabbit anti-mouse IgG [Nordic Laboratories-Tilburg, The -
Netherlands] or fluorescein-conjugated goat anti-human IgGl
[Southern Biotechnology, Birmingham, Alabama, USA] was
added, and the incubation was repea~ed. After further
washing, cells were mounted in glycerol and examined under
W light.
- 25 The results of comparative immunofluorescence for the
humanized antibody, RSHZl9, and the murine antibody RSVl9
indicated that 100% of clinical isolates are recognized by
both the humanized and murine antibodies. Such data
demonstrated that the humanized antibody has the potential
for recognition of most clinical isolates comprising both of
t~e major RSV subgroups.
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The humanized antibody, RSHZl9, was next tes~ed for
biological activity in vitro in a fusion inhibition assay.
A suspension of MA104 cells was i~fected with RSV at an
m.o.i. (multiplicity of infection) of 0.01 PFU (pla~ue
5 forming units) per cell. After l hour at 37C, 2 ml of ;
cells at 105/ml were distributed tO glass coverslips in ~-~
tubes. After a further 24 hours at 37C, the culture medium
was replaced by medium containing dilutions of humanized j;
antibody, RSHZ19. Twenty-four hours later, coverslip
10 cultures were fixed in methanol for 10 minutes and stained --
with May Grunwald stain [BDH Chem'cals Ltd., Poole, UK]. `~
The effect of increasing concentrations of RSHZl9 in `~
inhibiting the frequency of giant cells demonstrates the
biological acti~ity of the humanized antibody RSHZ19 in
inhibiting Type A RSV induced cell fusion. Additional
studies showed that the fusion inhibition titres for RSVl9 ~
versus RSHZ19 wexe comparable, providing additional evidence `j-
that affinity for the native viral antigen was fully
restored in the humanized RSHZl9. The humanized antibody ~`~
RSHZ19 has also been shownt using methodology analogous to
that utilized above for showing inhibition of Type A RSV ~;
induced cell fusion, to exhibit a dose dependent inhibition
of Type B RSV (strain 8/60) induced giant cell fusion.
The humanized antibody, RSH719, was next tested for
biological activity in vitro in an RSV-mouse infection
model. BALB/c mice [Charles Rivers: specific pathogen free
category 4 standard] were challenged intranasally with 104
PFU of the A2 strain of human RSV [Taylor et al., Infect.
Imm~n_, ~3:649-655 (1984)]. Grou~s of mice were
30 administered with 25~g of humani~ed antibody either one day `~
prior to virus infection or 4 days following infection. ~`
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Administration of antibody was either by the intranasal
(i.n.) or intraperitoneal (i.p.) routes. 5 days after RSV
infection, mice were sacrificed and lungs were assayed for
RSV PFU [see, Taylor et al., cited above]. The dat~ showed
that RSHZl9 at a single dose of 25 ~g per mouse is extremely
effective in prevention and txeatment of RSV infection.
RSHZl9 was also shown to be active in vivo when
administered prophylactically to mice challenged with Type B
RSV (strain 8/60) using methodology similar to that
described above. In addition, the humanized antibody
HuRSVl9VH/VK was also shown to be active in vlvo when
administered prophylactically to mice challenged with Type B
RSV (strain 8~60) using methodology similar to that
descri~ed above.
ExamDle 14 - Comp~risQn of blood levels of RS~Zl9 after i.~.
or ; p . Tnocul at; o-l of Mice
Five female BALB/c mice (weighing approximately 20g)
were inoculated i.p. with 50 ~g RSHZl9 tCHO~ and another 5
were inoculated intravenously (i.v.) with 50 ~g RSHZl9
20 tCHO). Mice were bled from the tail 2 hours, l, 4, 7, 14,
21 and 46 days later and the levels of RSHZl9 in the sera
were determined using two different ELISAs as follows.
(1) Plates were coated with a lysate of either RSV
(strain) A2-infected or uninfected Hep-2 cells,
followed by dilutions of mouse sera and HRP-anti
human IgG.
(ii) Plates were coated with 200ng of anti-idiotypic "
mAb Bl2, followed by mouse sera and HRP-anti human ~`
IgG.
Both assays gave essentially the same results, although
~- the B~2 ELISA appeared to be more sensitive. Two hours
a~ter inoculation the serum level of RSHZ19 was 5-fo1d
:
.
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greater in mice inoculated i.v. compared with those -
inoculated i.p.. However, titres of RSHZ19 were equivalent
in both groups of mice by 24 hours after inoculation. The
level of RSHZ19 remained constant for at least 4 da~s after .
inoculation and was beginning to decline at 7 days. After
this time, there was a rapid decline in serum levels of
RSHZ19 in mice inoculated i.v., whereas the level of RSHZ19
declined more slowly in mice inoculated i.p. These results r `~
are summarized in Table 5. .
--___--____~
Table 5
Comparison of Serum Levels of RSHZl9 (CHO)
After IV or IP Inoculation of Mice
`:
log~ ELTSA titre in mlç~ i~Qs~~
TV _ Tp _ --
Day.s B5_ELI~ LI~ RS ELISA ~12_~LI~
0.13.5 ~ 0.2 4.6 1 0.2 3.~ + 0.1 4.2 + 0.1 `:
1 3.1 + 0.2 4.2 + 0.04 3.3 ~ 0.04 4.3 i 0.1 .-
4 3.2 + 0.1 4.2 + 0.1 3.3 + 0.2 4.2 + 0~04
7 3.1 + 0.2 3.7 + 0.3 3.6 + 0.2 3.9 + 0.1 ;~
14 < 1.5 < 1.5 301 + 0.2 3.8 + 0.1
21 < 1.5 < 1.5 2.3 + 1.3 3~5 + 0.2 :
46 ND < 1.5 ND 3.3 + 0.1
To investigate if the rapid decline in RSHZl9 between ~.
days 7 and 14 in mice inoculated i.v. was due to an immune ,:
response to RSHZ19, the sera were tested for antibody to
RSHZ19 in an ELISA. Plates were coated with 50ng of RSHZ19,
40 followed by D21 mouse sera and HRP-anti mouse IgG. As seen ~.
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in Table 6, mice inoculated i.v. -eveloped antibody to
RSHZl9 at day 21, whereas mice i~.~culated i.p. had no
detectable antibody to RSHZl9. ~:~ese results suggest that
tolerance to RSHZl9 developed fo`~owing i.p., bu~ n~t i.v.
inoculation of mice with this an, body. Mice are inoculated
i.p. or i.v. with RSHZl9 producec from CH0 or myeloma cells
to further confirm these results.
;
-- ----_____________________ ~
Table 6
Antibody Response to RSHZl9 in Sera of Mice Inoculated
`
i.v. or i.p. with 50 ~g RSHZl9 (CH0) .
Mice log,0 ELISA
Tnoculated titre*
i.v. 2.5 + G.2 `~
~: :
- :
* Plates coated with 50ng RSHZl9 (CH0)
Yam~le 15 - Recoanition of Clin;-al Tsol ates
Preliminary experiments using biotin-labeled, RSHZl9
BOl, 2.~5 ~g~ml; 9/29~92 from Smi~hKline Beecham) and FITC-
,
30~ streptavidin (~igma) on RSV-infec~ed and uninfected calf
testes cells~showed that biotin-RSHZl9 at l/40 with FITC-
,
streptavidin at~l/80 gave specific fluorescence of RSV-
- infected cells~.
~- ~ Nine slides of nasopharyngeal aspirates from children
hospitalized with RSV infection ~-ere obtained from the WH0
Collaborating Centre for Reference and Research on Rapid
Laboratory Viral ~iagnosis, the Royal Victoria Infirmary,
~ -
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Newcastle-upon-Tyne, England. Each slide consisted of 3
replicate samples in separate chambers. One sample was
stained with Imagen~ RSV, (Novo Nordisk Diagnostics Ltd,
Cambridge CB4 4WS, UK) as instructed in the technical data.
Another sample was stained with a 1:40 dilution of
biotinylated RSHZ19 fox lh at room temperature, washed 3x ~-
with PBS, and incubated with FITC-Streptavidin for lh at
room temperature. The third sample was stained with FITC~
Streptavidin only. After washing 3x with PBS, the samples `
10 stained with FITC-Streptavidin were counterstained with ---
0.01% Evans blue for 5 min. washed and moun~ed in 80%
glycerol. ~SV-infected cells in the nasopharyngeal aspirate
samples stained using IMAGEN~ RSV showed discrete ;~
. -,;
fluorescent intracellular cytoplasmic inclusions typical of
infected cells stained with mAb to the N protein of RSV. In
contrast, nasopharyngeal aspirate cells stained with
biotinylated-RSHZ19 and FITC-Streptavidin showed more
generalized granular cytoplasmic staining, typical for the F~;
protein. There was no fluorescence of samples stained with
FITC-Streptavidin alone.
The results are illustrated in Table 7. Biotinylated `
RSHZ19 recognized RSV in all the nasopharyngeal aspirates
studied. The intensity of fluorescence in samples stained
with biotinylated RSHZ19 was less than in those stained with
IMAGEN~ RSV; however, the numbers of stained cells appeared
to be similar in both samples.
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______________________________________________________ .
Table 7
Binding of RSHZ19 to RSV in Nasopharyngeal Aspirates
Date __ Fluo~sc~n~ : _
Spec. Specimen Sub- ImagenTM FITC- Biot. RSHZ19
Rece1vedtype ~Y ~itrept. + FITC-Strel?t.
6513 02/02/88A +~++ - +~+
7430 15/03/88A +++ - ++
9997 16/07/85B ++++ - ++
7920 22/03/85B ++ - . +
81g5 20/11/91ND++++ - +++
8818 13/12/91ND~+++ - ++~
8845 14/12/91ND ++
9495 16/01/92ND +~+ - ++
9575 08/01/92ND ++~ - +~+
.
These studies indicate that RSHVl9 recognizes all
clinical isolates of RSV examined so far.
Exa~~ fi~ r;oph~1~gcl__ell~=_nf bovine m~b B4 on RSV~
;nfection in calves
Three 1 to 2 week old gnotobiotic calves, weighing 43
to 55 kg, were inoculated intratracheally (i.t.) with 15 mg
of purified bovine mAb, ~4, and three were inoculated i.t.
with PBS. Twenty-four hours later, all cal~es were -
challenged i.n. and i.t. with approximately 105 pfu of the
Snook strain of bovine RSV. The Snook strain of bo~ine RSV ~`
was isolated from the lung of a calf which died of pneumonia ;--
[Thomas et al., Br;t J~ Ex~. Pat~Ql.~ 65:19-28 (1984) ], and ~-
grown in secondary CK cells. Nasopharyngeal swabs were ~
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obtained daily after infection and calves were killed on day
7 of infection. Lung washings were obtained at post-mortem
by filling the lungs with 800 ml of PBS. Lung washings were ,~
centrifuged at 1300 g and the cell pellet resuspended in 5
ml of medium. All samples were assayed for RSV on secondary
C~ monolayers.
Treatment of calves with mAb B4 24 hours prior to
challenge with the bovine strain of RSV had no effect on
virus shedding from the nasopharynx throughout the 7 days of
10 infection. However, as reported in Table 8 below, little or :-~
no virus was recovered from the lungs of the calves treated
with B4, 7 days after RSV challenge. In contrast, between ~
103 and 109 pfu/ml was recovered from the lungs of the ~;
control calves. Calves given mAb B4 did not develop
pneumonic lesions, whereas the lungs of the control animals
were pneumonic.
Table 8
Prophylactic effects of bovine m~b B4 on
- 20RSV infection in calves
D7 Virus Titre
(log10 PFU~ml)
25 Treatment Calf No. Nose Lung Wash% Pneumonic ~`
Iesionq
B4 d-1 1097 2.4 <0.7 <1
1230 3.5 0.7 0 `
- 30 1242 3.6 <0.7 <1
None 1098 2.2 3.2 9 `
1231 <0.7 3.2 6
1245 2.1 4.2 6
,.
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F.~am~le 17 - Prophylactic Ef~ects of Bovlne mAbs on RSV
infe~tion in Calves
Calves were also treated i.t. with 15 mg B13 or 15 mg
Bl 24 hours prior to challen~e with bovine RSV (BRSV). MAb
B1 is an anti-F antibody that is non-neutralizing, non-
protective in mice but fixes complement (Kennedy et al,
(1988)). Although there was a reduction in the titre of
~irus in the lungs of calves given B13, the difference in
titre of virus compared with control calves given PBS was
not statistically significar.t (p = 0.07) (Table 8).
However, there was a statistically significant reduction in
the severity of pneumonic lesions in calves given B13 when
compared with controls. There were no slgnificant
differences in either the level of virus in the lungs or the
severity of pneumonia in calves given B1 when compared with
controls (Table 9).
These studies indicate that B4 is more protective
against BRSV infection in the calf than B13. Further, a
non-protective, complement fixing mAb, whilst not protective
in the calf, does not exacerbate pneumonic lesions.
'
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.. "`::.
______________________ ________________________________ ~,:
Table 9
Prophylactic Effects of Bovine mAbs on
RSV infection in Calves
~asal ~e~ina Luna ~irus
Mean Lung %
10 Treatmt No. Duration peak No. Wash Pneumonic
calves ~days) tit~ed Infec. titre~ leslQn~ _
B4 d-l 3 5.0 + 0 3.9 + 0.7 1 <o 7b <
B13 d-1 4 4.5 + 0.6 2.9 + 0.2 2 1.3~1.S' 2+2.6C ~
Bl d-l 4 9.8 + 0.5 3.2 + 0.7 4 2.6+1.8 5.5+2.4 ~;-
PBS d-l 9 4.4 + 1.2 3.0 + 0.5 9 3.1+1.5 10.5+7.0
a logIu PFU/ml
Probability that passively immunized animals are
significantly different from controls. p<0.01;
c p=0.07; t NS; p<0-05
,
F.xample 18 - Clon1na a~d Se~uencina Qf B4. B13 ~nd ~14
Cy~oplasmic RNA was prepared by the method of Favaloro
et al., eth. Fnzymol., h~:718-749 (1980) from B4, B13 and
30 B14 hybridoma cell lines. The primers `
BCGlFOR: 5'TTGAATTCAGACTTTCGGGGCTGTGGTGGAGG 3' ~SEQ ID NO:
29], which is based on se~uence complementary to the 5' end
of bovine ~-1 and y-2 constant region genes, and
BCLlFOR: 5'CCGAATTCGACCGAGGGTGGGGACTTGGGCTG 3' [SEQ ID NO:
30], which is complementary to the 5' end of the bovine
lambda constant region gene, were used in the synthesis of
Ig heavy (VH) and light (VL) chain variable region cDNAs, ;`
respectively.
cDNA synthesis reactions consisted of 20~g RNA, 0.4~M
BCGlFOR or BCLlFOR, 250~M each of dATP, dCTP, dGTP and dTTP,
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50mM Tris-HCl pH 7.5, 75mM KCl, 10mM DTT, 3mM MgCl2 and 27
units RNase inhibitor [Pharmacia, Milton Keynes, United
Kingdom] in a total volume of 50~1. Samples were heated at
70C for 10 minutes and slowly ccoled to 42C over a~period
S of 30 minutes. Then, 100~ MMLV reverse transcriptase [Life
Technologies, Paisley, United Kingdom~ was added and
incubation at 42C continued for 1 hour.
VH and VK cDNAs were then amplified using the
polymerase chain reaction (PCR) as described by Saiki et
al., Sc-ence, 2~:487~991 (1988). For the PCR, the primers -~
used were BCGlFOR, BCLlFOR,
~SEQ ID NO: 31] VHlBACK~
S'AGGT(S)(M)(R)CTGCAG(S)AGTC(W)GG 3'
[SEQ ID NO: 32] VL2BACK~
S'TTGACGCTCAGTCTGTGGTGAC(K)CAG(S)(M)GCCCTC 3'
VHlBACK is describe`d by Orlandi et al ., proC~ Nat'
C~ Sci.f U~, 86:3833-3937 ~(1989). The sequence of
VL2BACK was based on nucleotide sequences listed for the 5';~
end of human lambda variable regions [Kabat et al., (1987),
`` 20 ~cited above].
For~PCR~amplification of V~, DNA/primer mixtures
consisted~of S~1 RNA/cDNA hybrid and 0.5~M BCGlFOR and
V~lBACK primers. For PCR amplifications of VL, DNA/primer
mixtures consisted of 5~1 RNA/cDNA hybrid and 0.5~M BCLlFOR`~
.-~ 25~ and VL2:BAC~K primers. To these mixtures was added 250~M each
of dATP~ dCTP, dGTP and dTTP, lOmM Tris-HCl pH 8.3, 5OmM ~-
KC1, 1.~5mM~MgCl2, 0.01~ (w/v)~ gelatin, 0.01~ (v/~) Tween 20,
0.01% (v/v) Nonidet~P40~and 5 units AmpliTaq [Cetus].
` :Samples::were~subj:ected to 25 thermal cycles of PCR at 94C, "
30 seconds; 55C, 30 seconds; 72C, 45 seconds~; ending with -``
-r~ S minutes at~ 72C. For cloning and sequencing, amplified VH
~ DNA~was purified on a low melting point agarose gel and by ! '
., . ~ .
' ",
, ~ ~ `,''
:~ ~ ` : ,',,'
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Elutip-d column chromatography [Schleicher and Schuell-
Dussel, Germany] and cloned into phage M13 [Pharmacia-Milton ;
Keynes, United Kingdom]. The general cloning and ligation
methodology was as described in Maniatis et al., ci~ed
above.
VH DNA was cloned as PstI-EcoRI fragments into
similarly-digested M13mpl8/19 [Pharmacia-Milton Keynes, UK].
VL DN~ was cloned as SstI-EcoRI fragments into M13mpl8/19
digested with the same enzymes. Representative clones were
sequenced by the dideoxy method [Sanger et al., Proc. Nat ' 1.
~cad. ~ S~, 74:5463-5467 (1977)] using T7 DNA
polymerase [Pharmacia].
The amino acid sequences obtained by translation of the
variable region gene inserts were aligned with known VH and
VL sequences to allow identification of the CDRs.
The VL and VH amino acid sequences of B4 and the
apparently substantially identical B13 and B14 antibodies
are reported in Figs. 3A and 3B (VL), and 4A and 4B (VH3.
The B4 sequences are reported above the B13/Bl4 sequences to
demonstrate the homologies therebetween.
am~ 9 - ~himsxic B4 ~ntl~ody
To construct the B4 chimeric heavy chain expression
vector, the B4VH ~ene was amplified from an M13 clone
(Example 18) by PCR with oligonucleotides VHlBACK (described
in Example 18) and VHlFOR 15i TGAGGAGACGGTGACCGTGGTCCCT
TGGCCCCAG 3' [SEQ ID NO: 43] described by Orlandi et al,
Pr~ at'l,_Acad. Sc;. US~, ~fi:3833-3937 (1989)). The PCR
mixture consisted Qf 0.5 ~l M13 phage supernatant 0.5 uM
each of the above primers, 250 uM each of dATP, dCTP, dGTP
and dTTP, 10 mM KCl, 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4,
2 mM MgSO4, 0.1% Triton X-100 and l unit Vent DNA polymerase
(New England Biolabs) in a volume of 50 ul. Samples were
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subjected to 15 rounds of amplification at 94C, 30 seconds;
50C, 30 seconds; 75C, 1 minute; ending with 5 minutes at
75C. Amplified DNA was puxified on a low melting point
agarose gel and by Elutip-d column chromato~raphy
lSchleicher and Schuell-Dussel, Germany). The DNA was
cloned as PstI-BstEII fragments into similarly-digested
M13VHPCRl (Orlandi et la, 1989, cited above). The integrity -
of a chosen clone was confirmed by nucleotide sequencing.
The B4VH was cloned into an expression vector as
described in Example 11 except that the human IgGl constant
region was already present in the vector. The plasmid was ;
termed pSVgptB4BoVHHuIgG1. ;
To create B4 chimeric light chain expression vector,
the vector M13VKPCR1 (Orlandi et al, 1989, cited above) was
first modified to allow it to accept a lambda, rather than
kappa, chain variable region. This was achieved by mutating
the 5' end of the existing VK gene using the oligonucleotide
;~ ~ 5' TGGGCTCTGGGTTAACACGGACTGGGAGTGGACACC 3'tSEQ ID NO: 44]
and the 3 ' end using the oligonucleotide 5'
20 ATTCTACTCACGACCCATGGCCACCACCTTGGT 3' [SEQ ID NO: 45], `
introducing HpaI and NcoI restriction sites respectively.
The;~existing Nc~oI site in the vector was deleted using the
oligonucleotide 5' CTCCATCCCATGCTGAGGTCCTGTG 3' [SEQ ID NO:
4~].
M13VKPCRl was grown in E. coli RZ1032 (dut~ung~) to
give single-stranded template DNA containing uracil in plaee
of thymine. 0.5 ug DNA was mixed with 1 pmol each of the
three phosphorylated oligonucleotides above and 1 pmol of an
oligonucleotide VKPCRFOR (5' GCGGGCCTCTTCGCTATTACGC 3') [SEQ -~
ID NO: 47] which anneals to the M13 template downstream of
the insert DNA. The oligonucleotides were annealed to the
template in 20 ul of 50 mM Tris-HCl pH 7.5, 25 mM MgCl2, 63
- , ,'`-
.,
: ~:
~- ,"
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mM NaCl by heating to 80C for S minutes and cooling slowly
to room temperature. dATP, dCTP, dGTP and dTTP were added
to a 250 ~M final concentration, DTT to 7 mM, ATP to 1 mM
with 0.5 unit T7 DNA polymerase (USB) and 0.5 unit T4 DNA
ligase (BRL) in the same buffex. The 30 ~l reaction was
incubated at room temperature for one hour and the DNA
ethanol precipitated.
In order to nick the parental strand the DNA was
dissolved in 50 ~l of 60 mM Tris.HCl, pH 8.0, 1 mM EDTA, 1
mM DTT, 0.1 mg/ml BSA containing 1 unit uracil DNA
glycosylase and incubated at 37C for one hour before NaOH
was added to 0.2 M and incubation continued at room
temperature for 5 minutes. The DNA was ethanol
precipitated, dissolved in 20 ~l TE and the insert fragment
amplified by PCR. The reaction mixture contained 2 ~il
mutant DNA, 0.5 ~M each VKPCRFOR and VKPCRBACK (5' ~-
CTGTCTCAGGGCCAGGCGGTGA 3') [SEQ ID NO: 48], 250 ~M each of
dATP, dCTP, dGTP and dTTP, 10 mM Tris.HCl pH 8.3, 50 mM KCl,
1.5 mM MgCl2, 0.01% Tween-20, 0.01% gelatin, 0.01~ NP40 and
20 2 units Thermalase ~IBI) in 50 ul. Amplifica~ion wa~ -
achieved with 15 cycles of 94C, 30 seconds; 50C, 30
seconds; 72C, 1 minute; ending with 72C, 5 minutes.
The product DNA was cloned into M13mpl9 as a HindIII-
-BamHI fragment. Representative clones were sequenced and a
clone mutant in all three areas was chosen and named
M13VLPCR1.
HpaI and NcoI restriction sites were introduced at the
ends of the B4VL by amplifying the DNA from an Ml3 clone
~Example 18) using oligonucleotides VL3BACK (5'
TCTGTGTTAACGCAGGCGCCCTCCGTG 3') [SEQ ID NO: 49] and VLlFOR
(5' GGCTGACCCATGGCGATCAGTGTGGTC 3') [SEQ ID NO: 50~ and Vent
DNA polymerase as described above for the B4VH above. The
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product ~NA was purified, digested with HpaI and NcoI and
cloned into similarly-digested Mi3VLPCRl RF DNA. Clones ~
containing the B9VL were identified by sequencing and the -
HindIII-BamHI insert of one such clone used to construct an ;~
expression vector, pSVhygB4BoVLHuVK, as described in Example
The expression vectors were co-transfected into YB?/0
myeloma cells, transfectomas secreting antibody identified
and a chimeric antibody B4BoVH/BoVL purified as described in
Example 11. The chimeric antibody was compared to the B4
- bovine antibody for binding to RSV-infected cell lysate in
an ELISA. The method was essentially as described in
Example 11 except that RSV-infected and uninfected Hep2 cell
lysates were used. The reporter antibodies were goat anti- -
15 human IgG antibodies, HRPO-conjugated (Sera-Lab Ltd, Crawley -~
Down, UK) and rabbit anti-bovine IgG antibodies, HRPO-
conjugated ~Sigma, Poole, UK), used as 1 in 1000 dilutions. ;
The bovlne and chimeric (BoVH/BoVL) B4 antibodies bound
to the infected cell lysate whereas an irrelevant humanized
20 ~antibody~did not. None of the antibodies reacted against ~;
the control lysate. Tt is not possible to draw a direct `~;
comparison between the bovine and chimaeric antibodies from -;~
this experiment as different reporter antibodies were used.'`r`-''
- In a separate experiment comparing the conjugates,`~
25~ about 2.5 fo~ld more bo~ine antibody than human antibody was~;:
required to obtain the same ~D reading. Thus the bovine and
chimeric~ antibodies are approximately equivalent in binding.
F.~Tnpl e ~0~ qulnan 1 7ed: R4 . "
A~ ~YYJ
The B4VH~was humanized by transferring the bovine CDRs ;~
' onto human ~EWM VH frameworks (Saul et al, 1978, cited
above) using site-directed mutagenesis. The following
.
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bovine framework resldues (numbering 2S Kabat et al, (1987),
cited above) were incorporated into the humanized VH
alongside the CDRs (see Figure 10).
Phe27, Ser28, ~eu29 - while not being part of~the
5 hypervariable region, these residues are part of the .
structural loop of CDR1 (Chothia and Lesk, ~. Mo?. Biol~,
196:901-gl7 (1987)).
Leu48 - adjacent to CDR2, thls residue has affected the ~:
binding of other reshaped antibodies.
Arg71 - this resiàue has been shown to be important in
other reshaped examples and is involved in the packing of ~.
CDRs 1 and 2 (Tramontano et al., J. Mol. B;~L~ 21~:175-182
( 1 9 90 ) ~ .
Lys94 - the amino acid at this posi~ion can affect the
15 conformation of CDR3 by formation of a salt bridge (Chothia ::-
and Lesk (1987), cited above).
The template DNA was M13mpl9-based and contained a VH
gene comprising NEWM frameworks and irrelevant CDRs, similar
to that described by Riechmann et al., Na~u~e, 332:323-327
20 ~1988). The mutagenesis was carried out as described above :
for the construction of M13VLPCR1. The oligonucleotides
employed were: ~.
VHCDR1: 5' CTGTCTCACCCAGCTTACAGAATAGCTGCTCAATGAGA~G
CCAGACAC 3' [SEQ ID NO: 51]
~- 25 VHCDR2: 5' CATTGTCACTCTGG~TTTCAGGGCTGGGTTATAATATATGATT
. CCGCCATTGCTTGCGTCTCCAAGCCACTCAAGACC 3' [SEQ ID NO: 52]
VHCDR3: 5' CAAGGACCCTTGGCCCCAGGCGTCGACATACTCGCCCTTGC
GTCCAGTACAAGCATAACTTCCACTATCACCAACAGAACACTTTGCACAATA
ATAGACCGC 3' [SEQ ID NO: 53~
and the universal M13/pUC-20 primer, 5' GTAAAACGACGGCCAGT 3'
~SEQ ID NO: 54]. ;
SUBSTlT lTE SHEE~
WO93/20210 ~ b ~ ~ PCT/GB93/Q~72~
g4 ;~--
DNA encoding a VH containing all three B4 CDRs was ~;
subsequently excised from the Ml3 and cloned into the
expression vector described for the chimeric VH in Examples
ll and l9 and resulting in pSVgptBqHuVHHuIgGl.
pSVgptB4HuVHHuIgGl was co-transfected with the chimeric
light chain vector, pSVhygB4BoVLHuVK as described in Example
ll. The resulting partially humani~ed antibody B4HuVH/BoVL
therefore contains a humanized B4 heavy chain (B4HuVH) with ;~
a B4 light chain chimeric B4BoVLHuVK. Cells secreting
B4HuVH/BoVL antibody were expanded and antibody purified
from 400ml conditioned medium. ;~
The B4HuVH/BoVL antibody was compared to the chimeric ~-
antibody B4BoVH/BoVL in binding to RSV s~rain A2-infected
cell lysate in an ELISA. This allowed assessment of the i-
relative binding abilities of the chimeric and humanized
heavy chains.
The humanized heavy chain HuVH binds to RSV-infected
cell lysate, but is 2-3 fold deficient in binding rela~ive
to the chimeric heavy chain BoVH. `~
Additional murine residues were included to attempt to
increase binding. The HuVH gene was mutated to encode the !!~`
following changes: T at position 73, N at position 76 and F
at position 78 to NSV. These residues are part of a ~-turn `,~
which forms a fourth loop at the antigen binding surface.
A HuVHNSV/BoVL antibody was produced and tested for
binding to a lysate of cells infected with the Snook strain ~`
of RSV by ELISA. Inclusion of the RSV residues gave no
advantage over the original HuVH. `
One other difference between ~he BoVH and HuVH which
might affect binding is the region spanning amino acids #67-
70~ It is 'anticipated that the inclusion of the bovine B4
residues L at position 67 and I at position 69 are more
SUBSTITUTE SHEET
WO93/202l0 PCT/GB93/00725
likely to influence the antigen interaction as their side
chains pack inside the domain. Additionally the block -
change to L at position 67, G at position 68, I at posltion
69 and T at position 70 is also anticipated to be
advantageous.
B. B4 ~umanized Tiaht ~h~in
A humanized version of the B4 light chain B4HuVL was
constructed by site-directed mutagenesis of the bovine B4VL
frameworks to give frameworks of the human KOL lambda
variable region (see Figure ll). Cells were selected for
the presence of the gpt gene which is found on the heavy
chain expression vector.
Northern blotting was used ~o determine lf the HuVL RNA
was of the correct size. Total RNA was prepared from
BoVH/HuVL and BoVH/HuVL FR4 transfectomas and from BoVH/BoVL
transfectomas and untransfected YB2/0 as positive and
negative controls. Initial results using BoVL and HuVL
probes show bands of approximately the same size for all
three species of light chain. In a similar investigation
cDNA was prepared from each cell line and PCRs carried out
using a constant region primer and VL3BACK. Again the same
sized product was obtained for all three species of light
chain, indicating no major splicing problem.
Two more humanized light chain constructs - a human R~I
kappa framework-based version of the light chain and a CDR-
grafted light chain with frameworks of the human KIM46L
lambda chain, may be made using the actual nucleotide
sequence of the KIM46L VL gene (Cairns et al, J. Immunol.,
143:685-691 (1989)).
This is believed to be the first example of a bovine
antibody being humanized. The lack of bovine variable
region sequences in the databases meant that it was
SU BSTTTUTE SH EFT
W093/20210 r;J ~ PCT/GB93/00725
96
difficult to design primers for PCR amplification and thus ;~
to isolate DNA for the initial cloning and sequencing.
Ex~m~le 21 - Effect of RSHZl9 and RSBV04 adml~lstered
~ ed mis~
Groups of five mice were inoculated intranasally with
approximately lOs PFU of the A2 strain of RSV and were
treated on day 4 of infection with different amounts of ~:
RSBV04 administered intraperitoneally either alone or with -~
0.5 mg/kg RSHZl9, as shown in Table lO below. Mice were ;
lO killed five days after RSV challenge, and the level of virus '~
in the lungs determined on CK cells. The results are shown ;`~
in Table lO and indicated that the effect of combined
therapy with RSHZl9 and RSBV04 is additive rather than
synergistic.
1 5 ~ ~ ~~_ ~_ __ _ __ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ __ _ r~
TABLE lO
~- Dose (mg/kg)1 RSV titer in lungs ~-
,rouD R~Hzl9 R~RV04 loglO pFU/a _ ~-
A 0.5 -- 3.6 + 0.7
B 0.5 0.5 2.2 + 0.6
C 0.5 0.25 2.3 + 0.8 `-
- ~ D 0.5 0.125 2.6 + 0.9
E -- 1.0 2.l + 0.8
F --~ 0.75 2.3 + 0.6
G -- 0.625 2.6 + 0.7 -~
H -- -- 4.8 + O.l ~-
- ------------___________ ~;
~mAbs administered IP on day 4 of infection.
.'~
SUBSTlTUtTE SHEET ~;
WO93/20210 ~ J ~ ~ 2 PCT/CB93/00725
97
Numerous modifications and variations of the present
invention are included in the above-identified specification
and are expected to be obvious to one of skill in the art.
Such modification and alterations are believed to be
encompassed in the scope of the claims appended hereto.
SEQUENCE LISTING
10 (1) GENERAL INFORMATION: :
li~ APPLICANT: Taylor, Geraldine
Stott, Edward J.
(ii) TITLE OF INVENTION: Novel Antibodies for Treatment
and Prevention of Respiratory
Syncytial Virus Infection in Animals and
Man
(iii) NUMBER OF SEQUENCES: 53 ` ~
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRES~SEE: SmithKline Beecham Corporation
: Corporate Patents
(B) ST~EET: 709 Swedeland Road .-
~C) CITY: King of Prussia
(D) STATE: PA
~E) COUNTRY: USA ~
(F) ZIP: 19406 2799 .
30: ~
v) COMPUTER ~EADABLE FORM:
: ~A) MEDIUM TYPE: Floppy disk
~B) COMPUTER: IBM PC compatible
(C3 OPERATING SYSTEM: PC-DOStMS-DOS
~: 35 ~D) SOFTWARE: PatentIn Release #l.0, Version #1.25
.~ ~vi~ CURRENT APPLICATION DATA:
- (A) APPLICATION NUMBER: WO
(B) FILING DATE:
~ (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
A) APPLICATION NUMBER: GB 9207479.8
(B) FILING DATE: 06~APR-l992
SU8STITUTE SHEET
WO 93/20210 ~ r~ PCT/GB93/00725
98
(viii) ATTORNEY/AGENT INFORMATION~
(A) NAME: Jervis, Herbert H. ~-`
(B) REGISTRATION NUMBF.R: 31,171 ~
(C) REFERENCE/DOCKET NUMBER: P50153
S
(ix) TELECOMMUNICATION INFORMATION:
~A) TELEPHONE: 215-270-5019 ~
(B) TELFFAX: 215-270-5090 .`.
10 (2) INFORMATION FOR SEQ ID NO:1: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 amino acids `~
(B) TYPE: amino acid
15~D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUEMCE DESCRIPTION: SEQ ID NO~
Ser Val Val Thr Gln Glu Pro Ser Val Ser Gly Ser Leu Gly Gln
1 5 10 15 ~
Arg ~al Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Arg ~;
Trp Gly Val Asn Trp Tyr Gln Gln Val Pro Gly Ser Gly Leu Arg
35 40 45 :~
Thr Ile Ile Tyr Tyr Glu Ser Ser Arg Pro Ser Gly Val Pro Asp .
Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Thr ~eu Thr Ile .. ; 3065 7~ 75 -".'
Ser Ser Leu Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Thr ``
80 85 90
: Gly Asp Tyr Asn Ile Ala Val Phe Gly Ser Gly Thr Thr Leu Ile
100 105 `
35 Val Met Gly Gln Pro Lys Ser ~.
110
. -
~2) INFORMATION FOR :SEQ ID NO:2:
: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 116 amino acids :
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
! . ' (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ~ :
SUBSTmJTE SHEET
W093/20210 ~ . . PCT/GB93/00725
99
Ser Val Val Thr Gln Gln Pro Ser Val Ser Gly Ser Leu Gly Gln
1 5 10 15
Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Asp Asn Ile Gly Ile
Phe Ala Val Gly Trp Tyr Gln Gln Val Pro Gly Ser Gly Leu Arg
Thr Ile Ile Tyr Gly Asn Thr Lys Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile
65 70 75
Asn Ser Leu Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Val Cy5
Gly Glu Ser Lys Ser Ala Thr Pro Val Phe Gly Gly Gly Thr Thr
95 100 105
15 Leu Thr Val Leu Ser Gln Pro Lys Ser Pro Pro
ll~ 115
~2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 137 amino acids
(B) TYPE: amino acid
~D) TOPOLOGY: unknown :
(ii) MOLECULE TYPE: protein
(xi) S~QUENCE DESCRIPTION: SEQ ID NO:3: ~.
: 30 Gln Val Xaa Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser
1 S 10 15
Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser
Ser Tyr Ser Val Ser Trp Val Arg Gln Ala Pro Gly Lys Thr Leu
3535 40 45
Glu Trp Leu Gly Asp Ala Ser Asn Gly Gly Ile Ile Tyr Tyr Asn
50 55 60
. ~ro Ala Leu Lys Ser Arg Leu Gly Ile Thr Arg Asp Asn Ser Lys
65 . 70 75
Ser Gln Val Ser Leu Ser Leu Asn Thr Ile Thr Pro Glu Asp Thr
85 85 90
Ala Thr Tyr Tyr Cys Ala Lys Cys Ser Val Gly Asp Ser Gly Ser
95 100 10S
Tyr Ala Cys Thr Gly Arg Lys Gly Glu Tyr Val Asp Ala Trp Gly
451 110 llS ~ 120
Gln Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro
125 130 13S
SU8S~UTE SHEET
WO93/20210 `` ~ b ~ 2 PCT/GB93/00725
100
Lys Val
(2) INFORMATION FOR SEQ ID NO:4~ `
5(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 141 amino acids
. 5B) TYPE: amino acid
(D) TOPOLOGY: unknown
10(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: -
Gln Val Xaa Leu Gln Gln Ser Gly Pro Ser Leu Val Lys Pro Ser :~
151 5 10 15
Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Ser :
20 25 30
Asp His Asn Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Ala Leu :
35 40 45 `~.
Glu Trp Leu Gly Val Ile Tyr Lys Glu Gly Asp Lys Asp 'ryr Asn
50 55 60
Pro Ala Leu Lys Ser Arg Leu Ser Ile Thr Lys Asp Asn S.er Lys .-~
65 70 75 ~;:
Ser Gln Val Ser Leu Ser Leu Ser Ser Val Thr Thr Glu Asp Thr
2580 85 90 :~
Ala Thr Tyr Tyr Cys Ala Thr Leu Gly Cys Tyr Phe Val Glu Gly -~
95 100 105
Val Gly Tyr Asp Cys Thr Tyr Gly Leu Gln His Thr Thr Phe Xaa ~.
11~ 115 120 `~
30 Asp Ala Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser Ala Ser :
125 130 135
Thr Thr Ala Pro Lys Val -
140
(2) INFORMATION FOR SEQ ID NO-5~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 129 amino acids
4 0 (B ) TYPE: amino acid
(D ) TOPOLOGY: unknown :
(ii) MOLECULE TYPE: protein
! (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser
1 5 10 15
.
SUBSTITUTE SH EET
;~ 1 r~b ~
- WO93~20210 PCT/GB93/00725
.
101
Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser
Ser Tyr Ser Val Ser Trp Val Arg Gln Pro Pro Gly Arg 51y Leu
5 Glu Trp Leu Gly Asp Ala Ser Asn Gly Gly Ile Ile Tyr~Tyr Asn
Pro Ala Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys
Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr
- 80 85 90
Ala Val Tyr Tyr Cys Ala Lys Cys Ser Val Gly Asp Ser Gly Ser
100 105
Tyr Ala Cys Thr Gly Arg Lys Gly Glu Tyr Val Asp Ala Trp Gly
110 115 120
15 Gln Gly Thr Thr Val Thr Val Ser Ser
125
:'
(2~ INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109 amino acids
(B) TYPE: amino acid ~.
(D) TOPOLOGY: unknown
(ii~ MOLECULE TYPE: protein
. (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15 `.
Gly Asp Arg Val Thr Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile
20 25 30
Gly Arg Trp Gly Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
35 40 45
Pro Lys Leu Leu Ile Tyr Tyr Glu Ser Ser Arg Pro Ser Gly Val
~ 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe
65 70 ~ 75 ~
Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys
80 85 90
Ala Thr Gly Asp Tyr Asn Ile Ala Val Phe Gly Gln Gly Thr Lys
45 ~ ' 95 100 105
Leu Glu Ile Lys
SUBSTmJTE SHEET ~:
`~ ~J~
WO93~20210 PCT/GB93/00725
102
~2) INFORMATION FOR S~Q ID NO:7: /'.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 133 amino acids
(B) TYPE: amino acid .:~
~D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein `
1 0
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: `
Gln Val GLn Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser ~::
1 5 10 15 '~
Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Ser
Asp His Asn Val Gly Trp Val Arg Gln Pro Pro Gly Arg Gly Leu
Glu Trp Leu Gly Val Ile Tyr Lys Glu Gly Asp Lys Asp Tyr Asn
Pro Ala Leu Lys Ser Arg Val Thr Met Leu Lys Asp Thr Ser Lys -
65 70 75 ~:
Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr
80 85 90
Ala Val Tyr Tyr Cys Ala Thr Leu Gly Cys Tyr Phe Val Glu Gly
~5 100 105
Val Gly Tyr Asp Cys Thr Tyr Gly Leu Gln His Thr Thr Phe Xaa
Asp Ala Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 120
1~5 130 ~ .
(2) INFORMATION FOR SEQ ID NO:B: ;~
(i) SEQUENCE CHARACTERISTICS~
~A) LENGTH: 111 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein ~-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ::
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thx Ile Thr Cys Ser Gly Ser Sex Asp Asn IIe
Gly Ile Phe Ala Val Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala -
35 40 45
SU8STITUTE SHEET
W093/202l0 ` ~ PCT/GB93/00725
103
Pro Lys Leu Leu Ile Tyr Gly Asn Thr Lys Arg Pro Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys
8S ~ 90
Val Cys Gly Glu Ser Lys Ser Ala Thr Pro Val Phe Gly Gln Gly
100 105
Thr Lys Leu Glu Ile Lys
110
(2) INFORMATION FOR SEQ ID NO:9: -
lS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 348 base pairs
lB~ TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA ~genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CAGGTCCAGC TGCAGSAGTC WGGGACAGAG CTTGAGAGGT CAGGGGCCTC 50
AGTCAAGTTG TCCTG QCAG CTTCTGGCTT CAACATTAAA GACTACTATA 100 ,~-:
TGCACTGGAT~GAAGCAGAGG CCTGACCAGG GCCTGGAGTG GATTGGATGG 150
30 :
ATTGXTCCTG AGA~TGATGA TGTTCAATAT GCCCCGAAGT TCCAGGGCAA 200
~ .:
GGCCACTATG ACTGCAGACA CGTCCTCCAA CACAGCCTAC CTGCAGCTCA 250
CCAGCCTGAC ATTTGAGGAC ACTGCCGTCT ATTTCTGTAA TTCATGGGGG 300
AGTGAC$TTG ACCACTGGGG CCAAGGGACC ACGGTCACCG TCTCCTCA 348 :~:
:
,
~; 40 (2) INFORWATION FOR SEQ~ID NO:10:
(i) SEQUENCE ~HARACTERISTICS: ::
~- lA) LENGTH: 116 amino acids
(B) TYPE: amino acid
; (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
!
: ,
~.,
SUBSTI~UTE SH EET
.:`.
WO93/20210 , ~ b ~ PCT/GB93tO0725
104
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "amino acid at .
5position 6 can be either glu OL gln" ~-
(xi) SEQUENCE D~SCRIPTION: SEQ ID NO:l0: -:
Gln Val Gln Leu Gln Xaa Ser Gly Thr Glu Leu Glu Arg Ser Gly
l0l 5 l0 l5
Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys
20 25 30
Asp Tyr Tyr Met His Trp Met Lys Gln Arg Pro Asp Gln Gly Leu ~:
3~ 40 45
Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Asp Asp Val Gln Tyr
50 55 60
Ala Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser
65 70 75 -:
Ser Asn Thr Ala Tyr Leu Gln Leu Thr Ser Leu Thr Phe Glu Asp
20 80 85 90
Thr Ala Val Tyr Phe Cys Asn Ser Trp Gly Ser Asp Phe Asp His :~
95 l00 . 105
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser :
110 115
(2~ INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS: :~
(A) LENGTH: 337 base pairs ~-
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double .
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
-
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..333
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: .
GAC ATT CAG CTG ACC CAG TCT CCA CTC TCC CTG CCT GTC ACT 42
Asp Ile Gln Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
CTT GGA GAT CA~ GCC TCC ATC TCT TGC AGA TCT AGT CAG ACC 84
Leu Gly Asp Gln Ala Ser Ile Ser Cys ~rg Ser Ser Gln Thr
SU8STITUTE SHE~T
` WO93/20210 PCT/GR93/00725
6 6 2
!
105
CTT GTA CAT ACT GAT GGA AAC ACC TAT TTA GAA TGG TTT CTG 126
Leu Val His Thr Asp Gly Asn Thr Tyr Leu Glu Trp Phe Leu
CAG AAA CCA GGC CAG TCT CCA AAG CTC CTG ATC TAC AGA GTT 168
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Ar~ Val
TCC AAC CGA TTT TCT GGG GTC CCA GAC AGG TTC AGT GGC AGT 210 ;
Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
GGA TCA GGG ACA GAT TTC ACA CTC AAG ATC AGC AGA GTG GAG 252
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu
GCT GAG GAT CTG GGA GTT TAT TTC TGC TTT CAA GGT TCA CAT 294
Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln Gly Ser His
CTT CCT CGG ACG TTC GGT GGA GGG ACC AAG CTG GAG ATC TAAC 337
Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile ~
100 105 110 `.
.:
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS~
30 ~ :(A) LENGTH: 111 amino acids
(B)~ TYPE: amino acid
(D) TOPOLOGY: linear .`
- ~ii) MOLECULE TYPE: protein
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: `
:Asp Ile Gln Leu Thr~Gln Ser Pro Leu Ser Leu Pro Val Thr Leu
5 ~ 10 lS -:
40 :Gly Asp Gln Ala~Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu Val ..
`.
. .
, .
...
':
SUBSTITUTE SH EET
. - .
-~ :
i3 h ~. i
WO93/20210 PCT/GB93/0072
106 -~
His Thr Asp Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe~.
50 5~ 60 ~
5 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp . .
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val
Tyr Phe Cys Phe Gln Gly Ser Hls Leu Pro Arg Thr Phe Gly ~.ly
10 95 100 105
Gly Thr Lys Leu Glu Ile
110 ' '
~) INFORMATION FOR SEQ ID NO:13:
- :
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 116 amino acids
~B) TYPE: amino acid
~D) TOPOLOGY: unknown
~ii) MOLECULE TYPE: protein
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
25 Gln Val Gln Leu Gln Glu Ser Gly Thr Glu Leu Glu Arg Ser Gly .
1 5 10 15
- Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys
20 25 30 .:
Asp Tyr Tyr Met His Trp Met Lys Gln Arg Pro Asp Gln Gly Leu
35 40 45
Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Asp Asp Val Gln Tyr
50 55 60
Ala Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala ~sp Thr Ser
3565 70 75
Ser Asn Thr Ala Tyr L~u Gln Leu Thr Ser Leu Thr Phe Glu Asp
80 85 90
Thr Ala Val Tyr Phe Cys Asn Ser Trp Gly Ser Asp Phe Asp His
95 100 105 :-
40 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser :
110 llS
45 (l2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 116 amino acids
SUBSTITIJTE SHEET
; WO93/20210 ~ 2 PCT/GB93/00725
107 -
(B) TYPE: amino acld `-
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14~
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser
1 5 10 15
Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Ser
Asp Tyr Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu
Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Asp Asp Val Gln Tyr
1550 55 60 ~.
Ala Pro Lys Phe Gln Gly Arg Val Thr Asn Leu Val Asp Thr Ser
65 70 75 :~
Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp ... `
20~ 85 90
Thr ~la Val Tyr Tyr Cys Ala Arg Trp Gly Ser Asp Phe Asp His :.
95 10Q 105 ~`-
25 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser -`.
110 115 `.
(2) INFORMATION FOR SEQ ID NO:15:
(i) SE~UENCE CHARACTERISTICS~
(A) LENGTH: 116 amino.acids ~.
(B) TYPE: amino acid ,~
~D) TOPOLOGY: unknown .. `:
(ii) MOLECULE TYPE: protein ~:-
-~ (xi) SEQUENCE DES~CRIPTION: SEQ ID NO:15:
40 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser :
1 5 10 15 ~.
Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Ser E
20 25 30
Asp Tyr Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu .
45, 35 40 1 45 :
' Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Asp Asp Val Gln Tyr :-
50 55 60 :~
Ala Pro Lys Phe Gln Gly Arg Val Thr Met Leu Val As~ Thr Ser .`:
. -
.
SUBSTITIJTE Stl EET ~ ~
, .
WO93/20210 PCT~GB93/0072h i ~ U~ ~
108
Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Phe Cys Asn Ser Trp Gly Ser Asp Phe Asp His
95 100 105
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
110 115
(2) INFORMATION FOR SEQ ID NO:16:
'
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 116 amino acids
(B) TYPE: amino acid
(D~ TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser
1 5 10 15
Gln Thr Leu Ser Leu Thr C~s Thr Val Ser Gly Phe Asn Ile Lys
20 25 ~ 30
Asp Tyr Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu
2535 40 45
Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Asp Asp Val Gln Tyr
50 55 60
Ala Pro Lys Phe Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser
: 65 70 75
Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Phe Cys Asn Ser Trp Gly Ser Asp Phe Asp His
95 100 105
~ Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
:- 35 110 115
~2~ INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 112 amino acids
. (B) TYPE: amino acid
(D) TOPOLOGY: unknown
~ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
SUBSTITUTE SI~E~T
- WO93/20210 ~ 6 2 PCT/GB93/0072S
1 0 9 .
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Thr Leu Val
520 25 30 ~:~
His Thr Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln~Lys Pro
35 40 ~5 ;~
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe
50 55 60 ,-~
10 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp ~.:
65 70 75 .-
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr .
80 85 90 .~.
Tyr Tyr Cys Phe Gln Gly Ser His Leu Pro Arg Thr Phe Gly Gln :
15 95 100 105
Gly Thr Lys Val Glu Ile Lys
110 - ~
,'.~,'
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACT~RISTICS:
(A) LENGTH: 1899 base pairs
(B) TYPE: nucleic acid
(C~ STRANDEDNESS: double ~
(D) TOPOL~GY: unknown
(ii) MOLECULE TYPE: DNA (genomie)
~ix) F~ATURE:
(A) NAME/KEY: CDS :-
(B) LOCATIO~: 14.. 1735 -~
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
;-~
GGGGCAAATA ACA ATG GAG TTG CTA ATC CTC AAA GCA AAT GCA ATT46 ;.
:: Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile
1 5 10 ~`
ACC ACA ATC CTC ACT GCA GTC ACA TTT TGT TTT GCT TCT GGT 88
Thr Thr Ile Leu Thr Ala Val Thr Phe Cys Phe Ala Ser Gly
15 -20 25 ~:
CAA AAC ATC ACT GAA GAA TTT TAT CAA TCA ACA TGC AGT GCA 130 ~:.
Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala .
30 35 ~
45 GTT AGC AAA GGC TAT CTT AGT GCT CTG AGA ACT GGT TGG TAT 172 ~.
! ` Val Ser Lys! Gly Tyr Leu Ser Ala Leu Ar~ Thr Gly Trp Tyr
40 45 ~.
~,'.
,;
SUBSrITUTE SHEET ~ "
W 0 93/Z0210 ~ ~ PC-r/G D93/0072
110
ACC AGT GTT ATA ACT ATA GAA TTA AGT AAT ATC AAG GAA AAT 214
Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn
AAG TGT AAT GGA ACA GAT GCT AAG GTA AAA TTG ATA AAA CAA 256
Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys Gln
GAA TTA GAT AAA TAT A~A AAT GCT GTA ACA GAA TTG CAG TTG 298 :.
Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Lèu
9G 95
CTC ATG CAA AGC ACA CCA CCA ACA AAC AAT CGA GCC AGA AGA 340
Leu Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg
lS100 1~5
GAA CTA CCA AGG TTT ATG AAT TAT ACA CTC AAC AAT GCC AAA 382
Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys
110 115 120
AAA ACC AAT GTA ACA TTA AGC AAG AAA AGG AAA AGA AGA TTT 424
Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg ~he
125 130 135
25 CTT GGT TTT TTG TTA GGT GTT GGA TCT GCA ATC GCC AGT t;GC 4 66
Leu Gly Phe Leu Leu Gly Val Gly Ser Ala I le Ala Ser Gly
140 145 lS0
GTT GCT GTA TCT AAG GTC CTG CAC CTA GAA GGG GAA GTG AAC 508
30Val Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn
- 15~ 160 165
A~G ATC AAA AGT GCT CTA CTA TCC ACA AAC AAG GCT GTA GTC 550
Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val
35170 175
AGC TTA TCA AAT GGA GTT AGT GTC TTA ACC AGC AAA GTG TTA 592
Ser Leu Ser Asn Gly Val Ser Yal Leu Thr Ser Lys Val Leu
180 185 190
GAC CTC AA~ AAC TAT ATA GAT AAA CAA TTG TTA CCT ATT GTG 634
Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu I,eu Pro Ile Val
195 200 205
AAC AAG CAA AGC TGC AGC ATA TCA AAT ATA GAA ACT GTG ATA 676
Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile ,`
210 215 220
GAG TTC CAA CAA AAG AAC AAC AGA CTA CTA GAG ATT ACC AGG 718
Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg
225 230 235
SUBSTITUTE SI~EET
W093~20210 h ~ b `~ PCT/GB93/0072~ :
111 ~
GAA TTT AGT GTT AAT GCA GGT GTA ACT ACA CCT GTA AGC ACT 760
Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr
2~0 245
.~:~
TAC ATG TTA ACT AAT AGT GAA TTA TTG TCA TTA ATC AAT~GAT 802 :
Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp ~
250 2~5 260 ;~;
ATG CCT ATA ACA AAT GAT CAG AA~ AAG TTA ATG TCC A~C AAT 844
Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn -.
265 ~70 275 ~;
GTT CAA ATA GTT A5A CAG CAA AGT TAC TCT ATC ATG TCC ATA 886 .-
15 Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile -;
280 285 290
ATA AAA GAG GAA GTC TTA GCA TAT GTA GTA CAA TTA CCA CTA 928
Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu :~
295 300 305
TAT GGT GTT ATA GAT ACA CCC TGT TGG AAA CTA CAC ACA TCC 970 ~-
Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser ~
310 315 -
.-
CCT CTA TGT ACA ACC AAC ACA AAA GAA GGG TCC AAC ATC TGT 1012 `~
Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys
320 32S 330 .
30 TTA ACA AGA ACT GAC AGA GGA TGG TAC TG. GAC A~T GCA GGA lOS4 ;~
Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly
335 340 :
TCA GTA TCT TTC TTC CCA CAA GCT GAA ACA TGT AAA GTT CAA 1096 ~-.
35 Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys ~ys Val Gln ;-
350 355 360
TCA AAT CGA GTA TTT TGT GAC ACA ATG AAC AGT TTA ACA TTA 1038
Ser Asn ~rg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu `~
365 370 375
CCA AGT GAA ATA A~T CTC TGC AAT GTT GAC ATA TTC AAC CCC 1180
Pro Ser Glu Ile Asn Leu Cys Asn Val Asp Ile Phe Asn Pro
380 385
:
AAA TAT GAT TGT AAA ATT ATG ACT TCA AAA ACA GAT GTA AGC 1222
Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser
390 395 400
AGC TCC GTT ATC ACA TCT CTA GGA GCC ATT GTG TCA TGC TAT 1264 :.
S0 Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr ~:`
405 410 415 ~
SUBSTITUTE SHEEl' ~ .
WO 93/20210 PCr/GB93/007i~
112
GGC AAA ACT AL~ TGT ACA GCA ~ CC AAT AAA AAT CGT GGA ATC 1 3 0 6 ::
Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile
420 425 430
S ATA AAG ACA TTT TCT AAC GGG TGC GAT TAT GTA TCA AAT AAA 1348
Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser As~ Lys
435 440 445
GGG ATG GAC ACT GTG TCT GTA GGT AAC ACA TTA TAT TAT GTA 1390
10 Gly Met Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val
450 455
AAT AAG CAA GAA GGT AAA AGT CTC TAT GTA AAA GGT GAA CCA 1432
Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro
1~ 460 465 470
ATA ATA AAT TTC TAT GAC CCA TTA GTA TTC CCC TCT GAT GAA 1474
Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu
475 480 485 .
TTT GAT GCA TCA ATA TCT CAA GTC AAC GAG AAG ATT AAC CAG 1516
Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln
490 495 500 ~
25 AGC CTA GCA TTT ATT CGT AAA TCC GAT GAA TTA TTA CAT AAT 1558 ~ ~`
Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu Leu His Asn
S05 510 515 ~ ~-
GTA AAT GCT GGT AAA TCC ACC ACA A~T ATC ATG ATA ACT ACT 1600
Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr Thr
520 525 :
ATA ATT A~A GTG ATT ATA GTA ATA TTG TTA TCA TTA ATT GCT 1642
Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala
530 535 540
GTT GGA CTG CTC TTA TAC TGT AAG GCC AGA AGC ACA CCA GTC 1684
Val Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val
S~S . 550 555
- 40
ACA CTA AGC AAA CAT CAA CTG AGT GGT ATA AAT AAT ATT GCA 1726
Thr Leu Ser Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala
: 560 5Ç5 570 :
TTT AGT AAC TAAATAAAAA TAGCACCTAA TCATGTTCTT ACAATGGTTT 1775
. Phe Ser Asn
SUBSTITU~E SHE~T
WO93/20210 ~C~ - 2 PCT/GB93/0072
113
ACTATCTGCT CATAGACAAC CCATCTGTCA TTGGATTTTC TTAAAATCTG 1825 ~:
AACTTCATCG AAACTCTCAT CTATAAACCA TCTCACTTAC ACTATTTAAG 1875 ~
5 TAGATTCCTA GTTTATAGTT ATAT 1899 .;
~`:
- . ~
~.
~ ,.
'` ':
SUBSTITUTE SHFET
WO 93/2Q210 PCI`/GB93/0072j'''
~1 1 S.~ ~ ' !
114
(2) INFORMATION FO~ SEQ ID NO:19:
(i) SEQUENCE CH~RACTERISTICS:
(A~ LENGTH: 574 amino acids
(B) TYPE: amino acid
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
~xi1 SEQUENCE DESCRIPTION: SEQ ID NO:19:
Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu
1 5 10 15
Thr Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu
20 25 30
Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu
Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu
. 20 Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys
65 70 75
Val Lys Leu I le Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val
80 85 90
Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Pro Thr Asn Asn
: :: 95 100 105
~A~g Ala: Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn
115 120
Asn Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg
125 130 135
30 Arg Phe Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser
140 l~ 150
~ .
~: ~ly Val~Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn
` 15S 160 165
:Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser
I70 175 180
Leu Ser Asn Gly Val Ser Val Leu: Thr Ser Lys Val Leu Asp Leu
~ 185 190 195
Lys Aæn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn Lys Gln
200 ~ 205 210
-: ~ Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln
- 2 1 5 2 2 0 2 2 5
Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn
230 235 240
Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser
245 250 255
i
t
I
SUBSTITUTE SHEET
-: .WO 93/20210 PCr/GB93/0072~
6 2
115
Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn ~sp Gln
260 265 270
Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser
275 280 285
Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val
290 295 300
Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys
305 310 315
Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser
15320 325 330
Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn
335 340 345
Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val
350 355 360
Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu
365 370 375
Pro Ser Glu Il~ Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys
380 385 390
Tyr Asp Cys Lys Ile MPt Thr Ser Lys Thr Asp Val Ser Ser Ser
25395 400 405
Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr
410 415 420
Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe
425 430 435
30 Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp Thr Val
440 445 450
Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly Lys455 460 465
Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro
470 475 480
Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val
g~5 490 495
Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp
500 505 510
Glu Leu Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile
451 ~ 51S 520 525
Met Ile Thr Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser
530 535 540
SUBSrITUTE SHFET
WO93/Z0210 .;:~v PCT/GB93/007
116
Leu Ile Ala Val Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr
545 550 555
Pro Val Thr Leu Ser Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile ,-
560 565 ~ 570
Ala Phe Ser Asn
10 (2) INFORMAT~O~ E~OR SEQ ID NO:20: ~-
(i~ SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
~D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic3
(ix) FEATURE:
~A) NAMEtKEY: CDS
(B) LOCATION: 1..39
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
. :~
TTC GGC ACA GGG ACC AAA GTG ACT GTC CTG GGT CGT GAG TAG 42
Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Arg Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii3 MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Arg Glu
(2) INFORMATION FOR SEQ ID NO:22:
.
`
SUBSTITlJTE SI~IEE7 ~-
~o 93/20210 ~ ~ 3 ~ ~ D ~ PCT/GB93/0072~
;- '
117
~i) SEQUENCE CHARACTERISTICS: :
~A) LENGTH~ 39 base pairs
(B) TYPEa nucleic acid
(C) STRANDEDNESS: unknown ~-
~D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) .
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
TTCGGAACTG GGACCAAGGT CACCGTCCTA GGTAAGTGG 39 -
(2) INFORM~TION FOR SEQ ID NO:23: ;.
(i) SE~UENCE CHARACTERISTICS: .
(A) LENGTH: 18 base pairs ~.
(B) TYPE: nucleic acid `.;
(C) STRANDEDNESS: single -
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ~
;.. :
ATCTGTTTTT GAAGTCAT 18
. .
(2) INFORMATION FOR SEQ ID NO:24: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(Bl TYPE: nucleic acid
(C) STRANDEDNESS: single ~:
(D) TOPOLOGY: unknown ;
(ii) MOLECULE TYPE: DNA (genomic) .
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
ACGATTTTAT TGGATGC 17 -~
SUBSTITUTE SHEET
WO 93/20210 ~ ~ ~b ~ ~ PCT/GB93/0~72~'
118
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid ~
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQU~NCE DESCRIPTION: SEQ ID NO:25:
TGCATAATCA CACCCGT 17
(2) INFORMATION FOR SEQ ID NO:26:
(i3 SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOL~CULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2Ç:
CAAATCATCA GAGGGG 16
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CH~RACTERISTICS:
. (A) LENGTH: 18 base pairs
: 35 (B) TYPE: nucleic acid
( C ) STR~NDEDNES S: s in~le
(D) TOPOLOGY: unknown
(ii) MOI.ECULE TYPE: DNA (genomic)
(xi) SEQUE~CE DESCRIPTION: SEQ ID NO:27:
AATTCATCGG ATTTACGA 18
SUBSTITUTE S~tEE~ ~
:W093/20~10 ~ 3 ~ 6~ PCT/GBg3/0072~
119 ,
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs ~;~
(B~ TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown :
1 0 .;,
~ii) MOLECULE TYPE: DNA (genomic) :-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:~8:
15 CTCAGTTGAT CCTTGCTTAG 20 ...
(2) INFORMATION FOR SEQ ID NO:29: .:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 32 base pairs -
(B) TYPE: nucleic acid : ..
(C3 STRANDEDNESS: single ..
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
txi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
30 TTGAATTCAG ACTTTCGGGG CTGTGGTGGA GG 32
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
SUBSTITUTE S~EET
WO93/20210 rt~ PCT/GB93/0072
120
CCGAATTCGA CCGAGGGTGG GGACTTGGGC TG 32
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 21 base pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: single
~D) TOPOLOGY: unknown .
~ii) MOLECULE TYPE: ~NA (genomic~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3I:
AGGTSMRCTG CAGSAGTCWG G 21
~2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 34 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: single
2S (D) TOPOLOGY: unknown
~ii) MOLECULE TYPE: DNA (genomic)
: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
3~ .
:TTGACGCTCA GTCTGTGGTG ACKCAGSMGC CCTC 34
,~
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE~CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid ~ :
(C) STRANDEDNESS: single
~: 40 (D) TOPOLOGY: unknown
lii) MOLECULE TYPE: DNA ~genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: :
SUBSTITUTE SHEET
~ .
WO93/20210 ~ 6 -'~ PCT~GB93/00725
'~'`
,
121
TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAG 34 .
(2) INFORMATION FOR SEQ ID NO:34: -~
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid ::
(C) STRANDEDNESS: single
(D3 TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
GTTAGATCTC CAGCTTGGTC CC 22
(2) INFORMATION FOR SEQ ID NO:35: -
.
(i) SEQUENCE CHARACTERI5TICS: :
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid :`
(C) STRANDEDNESS: single
gD~ TOPOLOGY: unknown
(ii) MOLECULE TYP~: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: -I
GACATTCAGC TGACCCAGTC TCCA 24
(2) INFORMATION FOR SEQ ID NO:36:
.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
SUBSTITUTE SHE{~T
WO93/202l0 '~ PCT/GB93/0072~
~J
122
CTGTCTCACC CAGTGCATAT AGTAGTCGCT GAAGGTGAAG CCAGACACGG T 51
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs .
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
tD) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: -
CATTGTCACT CTGCCCTGGA ACTTCGGGGC ATATGGAACA TCATCATTCT 50
CAGGATCAAT CCA 63
~2) INFORMATIO~ FOR SEQ ID NO:38:
(i) SEQUENC~ CHARACTERISTICS:
(A) LENGTH: ~5 base pairs ~;.
(B) TYPE: nucleic acid :
(C) STRANDEDNESS: single .~:
~D) TOPOIOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: .~
30 CCCTTGGCCC CAGTGGTCAA AGTCACTCCC CCATCTTGCA CAATA 45 ~.
. .
-: (2) INFORMATION FOR SEQ ID NO:39:
~i) SEQUENCE:CHARACTERISTICS: `~
: ~A:) LENGTH: 79 base pairs -:
: (B) TYPE:: nucleic acid -~.
(C)~STRANDEDNESS: single ,~
~ ~ ~D) TOPOLOGY: unknown
: 40 ~ :~
~ ii) MOLECULE TYPE- DNA ~genomic)
,~ i
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: ;.
` !
i '~.
~'''`' '
: . ' `' .
; 1 '
~ '
SU8STITUTE SHEET . ~:-
'~
; W093/20210 PCT/GB93/00725
123
CTGCTGGTAC CATTCTAAAT AGGTGTTTCC ATCAGTATGT ACAAGGGTCT 50
GACTAGATCT ACAGGTGATG GTCA 74
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUE~CE GHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
~D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA ~genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
GCTTGGCACA CCAGAAAATC GGTTGGAAAC TCTGTAGATC AGCAG 45 ~;`
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs :
~B) TYPE: nucleic acid :
(C3 STRANDEDNESS: single
(D) TOPOLOGY: unknown
~ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: ~:
CCCTTGGCCG AACGTCCGAG GAAGATGTGA ACCTTGAAAG CAGTAGTAGG T 51
35 (2) INFOR~ATION FOR SEQ ID NO:42: ;
(i) SEQUENCE CHARACTERISTICS: ~:
(A) LENGTH: 28 base pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
~ii) MOLECULE TYPE: DNA (genomic)
1 (xi) SEQUENGE DESCRIPTION: SEQ ID NO:42:
SUBSTITUTE SHEET
W093/202l0 PCT/GB93/0072s
124
CTCCCCCATG AATTACAGAA ATAGACCG 28
~2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDN~SS: single
(D3 TOPOLOGY: unknown .
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAG 34 :~
, .
(2) INFORM~TION FOR SEQ ID NO:94-
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown ;:~
,~,
(ii) MOLECULE TYPE: DNA (genomic) .-~
....
(xi) SE~UENCE DESCRIPTION: SEQ ID NO:44:
TGGGCTCTGG GTTA~CACGG ACTGGGAGTG GACACC 36
. ~:
: 35 (2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CXARACTERISTICS: -
(A~ LENGTH: 33 base pairs `:
~B) TYPE: nucleic acid
(C) STRANDEDNESS: single
- (D) TOPOLOGY: unknown ,
(ii) MOLECULE TYPE: DNA (genomic) .
: ~ ' ' '
'
'~
`..
,., ~
SVBSTlTUtE SHEET
.-WO93/20~10 ~ 3 ) ~ ~ PCT/GB93/0072~
125 .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: .:
ATTCTACTCA CGACCCATGG CCACCACCTT GGT 33
(2~ INFORMATION FOR SEQ ID NO:46: -~
i ) S EQUENOE C~ACTER I S T I C S:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid .
~C) STRAN~EDNESS: single
(D) TOPOLOGY: unknown
~ii) MOLECULE TYPE: DNA (genomic)
~xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 4 6:
CTCCATCCCA TGCTGAGGTC CTGTG 25
(2) INFORMATION FOR SEQ ID NO:47: ~
~i) SEQUENCE CHARACTERISTICS: :
~A) LENGTH: 22 base pairs
~B) TYPE: nucleic acid -
(C) STRANDEDNESS: single `
(D) TOPOLOGY: unknown :~
~ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE~DESCRIPTION: SEQ ID NO:47: ~.
GCGGGCCTCT TCGCTATTAC GC 22
~2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
.~ 45 I -
SU8STITlJTE S~E~T
~ ~:
WO93/20210 PCT/GB93/007~`
~ ~ v ~
126
(xi) SEQUENCE DESC~IPTION: SEQ ID NO:48:
CTGTCTCAGG GCCAGGCGGT GA 22
(2) INFO~MATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single ~:-
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
TCTGTGTTAA CGCAGGCGCC CTCCGTG ~7 ~;~
(2) INFORMATION FOR SEQ ID NO:50
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single -
(D) TOPOLOGY: unknown :~
(ii) MOLECULE TYPE: DNA (genomic)
:- ~ 30 ~ ~(xi) SEQUENCE DESC~IPTION: SEQ ID NO:50:
GGCTGACCCA TGGCGATCAG TGTGGTC 27
- ~ 35 (2) INFORMATION FOR SEQ ID NO:51~
(i) SEQUENCE CHARACTERISTICS: :
~A) LENGTH: 48 base pairs .-~
(B) TYPE:: nucleic acid .~`;
(C) STRANDEDNESS: single `~
~ ~ -(D) TOPOLOGY: unknown
- (ii) MOLECULE TYPE: DNA ~genomic)
45 , (xi)~SEOUENCE DESCRIPTION: SEQ ID NO:51: ~
` ~:
...
~.
'` '~
SUBSTITUTE SHEET
-.:;~093/20210 ~i L ~ PCT/GBg3/00725
127
CTGTCTCACC CAGCTTACAG A~TAGCTGCT CAATGAGAAG CCAGACAC 48
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single - .:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) ~:
~ xi) SEQUENCE DESCRIPTION:`SEQ ID NO:52:
CATTGTCACT CTGGATTTCA GGGCTGGGTT ATAATATATG ATTCCGCCAT 50
TGCTTGCGTC TCCAAGCCAC TCA~GACC 78
'~:
t2) INFORMATION FOR SEQ ID NO:S3:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l02 base pairs :-
(B) TYPE: nucleic acid -
(C) STRANDEDNESS: single
(D) TOPOLOGY: un~nown
5ii) MOLECULE TYPE: DNA (genomic)
-30 (xi) SEQUENCE DESCRIPTION SEQ ID NO:53: .
.CAAGGACCCT TGGCCCCAGG CGTCGACATA CTCGCCCTTG CGTCCAGTAC 5O
AAGCATA~CT TCCACTATCA CCAACAGAAC ACTTTGCACA ATA~TAGACC GCl02
: :
(2) INFORMATION FOR SEQ ID NO:54:
:
(i) SEQUENCE C~ARACTERISTICS:
~A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single ~:
(D) TOPOLOGY: unknown
~ii) MOLECULE TYPE: DNA (genomic)
'.
SUBSTITUTE SHEFT
,
WO93/20210 PCT/GB93~007
1~8
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
~TAAAACGAC GGCCAGT 17
:
(2) INFORM~TION FOR SEQ ID NO:55~
(i) SEQUENCE CHARACTERISTICS: ~:
10~A) LENGTH: 438 amino acids .:
(B) TYPE: amino acid ::
(D) TOPOLOGY: unknown ~
(ii) MOLECULE TYPE: protein
:~-
(xi~ S~QUENCE DESCRIPTION: SEQ ID NO:55: ;~
Phe Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly -.
1 5 10 15
- Val Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys `
20 25 30 -:
Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu
35 4~ 45 `.~:~
Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys:~
50 55 60
Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn Lys Gln Ser:-.
3065 70 75
Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys.~
80 ~ 85 30 ~:
Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn Ala:~
95 100 105 `~.
35 Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu ~:
110 115 120 -`~
Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys
125 130 135 `
Lys Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr ~:
40140 145 150 `i~
Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val
155 . 160 165
Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu
170 175 18Q
His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn
185 190 195
SUBSTI~UTE SHEET
r~,~ .L ~ ~
-: WO 93/20210 PCI/GB93/00725
129
Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala .
200 205 210
Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln
215 220 225
Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro
23~ 235 240
Ser Glu Ile Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr
~0245 250 255 ~:
Asp Cys Lys Ile Met Thr S~r Lys Thr Asp Val Ser Ser Ser Val
260 265 270
Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys
275 280 2~
Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser
290 295 300
Asn Gly CyS Asp Tyr Val Ser Asn Lys Gly Met Asp Thr Val Ser
305 310 315
Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser
320 325 330
Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu
33~ 340 345
Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn
350 35~ 360
Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu ~ ::
365 370 375
Leu Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn I le Met
: 3~0 385 390
Ile Thr Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser I,eu
:395 400 405
Ile Ala Val Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro
410 415 420
- ~ ~ 35 Val Thr Leu Ser Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala
425 430 435
Phe Ser Asn
`'~
40 (2) INFORMATION FOR SEQ ID NO:56:
~i) SEQUENCE C~IARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
!` 45 , ~ (D) TOPOLOGY: unlcnown
(ii) MOLECULE TYPE: protein
SUBSTITUTE SHFET ~:
wo 93~20210 ~ ) b ~ (~ PC~lGBg3/0072';:
. ,:
130
(ix) FEATURE~
(A) NAME/KEY: Modified-site
(B) LOCATION: 1 :
(D) OTHER INFORMATION: /note= "X can be A~a, Cys
Asp, Glu, Phe, Gly, His, Leu,
Pro, Gln, Arg, Ser, Thr, Val, Trp or
Tyr"
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:56: ~
Xaa Thr Asn Asp Gln Lys Lys Leu ~-
1 5 ~-
~:
(2) INFORMATION FOR SEQ ID NO:57
~i),SEQUENCE CHAR~CTERISTICS~
(A) LENGTH: 8 amino acids `~
(B) TYPE: amino acid
(D) TOPOLOGY: unknown `
(ii) MOLECUL~ TYPE: protein ~`~
~ix~ FEATURE~
tA) NAME/KEY: Modified-site ;-^~
(B) LOCATION: 5 `
(D) OTHER INFORMATION: /note= "X can be Asp, Glu, `~
Phe, Ile, Leu, Met, Arg, Ser, Thr,
Val, or Trp"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57
Ile Thr Asn Asp Xaa Lys Lys Leu
1 5
(2) INFORMATION FOR SEQ ID NO:58: -
~i) SEQUENCE CHAR~CTERISTICS: -:
~A) LENGTH: 8 amino acids `-
~B) TYPE: amino acid
~D) TOPOLOGY: unknown ;.
i (ii) MOLECULE TYPE: protein ;
SUBSTITUTE SHEFT ,`;
. WO93~20210 ~ i~ J ~ PCT/GB93~00725
131
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= ~'X can be Asp, Glu,
Phe, Ile, Leu, Met, Arg, Ser, Thr,
- Val, Trp, Tyr or Gln"
(xi) SEQUENCE ~ESCRIPTION: SEQ ID NO:58:
Ile Thr Asn Asp Gln Xaa Lys Leu
1 5
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TQE~OLOGY: unknown ~.
~ii) MOLECULE TYPE: protein -.
(ix) FEATURE:
(A) NAME/KEY: Modified-site :
(B) LOCATION~
(D) OTHER INFORM~TION: /note- "X can be Ala, Cys,
Asp or Glu" :
3Q (xi) SEQUENCE DESCRIPTION: SEQ ID NQ:59:
Ile Thr Asn Asp Gln Lys Lys Xaa
SUBSTITUTE SHEET
