CA2133662A1 - Antibodies for treatment and prevention of respiratory syncytial virus infection - Google Patents

Abstract

2133662 9320210 PCTABS00027 The present invention provides variable light chain and variable heavy chain sequences derived from bovine anti-RSV F protein monoclonal antibodies (mAbs), B4 and B13/B14, and CDR peptides therefrom, which may be employed in the design of fusion proteins (including altered antibodies) which are charaterized by the antigen binding specificity of these mAbs. Also described is a humanized antibody containing bovine variable chain sequences. Methods for producing and using these compositions, including pharmaceutical compositions are disclosed.

Description

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 ,''"` ~: ~ ` '`
.': .', : ~ :`~

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

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

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 `-~

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 SUBST~TUTE St~EET

~: 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 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:

' ~ ..
::

WO93/20210 ;~ 2 PCT/GB93/00725 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 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
~..

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 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 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 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
` .~?

- WO93/20210 .~ 3 ~ ~ 2 PCT/GBg3/00725 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 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 `~

~` WO93/20210 ~ 6 6 2 PCT/GB93/00725 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 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 ~ ~ `

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 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 `

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 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

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 6 2 ! : ~

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 '~

' WO93/20210 ~ i G ~ PCT/GB93/00725 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 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 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 ;

'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 ~ ~
. ,................................... ~
!

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
,...

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 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 W O 93/20210 ~ PC-r/GB93/00725 `~

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 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).

W093/20210 ~ 6~ 4~ ~ 6 ~ PCT/GB93tO072S

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 ~ '~

11~3 U -~:

U

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 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~;

~ .

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 - W093/20210 ~ V G ~ PCT/GB93/00725 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 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 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'`

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 ``:

,.:
,',':
: :.
: ' ,:

: `
` .

--' 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 SUBSTITUTE SHEET ~`:

;; J ~ `~ J ~ ::
WO 93/20210 PCr/GB93/00725 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.

SU8STtTUTE S~tEET

WO93/20210 ~ fJ 3 ~ 6 ~ PCT/GB93/00725 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
,~ , SUBSTITUTE SHEET
; `

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' ~

`!; 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~``

3 b ~

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
.

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 SUBSTlllJTE SHEET

`

- W093/202l0 PCT/GB93/00725 :~ .

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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- WO93/20210 ~ 6 2 PCT/GB93/0072~ ~
.-'. :~

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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WO93/20210 ~ PCT/GB93/0072 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 :

.
' SU8STlTl3TE SHEET
. , wo g3/202l0 ~ 6 2 PCT/GB93/00725 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 ~.

WO93/20210 ~J ~ PCT/GB93/00725 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, ~ -SUBSTlTtJTE SHEET

~ 5 ~ ~ ~
-- W O 93/20210 PC~r/GB93/00725 ~'';

83 -~-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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WO 93/20210 ~ PCI/GB93/00725 ______________________________________________________ .
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 ~

WO93/20~10 ~ 6 ~ PCT/GB93/00725 ~ ;

;~
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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WO93/20210 PCT/GB93/00725`

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.

' SUBSTlTUTE SHEET -`- WO~3/20210 ~ 6 62 PCT/GB93/00725 ~ -:

.. "`::.

______________________ ________________________________ ~,:
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, SUBSTTTUTE SHEET `~

W093J20210 ~ ~U ~ ~ PCT/GB93/0072S

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 ! ' ., . ~ .
' ", , ~ ~ `,'' :~ ~ ` : ,',,' SU8STt~VTE SHEET

`' '!`~0 93/20210 ~,i r~ ~ ~ 6 2 PCT/GB93/00725 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 SUBSTITVTE SHEET

WO93/20210 ~ 2 PCT/GB93/0072j 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 - , ,'`-., : ~:

~- ,"

SUBSTmJTE SHEET .

. ~.

- WO93/20210 ~ PCT/GB93/0072S

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 SUBSmlJTE SHEET

WO93/2021~ P PCT/GB93/007~ ~

, 92 ;
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 .

SU~STITUTE SHEET :

~093/20210 PCT/GB93/00725 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 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 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 WO 93/20210 ~ r~ PCT/GB93/00725 (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 ``

: 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 ~.

. -~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 Ser Val Val Thr Gln Gln Pro Ser Val Ser Gly Ser Leu Gly Gln 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 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 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 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 Glu Trp Leu Gly Asp Ala Ser Asn Gly Gly Ile Ile Tyr Tyr Asn . ~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 Ala Thr Tyr Tyr Cys Ala Lys Cys Ser Val Gly Asp Ser Gly Ser 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 SU8S~UTE SHEET

WO93/20210 `` ~ b ~ 2 PCT/GB93/00725 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 :~

Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Ser :

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 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 -~

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 :

Thr Thr Ala Pro Lys Val -(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 .

SUBSTITUTE SH EET

;~ 1 r~b ~
- WO93~20210 PCT/GB93/00725 .

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 Ala Val Tyr Tyr Cys Ala Lys Cys Ser Val Gly Asp Ser Gly Ser Tyr Ala Cys Thr Gly Arg Lys Gly Glu Tyr Val Asp Ala Trp Gly 15 Gln Gly Thr Thr Val Thr Val Ser Ser :' (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 Gly Arg Trp Gly Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala 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 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 ~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 `

(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 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 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 -W093/202l0 ` ~ PCT/GB93/00725 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 Thr Lys Leu Glu Ile Lys (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:

AGTCAAGTTG TCCTG QCAG CTTCTGGCTT CAACATTAAA GACTACTATA 100 ,~-:
TGCACTGGAT~GAAGCAGAGG CCTGACCAGG GCCTGGAGTG GATTGGATGG 150 30 :
ATTGXTCCTG AGA~TGATGA TGTTCAATAT GCCCCGAAGT TCCAGGGCAA 200 ~ .:

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 (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 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 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 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 :

(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: .

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 !

Leu Val His Thr Asp Gly Asn Thr Tyr Leu Glu Trp Phe Leu 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 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 His 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 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 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 .

- 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 Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Asp Asp Val Gln Tyr Ala Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala ~sp Thr Ser Ser Asn Thr Ala Tyr L~u Gln Leu Thr Ser Leu Thr Phe Glu Asp 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 (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 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

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~ ~

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 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (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 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 Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Asp Asp Val Gln Tyr 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 ~ 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 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 :

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 ~`

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 Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn 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 Leu Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg lS100 1~5 Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg ~he 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 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 Ser Leu Ser Asn Gly Val Ser Yal Leu Thr Ser Lys Val Leu 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 Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile ,`

Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg SUBSTITUTE SI~EET

W093~20210 h ~ b `~ PCT/GB93/0072~ :

111 ~

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 -;

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu :~

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 ~

.-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 ;-Ser Asn ~rg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu `~

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 :

Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser 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~

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 Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser As~ Lys 10 Gly Met Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro 1~ 460 465 470 Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu 475 480 485 .

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 Val Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val S~S . 550 555 Thr Leu Ser Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala : 560 5Ç5 570 :

. Phe Ser Asn SUBSTITU~E SHE~T

WO93/20210 ~C~ - 2 PCT/GB93/0072 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.~ ~ ' !

(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 Thr Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu 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 Val Lys Leu I le Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val 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 Asn Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg 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 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 Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser i t I

SUBSTITUTE SHEET

-: .WO 93/20210 PCr/GB93/0072~

Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn ~sp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Il~ Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile MPt Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe 30 Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp Thr Val 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 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 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 SUBSrITUTE SHFET

WO93/Z0210 .;:~v PCT/GB93/007 Leu Ile Ala Val Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr 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:
. :~

Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Arg Glu (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~

;- ' ~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:

(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: ~
;.. :

. .
(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~' (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:

(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:

(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:

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:

(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 (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:

~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 '~'`
, 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:

(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

(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

(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: -~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 (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: ~:

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 ~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: .:

(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:

(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: ~.

~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 ~

(xi) SEQUENCE DESC~IPTION: SEQ ID NO:48:

(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:

- ~ 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 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:

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 -.

- 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:~

Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn Lys Gln Ser:-.

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 His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn SUBSTI~UTE SHEET

r~,~ .L ~ ~
-: WO 93/20210 PCI/GB93/00725 Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala .

Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln 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 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 Asn Gly CyS Asp Tyr Val Ser Asn Lys Gly Met Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser 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 ~ ::

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 - ~ ~ 35 Val Thr Leu Ser Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala 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';:

. ,:

(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 (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 (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 (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

Claims (33)

WO 93/20210 PCT/GB93/0072?

WHAT IS CLAIMED IS:

1. A fusion protein comprising an amino acid sequence having the antigen specificity of an anti-RSV antibody fused to a selected second peptide or protein sequence.

2. The protein according to claim 1 wherein said second sequence is heterologous to said sequence having anti-RSV antigen specificity.

3. The protein according to claim 1 wherein said antigen specificity is directed against the amino acid sequence of the F protein SEQ ID NO: 19 spanning amino acid #266 through #273 and analogs thereof.

4. The protein according to claim 1 wherein said antibody is a bovine antibody.

5. The protein according to claim 4 wherein said antibody is selected from the group consisting of bovine monoclonal antibody B4 and the bovine anti-RSV antibody B13/B14.

6. The protein according to claim 1 wherein said amino acid sequence is selected from the group consisting of the variable heavy chain of said antibody, the variable light chain of said antibody, at least one CDR from said variable heavy chain, at least one CDR from said variable light chain, a functional fragment or analog thereof.

7. The protein according to claim 1 wherein said amino acid sequence has the formula selected from the group consisting of (a) SEQ ID NO: 56: , wherein X is selected from the amino acids consisting of , (b) SEQ ID NO: 57: , wherein Y is selected from the amino acids consisting of , and Tyr, (c) SEQ ID NO: 58: , wherein Z is selected from the amino acids consisting of , and (d) SEQ ID NO: 59: , wherein W is selected from the amino acids consisting of .

8. The protein according to claim 1 wherein said amino acid sequence is selected from the group consisting of (a) a sequence, comprising a variable heavy chain sequence of Fig.
4A and 4B SEQ ID NO: 3, (b) a sequence, comprising a variable light chain sequence of Fig. 3A and 3B SEQ ID NO:
4, and (c) a functional fragment or analog of (a) or (b).

9. The protein according to claim 1 wherein said amino acid sequence comprises one or more CDR peptides selected from the group consisting of (a) amino acids 31 through 35 of SEQ ID NO: 3:
;

(b) amino acids 50 through 65 of SEQ ID NO: 3:
;
(c) amino acids 100 through 122 of SEQ ID NO: 3:
, wherein X is any or no amino acid;
(d) amino acids 22 through 34 of SEQ ID NO: 1 and 2: ;

(e) amino acids 50 through 56 of SEQ ID NO: 1:
;
(f) amino acids 89 through 96 of SEQ ID NO: 1:
;
(g) amino acids 89 through 97 of SEQ ID NO: 1:

;
(h) amino acid 50 through 56 of SEQ ID NO: 2:
;
(i) amino acid 89 through 99 of SEQ ID NO: 2:
;
(j) amino acid 31 through 35 of SEQ ID NO: 4:
;
(k) amino acid 50 through 65 of SEQ ID NO: 4:
; and (l) amino acid 98 through 122 of SEQ ID NO: 4:
, wherein Y is any amino acid.

10. A fusion molecule comprising a first fusion partner nucleotide sequence encoding an amino acid sequence having the antigen specificity of an anti-RSV antibody operatively linked to a selected second fusion partner nucleotide sequence.

11. An anti-RSV CDR peptide selected from (a) through (l) of claim 9 and (m) a fragment thereof, or an analog thereof, characterized by the antigen specificity of any of the above peptides.

12. An isolated bovine anti-RSV antibody variable light chain amino acid sequence, a fragment or analog thereof sharing the anti-RSV antigen specificity of said sequence.

13. The antibody according to claim 12 wherein said light chain sequence is naturally occurring in said antibody or modified, and is selected from the group consisting of the sequences of Figs. 3A and 3B SEQ ID NOS: 1 and 2, Fig.
11 SEQ ID NO: 6, and Fig. 13 SEQ ID NO: 8.

14. An isolated bovine anti-RSV antibody variable heavy chain amino acid sequence, a fragment or analog thereof sharing the anti-RSV antigen specificity of said sequence.

15. The sequence according to claim 14 wherein said heavy chain sequence is naturally occurring in said antibody or modified, and is selected from the group consisting of the sequences of Figs. 4A and 4B SEQ ID NOS: 3 and 4, Fig.
10 SEQ ID NO: 5, and Fig. 12 SEQ ID NO: 7.

16. An isolated nucleic acid sequence encoding the variable heavy chain amino acid sequence or variable light chain amino acid sequence of a selected anti-RSV antibody, a functional fragment or analog thereof, optionally containing restriction sites to facilitate insertion into a desired antibody framework region or fusion with a selected fusion partner.

17. An altered antibody comprising an amino acid sequence in which at least parts of the heavy chain variable region of an acceptor antibody have been replaced by analogous parts of the heavy chain variable region of at least one donor antibody having specificity for respiratory syncytial virus, and a suitable light chain sequence, said acceptor antibody being heterologous to said donor antibody.

18. The antibody according to claim 16 wherein the variable heavy chain region of the donor antibody is intact and fused to the heavy chain constant region of the acceptor antibody.

19. The antibody according to claim 17 wherein the variable heavy chain CDR fragments of the donor antibody replace the heavy chain CDR fragments of the acceptor antibody.

20. The antibody according to claim 17 wherein the light chain is selected from the group consisting of (a) an variable light chain region of the donor antibody fused to the light chain constant region of the acceptor antibody;

(b) a light chain comprising light chain CDR
fragments of the donor antibody replacing the light chain CDR fragments of the acceptor antibody;
(c) the donor antibody light chain; and (d) a heterologous acceptor antibody light chain.

21. The antibody according to claim 17 wherein the variable light chain region of the donor antibody is that of Fig. 3A and 3B SEQ ID NO: 1or a functional fragment thereof and the variable heavy chain region of the donor antibody is that of Fig. 4A and 4B SEQ ID NO: 3, or a functional fragment thereof, wherein the resulting altered antibody is characterized by the antigen binding specificity of mAb B4.

22. The antibody according to claim 17 wherein the variable heavy chain region of the donor antibody is that of Fig. 3A and 3B SEQ ID NO: 2 or a functional fragment thereof and the variable heavy chain region of the donor antibody is that of Fig. 4A and 4B SEQ ID NO: 4, or a functional fragment thereof, wherein the resulting altered antibody is characterized by the antigen binding specificity of mAb B13/B14.

23. A humanized antibody comprising an amino acid sequence in which at least parts of the sequence of the heavy chain variable region of a human acceptor antibody have been replaced by analogous parts of the amino sequence of the heavy chain variable region of at least one bovine donor antibody, and a suitable light chain sequence, said humanized antibody characterized by the antigen specificity of the bovine donor antibody.

24. The antibody according to claim 23 wherein the antigen specificity is binding to an epitope of RSV, said antibody comprising a humanized heavy chain variable region sequence selected from the group consisting of the sequence of Fig. 10 SEQ ID NO: 5 and the sequence of Fig. 12 SEQ ID
NO: 7.

25. The antibody according to claim 24 characterized by a light chain selected from the group consisting of the humanized light chain sequence of Fig. 11 SEQ ID NO: 6, the humanized sequence of Fig. 13 SEQ ID NO: 8, a naturally occurring bovine monoclonal antibody light chain characterized by the light chain variable sequences of Figs.
3A and 3B SEQ ID NO: 1, and a chimeric bovine/human light chain characterized by the light chain variable sequences of Figs. 3A and 3B SEQ ID NO: 1 fused to the light chain constant regions of a human acceptor antibody.

26. An antibody, other than B4, which is capable of binding to the RSV peptide consisting essentially of the amino acid sequence of the F protein spanning amino acid #266 through #273 of SEQ ID NO: 19 and analogs thereof, a Fab fragment thereof, or an F(ab')2 fragment thereof, said antibody being a monoclonal antibody or an altered humanized antibody.

27. An anti-RSV antibody, a Fab fragment or a F(ab')2 fragment thereof produced by screening an antibody library comprising hybridoma products and libraries derived from any species immunoglobulin repertoires, with one or more antibodies selected from the group consisting of B4 and B13/B14.

28. A pharmaceutical composition comprising the or more of a fusion protein of claims 1-9, a CDR peptide of claim 11, the sequences of claim 12-15 or the antibodies of claim 17-27, and a pharmaceutically acceptable carrier or diluent.

29. A method of preventing or treating human RSV
infection in a human in need thereof which comprises administering to said human an effective dosage of a pharmaceutical composition of claim 28.

30. A recombinant plasmid comprising a nucleic acid sequence of claim 16 or a nucleic acid sequence of a fusion molecule of claim 10.

3$. A mammalian cell line transfected with the recombinant plasmid of claim 30.

32. A method for producing a fusion protein of claim 1 comprising culturing a suitable cell line transfected with a nucleic acid sequence encoding said protein under the control of regulatory sequences capable of directing the replication and expression of said protein and obtaining the expressed protein from the cell culture.

33. A method for producing a fusion protein of claim 1 or an altered antibody of claim 17 comprising producing the fusion protein or antibody in a transgenic animal.