?2578361 Summary - Canadian Patents Database (2024)

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
Bindina member towards Pneumolysin

The present invention relates to a binding member comprising at least one
binding
domain capable of specifically binding Pneumolysin, in particular to a binding
mem-
ber having at least two binding domains, to the use of said binding members in
di-
agnostic methods as well as for treatment. Further described are Pneumolysin
pep-
tides and vaccine compositions comprising Pneumolysin peptides.

Background
Streptococcus pneumoniae is one of the leading causes of life-threatening
bacterial
infection. In developing countries it has been estimated that several million
children
under 5 years of age will die of S. pneumoniae each year (anonymous, 1985). In
the
industrialized world, the incidence of S. pneumoniae pneumonia is 5-10 per
100.000
persons and the case-fatality rate is 5-7%. S. pneumoniae meningitis occurs in
1-2
per 100.000 persons with a case-fatality of 30-40% (Lee et al., 1997). S.
pneumo-
niae is one of the most frequent causes of bacteremia. S. pneumoniae is the
most
frequent organism isolated from children with otitis media. App. 75% of all
children
less than 6 years old will suffer from otitis media.
S. pneumoniae is a gram-positive bacterium that grows in pairs or short
chains. The
surface is composed of three layers: capsule, cell wall and plasma membrane.
The
capsule is the thickest layer and completely conceals the inner structures of
growing
S. pneumoniae. Polymers of repeating units of oligosaccharides
(polysaccharides)
dominate the capsule. Different serotypes contain ribitol, arabitinol or
phosphoryl-
choline as part of their capsule, resulting in chemical structures that are
serotype
specific. The cell wall consists of peptidoglycan but also teichoic acid and
lipotei-
choic acid. The plasma membrane is a double phospholipid membrane that encom-
passes the cell and anchors various molecules to its surface (Alonso De
Velasco,
1995).

At present 90 different types of S. pneumoniae are recognized based on the
diver-
sity of the S. pneumoniae capsule (Sorensen, 1995). The capsule is pivotal in
the
pathogenesis of S. pneumoniae infections. Antibodies raised against one
capsular
type offers protection from infection with this type but not against infection
with other

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
2
capsular types. The current 23-valent polysaccharide vaccine offers protection
from
more than 60-85% of the most frequent serotypes.

Pneumolysin is a major virulence factor of some gram-positive bacteria and is
a
member of a family of cholesterol-binding toxins (de los Toyos et al., 1996).
It is a
soluble protein that disrupts cholesterol-containing membranes of cells by
forming
ring-shaped oligomers (porins) (Bonev et al., 2001). Further, Pneumolysin
activates
the complement system in a non-specific manner through interaction with Fc and
complement proteins. The toxicity of Pneumolysin can be attenuated by site-
directed
mutagenesis (Trp-433 to Phe substitution) of the Pneumolysin gene, resulting
in the
expression of pneumolysoid (PdB) (Alexander et al., 1994).

Pneumolysin appears conserved among tested S. pneumoniae strains (Paton et
al.,
1983). The deduced amino acid sequence based on the Pneumolysin gene from
different strains of S. pneumoniae is >99% identical (Mitchell et al., 1990).

IgA to Pneumolysin is detectable in saliva from children (242 of 261) and
adults (17
of 17) (Simeil et al., 2001). Anti-Pneumolysin IgG was detectable by EIA in
most
children less than two years (803 of 1108) and all adults (325/325) (Rapola et
al.,
2000). Seroconversion was correlated to carrier status, i.e. children who had
been
infected with S. pneumoniae cultured from nasopharyngeal or middle ear
specimens
were more likely to be anti-Pneumolysin IgG positive. In a different study
using an
ELISA method, IgG was detected in 7 of 40 healthy adults, 17 of 32 patients
with
chronic obstructive pulmonary disease, and 13 of 31 patients with pneumococcal
pneumonia (Musher et al., 2001). Interestingly, significantly fewer patients
with
pneumonia and bacteremia had detectable IgG compared to patients with pneumo-
nia but without bacteremia (4/16 vs. 9/15). This suggests that anti-
Pneumolysin an-
tibodies may prevent pneumonia from progressing to bacteremia.

Summary

The present invention relates to an anti-haemolytic binding member comprising
at
least one binding domain capable of specifically binding Pneumolysin, wherein
the
binding member is suitable for use in a pharmaceutical composition for
preventing

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
3
and treating diseases and disorders related to Streptococcus, in particular
Strepto-
coccus pneumoniae.

Accordingly, in one embodiment the invention relates to an isolated binding
member
comprising at least one binding domain capable of specifically binding
Pneumolysin,
said binding domain having a dissociation constant Kd for Pneumolysin which is
less
than 1 x 10-6. Preferably the binding member comprising the binding domain has
the
dissociation constant Kd defined above.

Due to the high binding strength the binding member is suitable for use in a
phar-
maceutical composition. Further more binding members with anti-haemolytic
activity
are particular useful.

In another aspect the invention relates to an isolated binding member
comprising at
least a first binding domain and a second binding domain, said first binding
domain
being capable of specifically binding Pneumolysin.

The binding member according to the invention is preferably an antibody or a
frag-
ment of an antibody. The antibody may be produced by any suitable method known
to the person skilled in the art, however it is preferred that at least a part
of the bind-
ing member is produced through a recombinant method. Accordingly, the present
invention relates in one aspect to an isolated nucleic acid molecule encoding
at least
a part of the binding member as defined above, as well as to a vector
comprising the
nucleic acid molecule defined above, and a host cell comprising the nucleic
acid
molecule defined above.

The invention further relates to a cell line engineered to express at least a
part of the
binding member as defined above, and more preferably engineered to express the
whole binding member as defined above.
In a further aspect the invention relates to a method of detecting or
diagnosing a
disease or disorder associated with Pneumococcus in an individual comprising

- providing a biological sample from said individual,
- adding at least one binding member as defined above to said biological
sample

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
4
- detecting binding members bound to said biological sample, thereby detecting
or
diagnosing the disease or disorder.

Also, in the method the invention further relates to a kit comprising at least
one bind-
ing member as defined above, wherein said binding member is labelled, for use
in a
diagnostic method.

In yet another aspect the invention relates to a pharmaceutical composition
compris-
ing at least one binding member as defined above.
Furthermore, the invention relates to the use of a binding member as defined
above
for the production of a pharmaceutical composition for the treatment or
prophylaxis
of disorders or diseases associated with Streptococcus pneumoniae, such as
pneumonia, meningitis and/or sepsis.
In yet a further aspect the invention relates to a method for treating or
preventing an
individual suffering from disorders or diseases associated with Streptococcus
pneumoniae, such as pneumonia, meningitis and/or sepsis by administering an ef-

fective amount of a binding member as defined above.
Further aspects relates to a Pnemolysin peptide recognized by an anti-
haemolytic
binding member and a vaccine composition comprising such peptide.

Drawings
Figure 1. Schematic drawing of a Fab fragment.
Figure 2. Pneumolysin amino acid sequence having SEQ ID NO 11.
Figure 3. Anti-Pneumolysin light chain and heavy chain variable segment.
Figure 4. Survival diagram for mice inoculated with Pneumococcus and antibody.
Figure 5. Antihaemolytic activity of Pneumolysin antibodies
Figure 6 Peptides for epitope mapping.
Figure 7 Graphic illustration of determination of Pneumolysin antibody
epitopes.
Figure 8 Isolation of 26-5F12 clones
Figure 9 Isolation of 26-23 C2 clones
Figure 10 Isolation of 22 1 C11 clones

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
Figure 11 CDR sequences of 26-5F1 2, 26-23C2 and 22-1 C11.

Sequence listing
5
SEQ ID NO 1: Amino acid 425-436 of Pneumolysin
SEQ ID NO 2: Amino acid 423-438 of Pneumolysin
SEQ ID NO 3: Variable light chain 26-5F12.1
SEQ ID NO 4: Variable heavy chain 26-5F12.1
SEQ ID NO 5: CDR 1 light chain 26-5F12.1
SEQ ID NO 6: CDR 2 light chain 26-5F12.1
SEQ ID NO 7: CDR 3 light chain 26-5F12.1
SEQ ID NO 8: CDR 1 heavy chain 26-5F12.1
SEQ ID NO 9: CDR 2 heavy chain 26-5F12.1
SEQ ID NO 10: CDR 3 heavy chain 26-5F12.1 and 26-23C2.2
SEQ ID NO 11: Pneumolysin sequence
SEQ ID NO 12: Variable light chain 26-23C2.2
SEQ ID NO 13: Variable heavy chain 26-23C2.2
SEQ ID NO 14: CDR 1 light chain 26-23C2.2
SEQ ID NO 15: CDR 2 light chain 26-23C2.2
SEQ ID NO 16: CDR 3 light chain 26-23C2.2
SEQ ID NO 17: CDR 1 heavy chain 26-23C2.2
SEQ ID NO 18: CDR 2 heavy chain 26-23C2.2
SEQ ID NO 19: Variable light chain 22-1C11
SEQ ID NO 20: Variable heavy chain 221 C11
SEQ ID NO 21: CDR 1 light chain 22-1C11
SEQ ID NO 22: CDR 2 light chain 22-1 C11
SEQ ID NO 23: CDR 3 light chain 22-1C11
SEQ ID NO 24: CDR 1 heavy chain 22-1C11
SEQ ID NO 25: CDR 2 heavy chain 22-1 C11
SEQ ID NO 26: CDR 3 heavy chain 22-1C11
Detailed description of the invention

Definitions
Affinity: the strength of binding between receptors and their ligands, for
example
between an antibody and its antigen.

Avidity: The functional combining strength of an antibody with its antigen
which is
related to both the affinity of the reaction between the epitopes and
paratopes, and
the valencies of the antibody and antigen

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
6
Amino Acid Residue: An amino acid formed upon chemical digestion (hydrolysis)
of
a polypeptide at its peptide linkages. The amino acid residues described
herein are
preferably in the "L" isomeric form. However, residues in the "D" isomeric
form can
be substituted for any L-amino acid residue, as long as the desired functional
prop-
erty is retained by the polypeptide. NH2 refers to the free amino group
present at the
amino terminus of a polypeptide. COOH refers to the free carboxy group present
at
the carboxy terminus of a polypeptide. In keeping with standard polypeptide,
abbre-
viations for amino acid residues are shown in the following Table of Correspon-

dence:
TABLE OF CORRESPONDENCE
SYMBOL
1-Letter 3-Letter Amino acid
Y Tyr t rosine
G Gly glycine
F Phe hen lalanine
M Met methionine
A Ala alanine
S Ser serine
I Ile isoleucine
L Leu leucine
T Thr threonine
V Val valine
P Pro proline
K Lys lysine
H His histidine
Q Gln glutamine
E Glu glutamic acid
Z Gix Glu and/or GIn
W Trp tryptop han
R Arg arginine
D Asp aspartic acid
N Asn as ara ine
B Asx Asn and/or As
C Cys c steine
X Xaa unknown or other

It should be noted that all amino acid residue sequences represented herein by
for-
mulae have a left-to-right, orientation in the conventional direction of amino
terminus
to carboxy terminus. In addition, the phrase "amino acid residue" is broadly
defined
to include the amino acids listed in the Table of Correspondence as well as
modified
and unusual amino acids. Furthermore, it should be noted that a dash at the
begin-
ning or end of an amino acid residue sequence indicates a peptide bond to a
further

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
7
sequence of one or more amino acid residues or a covalent bond to an amino-
terminal group such as NH2 or acetyl or to a carboxy-terminal group such as
COOH.
Antibody: The term antibody in its various grammatical forms is used herein to
refer
to immunoglobulin molecules and immunologically active portions of
immunoglobulin
molecules of the compositions of this invention, i.e., molecules that contain
an anti-
body combining site or paratope. Exemplary antibody molecules are intact immu-
noglobulin molecules, substantially intact immunoglobulin molecules and
portions of
an immunoglobulin molecule, including those portions known in the art as Fab,
Fab',
F(ab')2 and Fv. A schematic drawing of Fab is shown in Figure 1. The term
"anti-
body" as used herein is also intended to include human, single chain and human-

ized antibodies, as well as binding fragments of such antibodies or modified
ver-
sions of such antibodies, such as multispecific, bispecific and chimeric
molecules
having at least one antigen binding determinant derived from an antibody
molecule.
Antibody Classes: Depending on the amino acid sequences of the constant domain
of their heavy chains, immunoglobulins can be assigned to different classes.
There
are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and
IgM,
and several of these may be further divided into subclasses (isotypes), e.g.
IgG-1,
IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The heavy chains constant domains
that
correspond to the different classes of immunoglobulins are called alpha (a),
delta
(8), epsilon (s), gamma (y) and mu ( ), respectively. The light chains of
antibodies
can be assigned to one of two clearly distinct types, called kappa (ic) and
lambda
(7~), based on the amino sequences of their constant domain. The subunit
structures
and three-dimensional configurations of different classes of immunoglobulins
are
well known.

Antibody Combining Site: An antibody combining site is that structural portion
of an
antibody molecule comprised of a heavy and light chain variable and
hypervariable
regions that specifically binds (immunoreacts with) an antigen. The term
immunore-
act in its various forms means specific binding between an antigenic
determinant-
containing molecule and a molecule containing an antibody combining site such
as
a whole antibody molecule or a portion thereof. Alternatively, an antibody
combining
site is known as an antigen binding site.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
8
Anti-haemolytic: Capability to inhibit haemolysis. Here by inhibition of the
haemolytic
activity of Pneumolysin on erythrocytes.

Base Pair (bp): A partnership of adenine (A) with thymine (T), or of cytosine
(C) with
guanine (G) in a double stranded DNA molecule. In RNA, uracil (U) is
substituted for
thymine.

Binding member: a polypeptide that can bind to an epitope on a Streptococcus
pneumoniae protein, in particular capable of binding specifically to
Pneumolysin.
Binding domain: An antigen binding site which specifically binds an antigen. A
bind-
ing member may be multispecific and contain two or more binding domains which
specifically bind two immunologically distinct antigens.

Chimeric antibody: An antibody in which the variable regions are from one
species
of animal and the constant regions are from another species of animal. For
example,
a chimeric antibody can be an antibody having variable regions which derive
from a
mouse monoclonal antibody and constant regions which are human.

Complementary Bases: Nucleotides that normally pair up when DNA or RNA adopts
a double stranded configuration.

Complementarity determining region or CDR: Regions in the V-domains of an anti-

body that together form the antibody recognizing and binding domain.
Complementary Nucleotide Sequence: A sequence of nucleotides in a single-
stranded molecule of DNA or RNA that is sufficiently complementary to that on
an-
other single strand to specifically hybridize to it with consequent hydrogen
bonding.

Conserved: A nucleotide sequence is conserved with respect to a preselected
(ref-
erence) sequence if it non-randomly hybridizes to an exact complement of the
pre-
selected sequence.

Conservative Substitution: The term conservative substitution as used herein
de-
notes the replacement of an amino acid residue by another, biologically
similar resi-

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
9
due. Examples of conservative substitutions include the substitution of one
hydro-
phobic residue such as isoleucine, valine, leucine or methionine for another,
or the
substitution of one polar residue for another, such as the substitution of
arginine for
lysine, glutamic for aspartic acids, or glutamine for asparagine, and the
like. The
term conservative substitution also includes the use of a substituted amino
acid in
place of an unsubstituted parent amino acid provided that molecules having the
substituted polypeptide also have the same function.

Constant Region or constant domain or C-domain: Constant regions are those
struc-
tural portions of an antibody molecule comprising amino acid residue sequences
within a given isotype which may contain conservative substitutions therein.
Exem-
plary heavy chain immunoglobulin constant regions are those portions of an
immu-
noglobulin molecule known in the art as CH1, CH2, CH3, CH4 and CH5. An exem-
plary light chain immunoglobulin constant region is that portion of an
immunoglobu-
lin molecule known in the art as CL.

Diabodies: This term refers to a small antibody fragments with two antigen-
binding
sites, which fragments comprise a heavy chain variable domain (VH) connected
to a
light chain variable domain (VL) in the same polypeptide chain (VH-VL). By
using a
linker that is too short to allow pairing between the two domains on the same
chain,
the domains are forced to pair with the complementary domains of another chain
and create two antigen-binding sites. Diabodies are described more fully in,
for ex-
ample, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad Sci.
USA
90: 6444-6448 (1993).
Dissociation constant, Kd: A measure to describe the strength of binding (or
affinity
or avidity) between receptors and their ligands, for example an antibody and
its anti-
gen. The smaller Kd, the stronger binding.

Downstream: Further along a DNA sequence in the direction of sequence
transcrip-
tion or read out, that is travelling in a 3'- to 5'-direction along the non-
coding strand
of the DNA or 5'- to 3'-direction along the RNA transcript.

Duplex DNA: A double-stranded nucleic acid molecule comprising two strands of
substantially complementary polynucleotides held together by one or more
hydrogen

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
bonds between each of the complementary bases present in a base pair of the du-

plex. Because the nucleotides that form a base pair can be either a
ribonucleotide
base or a deoxyribonucleotide base, the phrase "duplex DNA" refers to either a
DNA-DNA duplex comprising two DNA strands (ds DNA), or an RNA-DNA duplex
5 comprising one DNA and one RNA strand.

Fusion Polypeptide: A polypeptide comprised of at least two polypeptides and a
link-
ing sequence to operatively link the two polypeptides into one continuous
polypep-
tide. The two polypeptides linked in a fusion polypeptide are typically
derived from
10 two independent sources, and therefore a fusion polypeptide comprises two
linked
polypeptides not normally found linked in nature.

Fv: dual chain antibody fragment containing both a VH and a VL.

Gene: A nucleic acid whose nucleotide sequence codes for an RNA or
polypeptide.
A gene can be either RNA or DNA.

Human antibody framework: A molecule having an antigen binding site and essen-
tially all remaining immunoglobulin-derived parts of the molecule derived from
a hu-
man immunoglobulin.

Humanised antibody framework: A molecule having an antigen binding site
derived
from an immunoglobulin from a non-human species, whereas some or all of the re-

maining immunoglobulin-derived parts of the molecule is derived from a human
im-
munoglobulin. The antigen binding site may comprise: either a complete
variable
domain from the non-human immunoglobulin fused onto one or more human con-
stant domains; or one or more of the complementarity determining regions
(CDRs)
grafted onto appropriate human framework regions in the variable domain. In a
hu-
manized antibody, the CDRs can be from a mouse monoclonal antibody and the
other regions of the antibody are human.

Hybridization: The pairing of substantially complementary nucleotide sequences
(strands of nucleic acid) to form a duplex or heteroduplex by the
establishment of
hydrogen bonds between complementary base pairs. It is a specific, i.e. non-

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
11
random, interaction between two complementary polynucleotides that can be com-
petitively inhibited.

Immunoglobulin: The serum antibodies, including IgG, IgM, IgA, IgE and IgD.
Immunoglobulin isotypes: The names given to the Ig which have different H
chains,
the names are IgG (IgG1,2,3,4), IgM, IgA (IgA1,2), sIgA, IgE, IgD.

Immunologically distinct: The phrase immunologically distinct refers to the
ability to
distinguish between two polypeptides on the ability of an antibody to
specifically bind
one of the polypeptides and not specifically bind the other polypeptide.

Individual: A living animal or human in need of susceptible to a condition, in
particu-
lar an infectious disease" as defined below. The subject is an organism
possessing
leukocytes capable of responding to antigenic stimulation and growth factor
stimula-
tion. In preferred embodiments, the subject is a mammal, including humans and
non-human mammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats,
and mice. In the most preferred embodiment, the subject is a human.

Infectious disease: a disorder caused by one or more species of Streptococcus,
in
particular Streptococcus pneumoniae.

Isolated: is used to describe the various binding members, polypeptides and
nucleo-
tides disclosed herein, that has been identified and separated and/or
recovered from
a component of its natural environment. Contaminant components of its natural
en-
vironment are materials that would typically interfere with diagnostic or
therapeutic
uses for the polypeptide, and may include enzymes, hormones, and other
proteina-
ceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide
will
be purified.
Label and indicating means: refer in their various grammatical forms to single
atoms
and molecules that are either directly or indirectly involved in the
production of a
detectable signal to indicate the presence of a complex

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
12
Monoclonal Antibody: The phrase monoclonal antibody in its various grammatical
forms refers to a population of antibody molecules that contains only one
species of
antibody combining site capable of immunoreacting with a particular antigen. A
monoclonal antibody thus typically displays a single binding affinity for any
antigen
with which it immunoreacts. A monoclonal antibody may contain an antibody mole-

cule having a plurality of antibody combining sites, each immunospecific for a
differ-
ent antigen, e.g., a bispecific monoclonal antibody.

Multimeric: A polypeptide molecule comprising more than one polypeptide. A mul-

timer may be dimeric and contain two polypeptides and a multimer may be
trimeric
and contain three polypeptides. Multimers may be hom*omeric and contain two or
more identical polypeptides or a multimer may be heteromeric and contain two
or
more non-identical polypeptides.

Nucleic Acid: A polymer of nucleotides, either single or double stranded.

Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety (pen-
tose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to
the
sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that
combina-
tion of base and sugar is a nucleoside. When the nucleoside contains a
phosphate
group bonded to the 3' or 5' position of the pentose it is referred to as a
nucleotide.
A sequence of operatively linked nucleotides is typically referred to herein
as a
"base sequence" or "nucleotide sequence", and their grammatical equivalents,
and
is represented herein by a formula whose left to right orientation is in the
conven-
tional direction of 5'-terminus to 3'-terminus.

Nucleotide Analog: A purine or pyrimidine nucleotide that differs structurally
from A,
T, G, C, or U, but is sufficiently similar to substitute for the normal
nucleotide in a
nucleic acid molecule.
Pneumococcus: is used synonymously with Streptococcus pneumoniae.

Polyclonal antibody: Polyclonal antibodies are a mixture of antibody molecules
rec-
ognising a specific given antigen, hence polyclonal antibodies may recognise
differ-
ent epitopes within said antigen.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
13
Polynucleotide: A polymer of single or double stranded nucleotides. As used
herein
"polynucleotide" and its grammatical equivalents will include the full range
of nucleic
acids. A polynucleotide will typically refer to a nucleic acid molecule
comprised of a
linear strand of two or more deoxyribonucleotides and/or ribonucleotides. The
exact
size will depend on many factors, which in turn depends on the ultimate
conditions
of use, as is well known in the art. The polynucleotides of the present
invention in-
clude primers, probes, RNA/DNA segments, oligonucleotides or "oligos"
(relatively
short polynucleotides), genes, vectors, plasmids, and the like.
Polypeptide: The phrase polypeptide refers to a molecule comprising amino acid
residues which do not contain linkages other than amide linkages between
adjacent
amino acid residues.

Receptor: A receptor is a molecule, such as a protein, glycoprotein and the
like, that
can specifically (non-randomly) bind to another molecule.

Recombinant DNA (rDNA) molecule: A DNA molecule produced by operatively link-
ing two DNA segments. Thus, a recombinant DNA molecule is a hybrid DNA mole-
cule comprising at least two nucleotide sequences not normally found together
in
nature. rDNA's not having a common biological origin, i.e., evolutionarily
different,
are said to be "heterologous".

Specificity: The term specificity refers to the number of potential antigen
binding
sites which immunoreact with (specifically bind to) a given antigen in a
polypeptide.
The polypeptide may be a single polypeptide or may be two or more polypeptides
joined by disulfide bonding. A polypeptide may be monospecific and contain one
or
more antigen binding sites which specifically bind an antigen or a polypeptide
may
be bispecific and contain two or more antigen binding sites which specifically
bind
two immunologically distinct antigens. Thus, a polypeptide may contain a
plurality of
antigen binding sites which specifically bind the same or different antigens.
Serotype: Identification of bacteria within species of Streptococcus that
consist of
many strains differing from one another in a variety of characteristics.
Commonly
used characteristics defining serotypes are particular antigenic molecules.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
14
Single Chain Antibody or scFv: The phrase single chain antibody refers to a
single
polypeptide comprising one or more antigen binding sites. Furthermore,
although
the H and L chains of an Fv fragment are encoded by separate genes, they may
be
linked either directly or via a peptide, for example a synthetic linker can be
made
that enables them to be made as a single protein chain (known as single chain
anti-
body, sAb; Bird et al. 1988 Science 242:423-426; and Huston et al. 1988 PNAS
85:5879-5883) by recombinant methods. Such single chain antibodies are also en-

compassed within the term "antibody", and may be utilized as binding
determinants
in the design and engineering of a multispecific binding molecule.

Upstream: In the direction opposite to the direction of DNA transcription, and
there-
fore going from 5' to 3' on the non-coding strand, or 3' to 5' on the mRNA.

Valency: The term valency refers to the number of potential antigen binding
sites,
i.e. binding domains, in a polypeptide. A polypeptide may be monovalent and
con-
tain one antigen binding site or a polypeptide may be bivalent and contain two
anti-
gen binding sites. Additionally, a polypeptide may be tetravalent and contain
four
antigen binding sites. Each antigen binding site specifically binds one
antigen. When
a polypeptide comprises more than one antigen binding site, each antigen
binding
site may specifically bind the same or different antigens. Thus, a polypeptide
may
contain a plurality of antigen binding sites and therefore be multivalent and
a poly-
peptide may specifically bind the same or different antigens.

V-domain: Variable domain are those structural portions of an antibody
molecule
comprising amino acid residue sequences forming the antigen binding sites. An
ex-
emplary light chain immunoglobulin variable region is that portion of an immu-
noglobulin molecule known in the art as VL.

VL: Variable domain of the light chain.
VH: Variable domain of the heavy chain.

Vector: A rDNA molecule capable of autonomous replication in a cell and to
which a
DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to
bring

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
about replication of the attached segment. Vectors capable of directing the
expres-
sion of genes encoding for one or more polypeptides are referred to herein as
"ex-
pression vectors". Particularly important vectors allow cloning of cDNA
(complemen-
tary DNA) from mRNAs produced using reverse transcriptase.
5

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
16
Description

As described above, the present invention relates to binding members, in
particular
antibodies or fragments thereof capable of specifically recognising and
binding to a
Streptococcus pneumoniae protein, more specifically to Pneumolysin. The
binding
members according to the invention are particularly useful in the treatment of
dis-
eases caused by Streptococcus pneumoniae, as well as for being employed in di-
agnostic methods and kits for detecting the bacteria. Pneumolysin is
preferably a
polypeptide having the amino acid sequence shown in Figure 2 (SEQ ID NO 11).
Thus, the binding member according to the invention should preferably be
immuno-
logically active, for example as an antibody, such as being capable of binding
to an
antigen and presenting the antigen to immunoactive cells, thereby facilitating
phago-
cytosis of said antigen.
In particular the binding member is an antibody, such as any suitable antibody
known in the art, in particular antibodies as defined herein, such as
antibodies or
immunologically active fragments of antibodies, or single chain antibodies.
Antibody molecules are typically Y-shaped molecules whose basic unit consist
of
four polypeptides, two identical heavy chains and two identical light chains,
which
are covalently linked together by disulfide bonds. Each of these chains is
folded in
discrete domains. The C-terminal regions of both heavy and light chains are
con-
served in sequence and are called the constant regions, also known as C-
domains.
The N-terminal regions, also known as V-domains, are variable in sequence and
are
responsible for the antibody specificity. The antibody specifically recognizes
and
binds to an antigen mainly through six short complementarity-determining
regions
located in their V-domains (see Fig. 1).

The antibodies according to the invention are especially useful, since they
have a
strong affinity towards Pneumolysin.

Accordingly, the binding members according to the invention have a binding
domain
having a dissociation constant Kd for Pneumolysin which is less than 1 x 10-6.
M.
More preferably the dissociation constant Kd for Pneumolysin is less than 1 x
10"7 M,
more preferably less than 1 x 10-$M, more preferably less than 5 x 10"$M, more
pref-

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
17
erably less than 1 x 10-9M, more preferably less than 5 x 10"9M, more
preferably less
than I x 10"10M.

The affinity of the binding member towards the Pneumolysin is preferably
measured
as described in Example 4.

The binding member is preferably an isolated binding member as defined above,
and more preferably an isolated, pure binding member.

Anti-haemolytic activity
It is further contemplated that binding members having anti haemolytic
activity are
particular suitable in the treatment of diseases caused by Streptococcus
pneumo-
niae. With out being bound by the theory it is believed that binding of an
anti-
haemolytic binding member to Pneumolysin prevents the attachment of Pneumo-
lysin to the membrane of the target cell. In vitro functional assay is
prefereably per-
formed as described in example 2 and 3.

It is preferred that the binding member according to the invention is capable
of inhib-
iting haemolysis at least 50 % at a concentration of 4000 ng/ml in an assay as
de-
scribed in example 3. More preferably the binding member inhibts haemolysis by
at
least 60 % such as 80, such as 85, most preferably such as 90 % at a
concentration
of 4000 ng/ml in an assay as described in example 3.

Most preferred the binding member according to the invention is capable of
inhibit-
ing haemolysis at least 50 % at a concentration of 160 ng/mI in an assay as de-

scribed in example 3. More preferably the binding member inhibits haemolysis
by at
least 60 % such as 80, such as 85, most preferably 90 % at a concentration of
160
ng/ml in an assay as described in example 3.

Complementarity-determining regions

Without being bound by theory it is believed that the high binding strength
and/or
anti-haemolytic activity is caused by incorporating into the binding domain an
amino
acid sequence having one or more of the following motifs of the sequences
shown
below.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
18
In an embodiment the binding domain comprises at least one of the amino acid
se-
quence sets selected from the group of:
- the amino acid sequence sets SEQ ID NO 5 or a hom*ologue thereof, SEQ ID
NO 6 or a hom*ologue thereof, and SEQ ID NO 7 or a hom*ologue thereof, or
- the amino acid sequence sets SEQ ID NO 14 or a hom*ologue thereof, SEQ
ID NO 15 or a hom*ologue thereof, and SEQ ID NO 16 or a hom*ologue
thereof, or

preferably, the binding domain comprises at least one of the amino acid
sequence
sets selected from the group of:
- the amino acid sequence sets SEQ ID NO 8 or a hom*ologue thereof, SEQ ID
NO 9 or a hom*ologue thereof, and SEQ ID NO 10 or a hom*ologue thereof.
- the amino acid sequence sets SEQ ID NO 17 or a hom*ologue thereof, SEQ
ID NO 18 or a hom*ologue thereof, and SEQ ID NO 10.

In the amino acid sequence sets above, the amino acid sequences are preferably
arranged in the binding domain as CDRI, CDR2 and CDR3, i.e. spaced apart by
other amino acid sequences.
More specifically the binding domain preferably comprises a CDR1 region
compris-
ing a sequence selected from SEQ ID NO 5 and SEQ ID NO 8 or a hom*ologue
thereof, and/or the binding domain preferably comprises a CDR2 region
comprising
a sequence selected from SEQ ID NO 6 and SEQ ID NO 9 or a hom*ologue thereof,
and/or the binding domain preferably comprises a CDR3 region comprising a se-
quence selected from SEQ ID NO 7 and SEQ ID NO 10 or a hom*ologue thereof.
Alternatively the binding domain preferably comprises a CDR1 region comprising
a
sequence selected from SEQ ID NO 14 and SEQ ID NO 17 or a hom*ologue thereof,
and/or the binding domain preferably comprises a CDR2 region comprising a se-
quence selected from SEQ ID NO 15 and SEQ ID NO 18 or a hom*ologue thereof,
and/or the binding domain preferably comprises a CDR3 region comprising a se-
quence selected from SEQ ID NO 16 and SEQ ID NO 10 or a hom*ologue thereof.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
19
The findings of the applicant described herein suggest that the sequence of
the
variable heavy chain may be important for haemolytic activity. Thus preferred
em-
bodiments include binding domains comprising one or more of the sequences se-
quence selected from the group of; SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 17,
SEQ ID NO 18 and SEQ ID NO 10 or a hom*ologue thereof. Especially preferred, is
a binding domain comprising SEQ ID NO 9 or SEQ ID NO 18 or a hom*ologue
thereof. Mostly preferred is a binding domain comprising SEQ ID NO 10 or a
hom*o-
logue thereof.

Thus it is particularly preferably, that the variable part of the binding
domain com-
prises a sequence selected from SEQ ID NO 3 and SEQ ID NO 4 or a hom*ologue
thereof, wherein a hom*ologue is as defined elsewhere herein.

Alternatively, the variable part of the binding domain comprises a sequence
selected
from SEQ ID NO 12 and SEQ ID NO 13 or a hom*ologue thereof, wherein a hom*o-
logue is as defined elsewhere herein.

In preferred specific embodiment the variable light chain of the binding
domain com-
prises a sequence selected from SEQ ID NO 3 and SEQ ID NO 12 or/and most
preferably the variable heavy chain of the binding domain comprises a sequence
selected from SEQ ID NO 4 and SEQ ID NO 13.

The hom*ology of any one of the hom*ologues described above preferably confers
the
binding domain comprising one or more hom*ologues with dissociation constant Kd
for Pneumolysin as defined above.

Identity and hom*ology

The term "identity" shall be construed to mean the percentage of amino acid
resi-
dues in the candidate sequence that are identical with the residue of a
correspond-
ing sequence to which it is compared, after aligning the sequences and
introducing
gaps, if necessary to achieve the maximum percent identity for the entire
sequence,
and not considering any conservative substitutions as part of the sequence
identity.
Neither N- or C-terminal extensions nor insertions shall be construed as
reducing
identity or hom*ology. Methods and computer programs for the alignment are well
known in the art. Sequence identity may be measured using sequence analysis

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
software (e.g., Sequence Analysis Software Package, Genetics Computer Group,
University of Wisconsin Biotechnology Center, 1710 University Ave., Madison,
Wis.
53705). This software matches similar sequences by assigning degrees of hom*ol-
ogy to various substitutions, deletions, and other modifications.
5
A hom*ologue of one or more of the sequences specified herein may vary in one
or
more amino acids as compared to the sequences defined, but is capable of
perform-
ing the same function, i.e. a hom*ologue may be envisaged as a functional
equiva-
lent of a predetermined sequence.
As described above a hom*ologue of any of the predetermined sequences herein
may be defined as:

i) hom*ologues comprising an amino acid sequence capable of recognising an
antigen also being recognised by the predetermined amino acid sequence,
and/or

ii) hom*ologues comprising an amino acid sequence capable of binding selec-
tively to an antigen, wherein said antigen is also bound selectively by a pre-
determined sequence, and/or

iii) hom*ologues having a substantially similar or higher binding affinity to
Pneu-
molysin as a binding domain comprising a predetermined sequence, such as
SEQ ID NO 3, 4 12 and 13.
Examples of hom*ologues comprises one or more conservative amino acid substitu-
tions including one or more conservative amino acid substitutions within the
same
group of predetermined amino acids, or a plurality of conservative amino acid
substi-
tutions, wherein each conservative substitution is generated by substitution
within a
different group of predetermined amino acids.

hom*ologues may thus comprise conservative substitutions independently of one
another, wherein at least one glycine (Gly) of said hom*ologue is substituted
with an
amino acid selected from the group of amino acids consisting of Ala, Val, Leu,
and
IIe, and independently thereof, hom*ologues, wherein at least one of said
alanines
(Ala) of said hom*ologue thereof is substituted with an amino acid selected
from the

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
21
group of amino acids consisting of Gly, Val, Leu, and lie, and independently
thereof,
hom*ologues, wherein at least one valine (Val) of said hom*ologue thereof is
substi-
tuted with an amino acid selected from the group of amino acids consisting of
Gly,
Ala, Leu, and Ile, and independently thereof, hom*ologues thereof, wherein at
least
one of said leucines (Leu) of said hom*ologue thereof is substituted with an
amino
acid selected from the group of amino acids consisting of Gly, Ala, Val, and
lie, and
independently thereof, hom*ologues thereof, wherein at least one isoleucine
(Ile) of
said hom*ologues thereof is substituted with an amino acid selected from the
group
of amino acids consisting of Gly, Ala, Val and Leu, and independently thereof,
hom*ologues thereof wherein at least one of said aspartic acids (Asp) of said
hom*o-
logue thereof is substituted with an amino acid selected from the group of
amino
acids consisting of Glu, Asn, and Gln, and independently thereof, hom*ologues
thereof, wherein at least one of said phenylalanines (Phe) of said hom*ologues
thereof is substituted with an amino acid selected from the group of amino
acids
consisting of Tyr, Trp, His, Pro, and preferably selected from the group of
amino
acids consisting of Tyr and Trp, and independently thereof, hom*ologues
thereof,
wherein at least one of said tyrosines (Tyr) of said hom*ologues thereof is
substituted
with an amino acid selected from the group of amino acids consisting of Phe,
Trp,
His, Pro, preferably an amino acid selected from the group of amino acids
consisting
of Phe and Trp, and independently thereof, hom*ologues thereof, wherein at
least
one of said arginines (Arg) of said fragment is substituted with an amino acid
se-
lected from the group of amino acids consisting of Lys and His, and
independently
thereof, hom*ologues thereof, wherein at least one lysine (Lys) of said
hom*ologues
thereof is substituted with an amino acid selected from the group of amino
acids
consisting of Arg and His, and independently thereof, hom*ologues thereof,
wherein
at least one of said aspargines (Asn) of said hom*ologues thereof is
substituted with
an amino acid selected from the group of amino acids consisting of Asp, Glu,
and
Gln, and independently thereof, hom*ologues thereof, wherein at least one
glutamine
(Gln) of said hom*ologues thereof is substituted with an amino acid selected
from the
group of amino acids consisting of Asp, Glu, and Asn, and independently
thereof,
hom*ologues thereof, wherein at least one proline (Pro) of said hom*ologues
thereof
is substituted with an amino acid selected from the group of amino acids
consisting
of Phe, Tyr, Trp, and His, and independently thereof, hom*ologues thereof,
wherein
at least one of said cysteines (Cys) of said hom*ologues thereof is substituted
with

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
22
an amino acid selected from the group of amino acids consisting of Asp, Glu,
Lys,
Arg, His, Asn, Gin, Ser, Thr, and Tyr.

Conservative substitutions may be introduced in any position of a preferred
prede-
termined sequence. It may however also be desirable to introduce non-
conservative
substitutions, particularly, but not limited to, a non-conservative
substitution in any
one or more positions.

A non-conservative substitution leading to the formation of a functionally
equivalent
hom*ologue of the sequences herein would for example i) differ substantially in
polar-
ity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp,
Val, IIe,
Leu, Phe or Met) substituted for a residue with a polar side chain such as
Gly, Ser,
Thr, Cys, Tyr, Asn, or Gln or a charged amino acid such as Asp, Glu, Arg, or
Lys, or
substituting a charged or a polar residue for a non-polar one; and/or ii)
differ sub-
stantially in its effect on polypeptide backbone orientation such as
substitution of or
for Pro or Gly by another residue; and/or iii) differ substantially in
electric charge, for
example substitution of a negatively charged residue such as Glu or Asp for a
posi-
tively charged residue such as Lys, His or Arg (and vice versa); and/or iv)
differ sub-
stantially in steric bulk, for example substitution of a bulky residue such as
His, Trp,
Phe or Tyr for one having a minor side chain, e.g. Ala, Gly or Ser (and vice
versa).
Substitution of amino acids may in one embodiment be made based upon their hy-
drophobicity and hydrophilicity values and the relative similarity of the
amino acid
side-chain substituents, including charge, size, and the like. Exemplary amino
acid
substitutions which take various of the foregoing characteristics into
consideration
are well known to those of skill in the art and include: arginine and lysine;
glutamate
and aspartate; serine and threonine; glutamine and asparagine; and valine,
leucine
and isoleucine.

In an embodiment the binding domain comprises a hom*ologue having an amino acid
sequence at least 60 % identical to a sequence selected from SEQ ID NO 5, 6,
7, 8,
9, 10, 14, 15, 16, 17 and 18. In a preferred embodiment the binding domain com-

prises a hom*ologue having an amino acid sequence at least 60 % indentical to a
sequence selected from SEQ ID NO 3, 4 12 and 13.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
23
More preferably the hom*ologue is at least 65 %, such as at least 70 %
identical,
such as at least 75 % identical, such as at least 80 % identical, such as at
least 85
% identical, such as at least 90 % identical, such as at least 95 % identical,
such as
at least 98 % identical to a sequence selected from selected from SEQ ID NO 5,
6,
7, 8, 9, 10, 14, 15, 16, 17 and 18 or preferably SEQ ID NO 3, 4 12 and 13.

In a more preferred embodiment the percentages mentioned above relates to the
identity of the sequence of a hom*ologue as compared to a sequence selected
from
SEQ ID NO 3, 4 12 and 13..
Epitopes
The anti-haemolytic binding member according to the present invention
preferably
recognize and bind to an epitope also recognized by an antibody having a
variable
part comprising a sequence selected from the group of SEQ ID NO 3, 4, 12 or
13.
In an embodiment the binding domain of the anti-haemolytic binding member, rec-

ognise an epitope in the N-terminal part of Pneumolysin. Preferably within the
N-
terminal part corresponding to amino acid 1-436 of Pneumolysin as identified
by
SEQ ID NO 11. It is further preferred that the epitope recognized by the
binding do-
main is within amino acid 50-436, or preferably amino acid 100-436 of
Pneumolysin
as identified by SEQ ID NO 11. In specific embodiment the epitope recognized
by
the binding member is with in amino acid 200-435 or 300-435 of Pneumolysin as
identified by SEQ ID NO 11.
The binding domain of the binding member of the invention preferably recognise
an
epitope comprised by the amino acid sequence identified by SEQ ID NO: 27. In a
prefered embodiment the binding domain recognises an epitope comprised by SEQ
ID NOs 28, 29, 30 and 31 more preferably an epitope comprised by SEQ ID 29 and
30.

It is further preferred that the epitope recognized by the binding domain is
within
amino acid 400-438, or preferably amino acid 420-436 of Pneumolysin as
identified
by SEQ ID NO 11. In specific embodiment the epitope recognized by the binding

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
24
member is with in amino acid 422-436 or 425-436 of Pneumolysin as identified
by
SEQ ID NO 11.

Serotypes

As described above, 90 different serotypes of Streptococcus pneumoniae have
been identified. It is preferred that the binding member according to this
invention is
capable of binding Pneumolysin from two or more different Pneumococcus sero-
types, such as from three or more different Pneumococcus serotypes, such as
from
four or more different Pneumococcus serotypes, such as from five or more
different
Pneumococcus serotypes. Most preferably the binding member according to the
invention is capable of recognising and binding Pneumococcus from essentially
all
serotypes.
Monoclonal/polyclonal antibodies

In one embodiment of the invention, the binding member is an antibody, wherein
the
antibody may be a polyclonal or a monoclonal antibody derived from a mammal or
mixtures of monoclonal antibodies. In a preferred embodiment the binding
member
is a monoclonal antibody or a fragment thereof. The antibody may be any kind
of
antibody; however it is preferably an IgG antibody. More preferably the
antibody is
an IgG1 antibody or a fragment thereof.

Monoclonal antibodies (Mab's) are antibodies, wherein every antibody molecule
is
similar and thus recognises the same epitope. Monoclonal antibodies are in
general
produced by a hybridoma cell line. Methods of making monoclonal antibodies and
antibody-synthesizing hybridoma cells are well known to those skilled in the
art. An-
tibody-producing hybridomas may for example be prepared by fusion of an
antibody-
producing B lymphocyte with an immortalized cell line.

A monoclonal antibody can be produced by the following steps. In all
procedures, an
animal is immunized with an antigen such as a protein (or peptide thereof) as
de-
scribed above for preparation of a polyclonal antibody. The immunization is
typically
accomplished by administering the immunogen to an immunologically competent
mammal in an immunologically effective amount, i.e., an amount sufficient to
pro-

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
duce an immune response. Preferably, the mammal is a rodent such as a rabbit,
rat
or mouse. The mammal is then maintained on a booster schedule for a time
period
sufficient for the mammal to generate high affinity antibody molecules as
described.
A suspension of antibody-producing cells is removed from each immunized mammal
5 secreting the desired antibody. After a sufficient time to generate high
affinity anti-
bodies, the animal (e.g., mouse) is sacrificed and antibody-producing
lymphocytes
are obtained from one or more of the lymph nodes, spleens and peripheral
blood.
Spleen cells are preferred, and can be mechanically separated into individual
cells
in a physiological medium using methods well known to one of skill in the art.
The
10 antibody-producing cells are immortalized by fusion to cells of a mouse
myeloma
line. Mouse lymphocytes give a high percentage of stable fusions with mouse
hom*o-
logous myelomas, however rat, rabbit and frog somatic cells can also be used.
Spleen cells of the desired antibody-producing animals are immortalized by
fusing
with myeloma cells, generally in the presence of a fusing agent such as
polyethyl-
15 ene glycol. Any of a number of myeloma cell lines suitable as a fusion
partner are
used with to standard techniques, for example, the P3-NS1/1-Ag4-1, P3-x63-
Ag8.653 or Sp2/O-Ag14 myeloma lines, available from the American Type Culture
Collection (ATCC), Rockville, Md.

20 Monoclonal antibodies can also be generated by other methods well known to
those
skilled in the art of recombinant DNA technology. An alternative method,
referred to
as the "combinatorial antibody display" method, has been developed to identify
and
isolate antibody fragments having a particular antigen specificity, and can be
utilized
to produce monoclonal antibodies.
Polyclonal antibodies is a mixture of antibody molecules recognising a
specific given
antigen, hence polyclonal antibodies may recognise different epitopes within
said
antigen. In general polyclonal antibodies are purified from serum of a mammal,
which previously has been immunized with the antigen. Polyclonal antibodies
may
for example be prepared by any of the methods described in Antibodies: A
Labora-
tory Manual, By Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press,
1988. Polyclonal antibodies may be derived from any suitable mammalian
species,
for example from mice, rats, rabbits, donkeys, goats, and sheep.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
26
Specificity

The binding member may be monospecific towards Pneumolysin, wherein
specificity
towards Pneumolysin means that the binding member immunoreacts with Pneumo-
lysin. In another embodiment, the binding member is bispecific or
multispecific hav-
ing at least one portion being specific towards Pneumolysin.

Monovalent antibodies

The monospecific binding member may be monovalent, i.e. having only one
binding
domain.

For a monovalent antibody, the immunoglobulin constant domain amino acid
residue
sequences comprise the structural portions of an antibody molecule known in
the art
as CH1, CH2, CH3 and CH4. Preferred are those binding members which are
known in the art as CL. Preferrred CL polypeptides are selected from the group
con-
sisting of Ckappa and Ciambda-

Furthermore, insofar as the constant domain can be either a heavy or light
chain
constant domain (CH or CL, respectively), a variety of monovalent binding
member
compositions are contemplated by the present invention. For example, light
chain
constant domains are capable of disulfide bridging to either another light
chain con-
stant domain, or to a heavy chain constant domain. In contrast, a heavy chain
con-
stant domain can form two independent disulfide bridges, allowing for the
possibility
of bridging to both another heavy chain and to a light chain, or to form
polymers of
heavy chains.

Thus, in another embodiment, the invention contemplates a composition
comprising
a monovalent polypeptide wherein the constant chain domain C has a cysteine
resi-
due capable of forming at least one disulfide bridge, and where the
composition
comprises at least two monovalent polypeptides covalently linked by said
disulfide
bridge.

In preferred embodiments, the constant chain domain C can be either CL or CH.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
27
Where C is CL, the CL polypeptide is preferably selected from the group
consisting of
Ckappa and Clambda,

In another embodiment, the invention contemplates a binding member composition
comprising a monovalent polypeptide as above except where C is CL having a cys-

teine residue capable of forming a disulfide bridge, such that the composition
con-
tains two monovalent polypeptides covalently linked by said disulfide bridge.

Multivalent
In another embodiment of the invention the binding member is a multivalent
binding
member having at least two binding domains. The binding domains may have speci-

ficity for the same ligand or for different ligands.

Multispecificity, including bispecificity

In a preferred embodiment the present invention relates to multispecific
binding
members, which have affinity for and are capable of binding at least two
different
entities. Multispecific binding members can include bispecific binding
members.
In one embodiment the multispecific molecule is a bispecific antibody (BsAb),
which
carries at least two different binding domains, at least one of which is of
antibody
origin.

A bispecific molecule of the invention can also be a single chain bispecific
molecule,
such as a single chain bispecific antibody, a single chain bispecific molecule
com-
prising one single chain antibody and a binding domain, or a single chain
bispecific
molecule comprising two binding domains. Multispecific molecules can also be
sin-
gle chain molecules or may comprise at least two single chain molecules.
The multispecific, including bispecific, antibodies may be produced by any
suitable
manner known to the person skilled in the art.

The traditional approach to generate bispecific whole antibodies was to fuse
two
hybridoma cell lines each producing an antibody having the desired
specificity. Be-

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
28
cause of the random association of immunoglobulin heavy and light chains,
these
hybrid hybridomas produce a mixture of up to 10 different heavy and light
chain
combinations, oniy one of which is the bispecific antibody. Therefore, these
bispeci-
fic antibodies have to be purified with cumbersome procedures, which
considerably
decrease the yield of the desired product.

Alternative approaches include in vitro linking of two antigen specificities
by chemi-
cal cross-linking of cysteine residues either in the hinge or via a
genetically intro-
duced C-terminal Cys as described above. An improvement of such in vitro assem-

bly was achieved by using recombinant fusions of Fab's with peptides that
promote
formation of heterodimers. However, the yield of bispecific product in these
methods
is far less than 100%.

A more efficient approach to produce bivalent or bispecific antibody
fragments, not
involving in vitro chemical assembly steps, was described by Holliger et al.
(1993).
This approach takes advantage of the observation that scFv's secreted from
bacte-
ria are often present as both monomers and dimers. This observation suggested
that the VH and VL of different chains could pair, thus forming dimers and
larger
complexes. The dimeric antibody fragments, also named "diabodies" by Hollinger
et
al., are in fact small bivalent antibody fragments that assembled in vivo. By
linking
the VH and VL of two different antibodies 1 and 2, to form "cross-over" chains
VH
1VL 2 and VH 2-VL 1, the dimerisation process was shown to reassemble both an-
tigen-binding sites. The affinity of the two binding sites was shown to be
equal to the
starting scFv's, or even to be 10-fold increased when the polypeptide linker
cova-
lently linking VH and VL was removed, thus generating two proteins each
consisting
of a VH directly and covalently linked to a VL not pairing with the VH. This
strategy of
producing bispecific antibody fragments was also described in several patent
appli-
cations. Patent application WO 94/09131 (SCOTGEN LTD; priority date Oct. 15,
1992) relates to a bispecific binding protein in which the binding domains are
de-
rived from both a VH and a VL region either present at two chains or linked in
an
scFv, whereas other fused antibody domains, e.g. C-terminal constant domains,
are
used to stabilise the dimeric constructs. Patent application WO 94/13804 (CAM-
BRIDGE ANTIBODY TECHNOLOGY/MEDICAL RESEARCH COUNCIL; first priority
date Dec. 4, 1992) relates to a polypeptide containing a VH and a VL which are
in-

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
29
capable of associating with each other, whereby the V-domains can be connected
with or without a linker.

Mallender and Voss, 1994 (also described in patent application WO 94/13806;
DOW
CHEMICAL CO; priority date Dec. 11, 1992) reported the in vivo production of a
single-chain bispecific antibody fragment in E. coli. The bispecificity of the
bivalent
protein was based on two previously produced monovalent scFv molecules pos-
sessing distinct specificities, being linked together at the genetic level by
a flexible
polypeptide linker. Traditionally, whenever single-chain antibody fragments
are re-
ferred to, a single molecule consisting of one heavy chain linked to one
(correspond-
ing) light chain in the presence or absence of a polypeptide linker is
implicated.
When making bivalent or bispecific antibody fragments through the "diabody" ap-

proach (Holliger et al., (1993) and patent application WO 94/09131) or by the
"dou-
ble scFv" approach (Mallender and Voss, 1994 and patent application WO
94/13806), again the VH is linked to a (the corresponding) VL.

The multispecific molecules described above can be made by a number of
methods.
For example, all specificities can be encoded in the same vector and expressed
and
assembled in the same host cell. This method is particularly useful where the
multi-
specific molecule is a mAb X mAb, mAb X Fab, Fab X F(ab')2 or ligand X Fab
fusion
protein. Various other methods for preparing bi- or multivalent antibodies are
de-
scribed for example described in U.S. Pat. Nos. 5,260,203; 5,455,030;
4,881,175;
5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

By using a bispecific or multispecific binding member according to the
invention the
invention offers several advantages as compared to monospecific/monovalent
bind-
ing members.

A bispecific/multispecific binding member has a first binding domain capable
of spe-
cifically recognising and binding a Streptococcus protein, in particular
Pneumolysin,
whereas the other binding domain(s) may be used for other purposes:

In one embodiment at least one other binding domain is used for binding to a
Strep-
tococcus protein, such as binding to another epitope on the same Streptococcus
protein as compared to the first binding domain. Thereby specificity for the
Strepto-

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
coccus species may be increased as well as increase of avidity of the binding
mem-
ber.

In another embodiment the at least one other binding domain may be used for
spe-
5 cifically binding a mammalian cell, such as a human cell. It is preferred
that the at
least other binding domain is capable of binding an immunoactive cell, such as
a
leucocyte, a macrophage, a lymphocyte, a basophilic cell, and/or an
eosinophilic
cell, in order to increase the effect of the binding member in a therapeutic
method.
This may be accomplished by establishing that the at least one other binding
do-
10 main is capable of specifically binding a mammalian protein, such as a
human pro-
tein, such as a protein selected from any of the cluster differentiation
proteins (CD),
in particular CD64 and/or CD89. A method for producing bispecific antibodies
having
CD64 specificity is described in US 6,071,517 to Medarex, Inc.

15 An "effector cell" as used herein refers to an immune cell which is a
leukocyte or a
lymphocyte. Specific effector cells express specific Fc receptors and carry
out spe-
cific immune functions. For example, monocytes, macrophages, neutrophils, eosi-

nophils, and lymphocytes which express CD89 receptor are involved in specific
kill-
ing of target cells and presenting antigens to other components of the immune
sys-
20 tem, or binding to cells that present antigens.

Humanised antibody framework

It is not always desirable to use non-human antibodies for human therapy,
since the
25 non-human "foreign" epitopes may elicit immune response in the individual
to be
treated. To eliminate or minimize the problems associated with non-human
antibod-
ies, it is desirable to engineer chimeric antibody derivatives, i.e.,
"humanized" anti-
body molecules that combine the non-human Fab variable region binding determi-
nants with a human constant region (Fc). Such antibodies are characterized by
30 equivalent antigen specificity and affinity of the monoclonal and
polyclonal antibod-
ies described above, and are less immunogenic when administered to humans, and
therefore more likely to be tolerated by the individual to be treated.

Accordingly, in one embodiment the binding member has a binding domain carried
on a humanised antibody framework, also called a humanised antibody.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
31
Humanised antibodies are in general chimeric antibodies comprising regions
derived
from a human antibody and regions derived from a non-human antibody, such as a
rodent antibody. Humanisation (also called Reshaping or CDR-grafting) is a
well-
established technique for reducing the immunogenicity of monoclonal antibodies
(mAbs) from xenogeneic sources (commonly rodent), increasing the hom*ology to a
human immunoglobulin, and for improving their activation of the human immune
system. Thus, humanized antibodies are typically human antibodies in which
some
CDR residues and possibly some framework residues are substituted by residues
from analogous sites in rodent antibodies.

It is further important that humanized antibodies retain high affinity for the
antigen
and other favourable biological properties. To achieve this goal, according to
a pre-
ferred method, humanized antibodies are prepared by a process of analysis of
the
parental sequences and various conceptual humanized products using three-
dimensional models of the parental and humanized sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to those skilled
in
the art. Computer programs are available which illustrate and display probable
three-dimensional conformational structures of selected candidate
immunoglobulin
sequences. Inspection of these displays permits analysis of the likely role of
certain
residues in the functioning of the candidate immunoglobulin sequence, i.e.,
the
analysis of residues that influence the ability of the candidate
immunoglobulin to
bind its antigen. In this way, FR residues can be selected and combined from
the
recipient and import sequences so that the desired antibody characteristic,
such as
increased affinity for the target antigen(s), is maximized, although it is the
CDR resi-
dues that directly and most substantially influence antigen binding.

One method for humanising MAbs related to production of chimeric antibodies in
which an antigen binding site comprising the complete variable domains of one
anti-
body are fused to constant domains derived from a second antibody, preferably
a
human antibody. Methods for carrying out such chimerisation procedures are for
example described in EP-A-0 120 694 (Celltech Limited), EP-A-0 125 023 (Genen-
tech Inc.), EP-A-0 171 496 (Res. Dev. Corp. Japan), EP-A-0173494 (Stanford Uni-

versity) and EP-A-0 194 276 (Celitech Limited). A more complex form of
humanisa-
tion of an antibody involves the re-design of the variable region domain so
that the

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
32
amino acids constituting the non-human antibody binding site are integrated
into the
framework of a human antibody variable region (Jones et al., 1986).

The humanized antibody of the present invention may be made by any method ca-
pable of replacing at least a portion of a CDR of a human antibody with a CDR
de-
rived from a non-human antibody. Winter describes a method which may be used
to
prepare the humanized antibodies of the present invention (UK Patent
Application
GB 2188638A, filed on Mar. 26, 1987), the contents of which is expressly
incorpo-
rated by reference. The human CDRs may be replaced with non-human CDRs using
oligonucleotide site-directed mutagenesis as described in the examples below.

As an example the humanized antibody of the present invention may be made as
described in the brief explanation below. The humanized antibodies of the
present
invention may be produced by the following process:
(a) constructing, by conventional techniques, an expression vector containing
an
operon with a DNA sequence encoding an antibody heavy chain in which the
CDRs and such minimal portions of the variable domain framework region that
are required to retain antibody binding specificity are derived from a non-
human
immunoglobulin, and the remaining parts of the antibody chain are derived from
a human immunoglobulin, thereby producing the vector of the invention;

(b) constructing, by conventional techniques, an expression vector containing
an
operon with a DNA sequence encoding a complementary antibody light chain in
which the CDRs and such minimal portions of the variable domain framework
region that are required to retain donor antibody binding specificity are
derived
from a non-human immunoglobulin, and the remaining parts of the antibody
chain are derived from a human immunoglobulin, thereby producing the vector
of the invention;
(c) transfecting the expression vectors into a host cell by conventional
techniques to
produce the transfected host cell of the invention; and

(d) culturing the transfected cell by conventional techniques to produce the
human-
ised antibody of the invention.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
33
The host cell may be cotransfected with the two vectors of the invention, the
first
vector containing an operon encoding a light chain derived polypeptide and the
sec-
ond vector containing an operon encoding a heavy chain derived polypeptide.
The
two vectors contain different selectable markers, but otherwise, apart from
the anti-
body heavy and light chain coding sequences, are preferably identical, to
ensure, as
far as possible, equal expression of the heavy and light chain polypeptides.
Alterna-
tively, a single vector may be used, the vector including the sequences
encoding
both the light and the heavy chain polypeptides. The coding sequences for the
light
and heavy chains may comprise cDNA or genomic DNA or both.

The host cell used to express the altered antibody of the invention may be
either a
bacterial cell such as Escherichia coli, or a eukaryotic cell. In particular a
mammal-
ian cell of a well defined type for this purpose, such as a myeloma cell or a
Chinese
hamster ovary cell may be used.

The general methods by which the vectors of the invention may be constructed,
transfection methods required to produce the host cell of the invention and
culture
methods required to produce the antibody of the invention from such host cells
are
all conventional techniques. Likewise, once produced, the humanized antibodies
of
the invention may be purified according to standard procedures as described
below.
Human antibody framework

In a more preferred embodiment the invention relates to a binding member,
wherein
the binding domain is carried by a human antibody framework, i.e. wherein the
anti-
bodies have a greater degree of human peptide sequences than do humanised anti-

bodies.

Human mAb antibodies directed against human proteins can be generated using
transgenic mice carrying the complete human immune system rather than the
mouse system. Splenocytes from these transgenic mice immunized with the
antigen
of interest are used to produce hybridomas that secrete human mAbs with
specific
affinities for epitopes from a human protein (see, e.g., Wood et al.
International
Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741;

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
34
Lonberg et al. International Application WO 92/03918; Kay et al. International
Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L.
et
al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad.
Sci.
USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al.
1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).
Such transgenic mice are available from Abgenix, Inc., Fremont, Calif., and
Meda-
rex, Inc., Annandale, N.J. It has been described that the hom*ozygous deletion
of the
antibody heavy-chain joining region (IH) gene in chimeric and germ-line mutant
mice
results in complete inhibition of endogenous antibody production. Transfer of
the
human germ-line immunoglobulin gene array in such germ-line mutant mice will
re-
sult in the production of human antibodies upon antigen challenge. See, e.g.,
Jako-
bovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al.,
Nature
362:255-258 (1993); Bruggermann et al., Year in Immunol. 7:33 (1993); and
Duchosal et al. Nature 355:258 (1992). Human antibodies can also be derived
from
phage-display libraries (Hoogenboom et al., J. Mol. Biol. 227: 381 (1991);
Marks et
al., J. Mol. Biol. 222:581-597 (1991); Vaughan, et al., Nature Biotech 14:309
(1996)).

Fragments

In one embodiment of the invention the binding member is a fragment of an anti-

body, preferably an antigen binding fragment or a variable region. Examples of
anti-
body fragments useful with the present invention include Fab, Fab', F(ab') 2
and Fv
fragments. Papain digestion of antibodies produces two identical antigen
binding
fragments, called the Fab fragment, each with a single antigen binding site,
and a
residual "Fc" fragment, so-called for its ability to crystallize readily.
Pepsin treatment
yields an F(ab') 2 fragment that has two antigen binding fragments which are
capable
of cross-linking antigen, and a residual other fragment (which is termed
pFc'). Addi-
tional fragments can include diabodies, linear antibodies, single-chain
antibody
molecules, and multispecific antibodies formed from antibody fragments.

The antibody fragments Fab, Fv and scFv differ from whole antibodies in that
the
antibody fragments carry only a single antigen-binding site. Recombinant
fragments
with two binding sites have been made in several ways, for example, by
chemical

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
cross-linking of cysteine residues introduced at the C-terminus of the VH of
an Fv
(Cumber et al., 1992), or at the C-terminus of the VL of an scFv (Pack and
Pluck-
thun, 1992), or through the hinge cysteine residues of Fab's (Carter et al.,
1992).

5 Preferred antibody fragments retain some or essential all the ability of an
antibody to
selectively binding with its antigen or receptor. Some preferred fragments are
de-
fined as follows:

(1) Fab is the fragment that contains a monovalent antigen-binding fragment of
an
10 antibody molecule. A Fab fragment can be produced by digestion of whole
anti-
body with the enzyme papain to yield an intact light chain and a portion of
one
heavy chain.

(2) Fab' is the fragment of an antibody molecule and can be obtained by
treating
15 whole antibody with pepsin, followed by reduction, to yield an intact light
chain
and a portion of the heavy chain. Two Fab' fragments are obtained per antibody
molecule. Fab' fragments differ from Fab fragments by the addition of a few
resi-
dues at the carboxyl terminus of the heavy chain CH1 domain including one or
more cysteines from the antibody hinge region.
(3) (Fab')2 is the fragment of an antibody that can be obtained by treating
whole
antibody with the enzyme pepsin without subsequent reduction. F(ab')2 is a
dimer of two Fab' fragments held together by two disulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigen
recogni-
tion and binding site. This region consists of a dimer of one heavy and one
light
chain variable domain in a tight, non-covalent association (VH -V L dimer). It
is in
this configuration that the three CDRs of each variable domain interact to
define
an antigen binding site on the surface of the VH -V L dimer. Collectively, the
six
CDRs confer antigen binding specificity to the antibody. However, even a
single
variable domain (or half of an Fv comprising only three CDRs specific for an
an-
tigen) has the ability to recognize and bind antigen, although at a lower
affinity
than the entire binding site.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
36
In one embodiment of the present invention the antibody is a single chain
antibody
("SCA"), defined as a genetically engineered molecule containing the variable
region
of the light chain, the variable region of the heavy chain, linked by a
suitable poly-
peptide linker as a genetically fused single chain molecule. Such single chain
anti-
bodies are also referred to as "single-chain Fv" or "sFv" antibody fragments.
Gener-
ally, the Fv polypeptide further comprises a polypeptide linker between the VH
and
VL domains that enables the sFv to form the desired structure for antigen
binding.
The antibody fragments according to the invention may be produced in any
suitable
manner known to the person skilled in the art. Several microbial expression
systems
have already been developed for producing active antibody fragments, e.g. the
pro-
duction of Fab in various hosts, such as E. coli (Better et al., 1988, Skerra
and
Pluckthun, 1988, Carter et al., 1992), yeast (Horwitz et al., 1988), and the
filamen-
tous fungus Trichoderma reesei (Nyyssonen et al., 1993) has been described.
The
recombinant protein yields in these alternative systems can be relatively high
(1-2 g/I
for Fab secreted to the periplasmic space of E. coli in high cell density
fermentation,
see Carter et al., 1992), or at a lower level, e.g. about 0.1 mg/I for Fab in
yeast in
fermenters (Horwitz et al., 1988), and 150 mg/I for a fusion protein CBHI-Fab
and
1 mg/I for Fab in Trichoderma in fermenters (Nyyssonen et al., 1993) and such
pro-
duction is very cheap compared to whole antibody production in mammalian cells
(hybridoma, myeloma, CHO).

The fragments can be produced as Fab's or as Fv's, but additionally it has
been
shown that a VH and a VL can be genetically linked in either order by a
flexible poly-
peptide linker, which combination is known as an scFv.

Isolated nucleic acid molecule/vector/host cell

In one aspect the invention relates to an isolated nucleic acid molecule
encoding at
least a part of the binding member as defined above. In one embodiment the
nucleic
acid molecule encodes a light chain and another nucleic acid encodes a heavy
chain. The two nucleic acid molecule may be separate or they may be fused into
one nucleic acid molecule, optionally spaced apart by a linker sequence. In
particu-
lar in relation to antibody fragments the nucleic acid molecule may encode the
whole
binding member; however, dependent on the design of the binding member, this

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
37
may also be relevant for some larger binding members. The nucleic acid
molecule is
preferably a DNA sequence, more preferably a DNA sequence comprising in its up-

stream end regulatory elements promoting the expression of the binding member
once the nucleic acid molecule is arranged in a host cell.
Accordingly, in one embodiment the invention relates to a polynucleotide
selected
from the group consisting of

i) a polynucleotide comprising a sequence selected from the nucleotide
sequence of Example 6,

a polynucleotide encoding a binding member comprising one or more of
the amino acid sequence selected from the group of SEQ ID NO 3, 4,12
or 13,
ii) a polynucleotide encoding a fragment of a polypeptide encoded by
polynucleotides i), wherein said fragment

a) is capable of recognising an antigen also being recognised by the
binding member of ii), and/or

b) is capable of binding selectively to an antigen, wherein said antigen is
also bound selectively by the binding member of ii), and/or

c) has a substantially similar or higher binding affinity to Pneumolysin as
a binding domain comprising a predetermined sequence, such as
SEQ ID NO 3, 4, 12 or 13,

iii) a polynucleotide, the complementary strand of which hybridizes under
stringent conditions, with a polynucleotide as defined in any of i), ii),
iii),
and encodes a polypeptide as defined in iii),

iv) a polynucleotide comprising a nucleotide sequence which is degenerate
to the nucleotide sequence of a polynucleotide as defined in any of i) -
iv),

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
38
and the complementary strand of such a polynucleotide.

The invention further relates to a vector comprising the nucleic acid molecule
as
defined above, either one vector per nucleic acid, or two or more nucleic
acids in the
same vector. The vector preferably comprises a nucieotide sequence which regu-
lates the expression of the antibody encoded by the nucleic acid molecule.

In yet another aspect the invention relates to a host cell comprising the
nucleic acid
molecule as defined above.

Also, the invention relates to a cell line engineered to express the binding
member
as defined above, this cell line for example being a hybridoma of a murine
lympho-
cyte and an immortalised cell line. The cell line may be any suitable cell
line, how-
ever the cell line P3 is preferred. In another embodiment a CHO cell line is
pre-
ferred.

Purification of binding members

After production the binding members according to the invention are preferably
puri-
fied. The method of purification used is dependent upon several factors
including the
purity required, the source of the antibody, the intended use for the
antibody, the
species in which the antibody was produced, the class of the antibody and,
when
the antibody is a monoclonal antibody, the subclass of the antibody.
Any suitable conventional methods of purifying polypeptides comprising
antibodies
include precipitation and column chromatography and are well known to one of
skill
in the purification arts, including cross-flow filtration, ammonium sulphate
precipita-
tion, affinity column chromatography, gel electrophoresis and the like may be
used.
The method of purifying an antibody with an anti-immunoglobulin antibody can
be
either a single purification procedure or a sequential purification procedure.
Methods
of single and sequential purification are well known to those in the
purification arts.
In a single-step purification procedure, the antibody is specifically bound by
a single
anti-immunoglobulin antibody. Non-specifically bound molecules are removed in
a

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
39
wash step and the specifically bound molecules are specifically eluted. In a
sequen-
tial purification procedure, the antibody is specifically bound to a first
anti-
immunoglobulin antibody, non-specifically bound molecules are removed in a
wash
step, and the specifically bound molecules are specifically eluted. The eluant
from
the first anti-immunoglobulin antibody is then specifically bound to a second
anti-
immunoglobulin antibody. The non-specifically bound molecules are removed in a
wash step, and the specifically bound molecules are specifically eluted. In a
pre-
ferred embodiment, the antibody is sequentially purified by a first and second
anti-
immunoglobulin antibody selected from the group consisting of antibodies which
specifically bind heavy and light chain constant regions.

A commonly used method of purification is affinity chromatography in which the
an-
tibody to be purified is bound by protein A, protein G or by an anti-
immunoglobulin
antibody. Another method of affinity chromatography, which is well known to
those
of skill in the art, is the specific binding of the antibody to its respective
antigen.
In particular for purifying a multispecific, including a bispecific antibody,
a sequential
purification procedure may be used, wherein the bispecific antibody comprising
two
or more variable domains is specifically bound to a first antigen and then to
a sec-
ond antigen.

In an alternative embodiment, a bispecific antibody comprising two or more
variable
regions is purified by sequential purification by specifically binding the
antibody to a
first antigen in a first purification step and to a second antigen in a second
purifica-
tion step.

The method of purifying an antibody with an anti-immunoglobulin antibody can
be
either a single purification procedure or a sequential purification procedure.
Methods
of single and sequential purification are well known to those in the
purification arts.
In a single-step purification procedure, the antibody is specifically bound by
a single
anti-immunoglobulin antibody. Non-specifically bound molecules are removed in
a
wash step and the specifically bound molecules are specifically eluted. In a
sequen-
tial purification procedure, the antibody is specifically bound to a first
anti-immuno-
globulin antibody, non-specifically bound molecules are removed in a wash
step,
and the specifically bound molecules are specifically eluted. The eluant from
the first

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
anti-immunoglobulin antibody is then specifically bound to a second anti-
immuno-
globulin antibody. The non-specifically bound molecules are removed in a wash
step, and the specifically bound molecules are specifically eluted. In a
preferred em-
bodiment, the antibody is sequentially purified by a first and second anti-
immuno-
5 globulin antibody selected from the group consisting of antibodies which
specifically
bind heavy and light chain constant regions. In a more preferred embodiment,
the
antibody is sequentially purified by a first and second anti-immunoglobulin
antibody
selected from the group consisting of antibodies which specifically bind the
heavy
chain constant region of IgG and light chain constant regions of kappa and
lambda.
10 In an even more preferred embodiment, the anti-immunoglobulin antibody is
se-
lected from the group consisting of antibodies which specifically bind the
light chain
constant regions of kappa and lambda.

Diagnostic Methods
The present invention also describes a diagnostic system, preferably in kit
form, for
assaying for the presence of Streptococcus, in particular Streptococcus pneumo-

niae, in a biological sample where it is desirable to detect the presence, and
pref-
erably the amount, of bacteria in a sample according to the diagnostic methods
de-
scribed herein.

The diagnostic system includes, in an amount sufficient to perform at least
one as-
say, a binding member composition according to the present invention,
preferably as
a separately packaged reagent, and more preferably also instruction for use.
The biological sample can be a tissue, tissue extract, fluid sample or body
fluid
sample, such as blood, plasma or serum.

Packaged refers to the use of a solid matrix or material such as glass,
plastic (e.g.,
polyethylene, polypropylene or polycarbonate), paper, foil and the like
capable of
holding within fixed limits a binding member of the present invention. Thus,
for ex-
ample, a package can be a glass vial used to contain milligram quantities of a
con-
templated labelled binding member preparation, or it can be a microtiter plate
well to
which microgram quantities of a contemplated binding member has been
operatively
affixed, i.e., linked so as to be capable of binding a ligand.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
41
"Instructions for use" typically include a tangible expression describing the
reagent
concentration or at least one assay method parameter such as the relative
amounts
of reagent and sample to be admixed, maintenance time periods for
reagent/sample
admixtures, temperature, buffer conditions and the like.

A diagnostic system of the present invention preferably also includes a label
or indi-
cating means capable of signalling the formation of a binding reaction complex
con-
taining a binding member complexed with the preselected ligand.
Any label or indicating means can be linked to or incorporated in an expressed
poly-
peptide, or phage particle that is used in a diagnostic method. Such labels
are them-
selves well-known in clinical diagnostic chemistry.

The labeling means can be a fluorescent labeling agent that chemically binds
to
antibodies or antigens without denaturing them to form a fluorochrome (dye)
that is
a useful immunofluorescent tracer. Suitable fluorescent labeling agents are
fluoro-
chromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante
(FITC), 5-
dimethylamine-l-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine iso-

thiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC)
and the like. A description of immunofluorescence analysis techniques is found
in
DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis, et
al.,
eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated
herein by
reference.
In preferred embodiments, the indicating group is an enzyme, such as
horseradish
peroxidase (HRP), glucose oxidase, or the like. In such cases where the
principal
indicating group is an enzyme such as HRP or glucose oxidase, additional
reagents
are required to visualize the fact that a receptor-ligand complex
(immunoreactant)
has formed. Such additional reagents for HRP include hydrogen peroxide and an
oxidation dye precursor such as diaminobenzidine. An additional reagent useful
with
glucose oxidase is 2,2'-amino-di-(3-ethyl-benzthiazoline-G-sulfonic acid)
(ABTS).
Radioactive elements are also useful labeling agents and are used
illustratively
herein. An exemplary radiolabeling agent is a radioactive element that
produces

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
42
gamma ray emissions. Elements which themselves emit gamma rays, such as 124 I
125 I, 128 I, 132 I and 51 Cr represent one class of gamma ray emission-
producing ra-
dioactive element indicating groups. Particularly preferred is 125 I. Another
group of
useful labeling means are those elements such as 11 C 1s F, 150 and 13 N which
themselves emit positrons. The positrons so emitted produce gamma rays upon
encounters with electrons present in the animal's body. Also useful is a beta
emitter,
such as 111 indium or 3 H.

The linking of labels, i.e., labeling of, polypeptides and proteins or phage
is well
known in the art. For instance, proteins can be labelled by metabolic
incorporation of
radioisotope-containing amino acids provided as a component in the culture me-
dium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981). The
tech-
niques of protein conjugation or coupling through activated functional groups
are
particularly applicable. See, for example, Aurameas, et al., Scand. J.
Immunol., Vol.
8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S.
Pat. No.
4,493,795.

The diagnostic systems can also include a specific binding agent, preferably
as a
separate package. A "specific binding agent" is a molecular entity capable of
selec-
tively binding a binding member species of the present invention or a complex
con-
taining such a species, but is not itself a binding member of the present
invention.
Exemplary specific binding agents are antibody molecules, complement proteins
or
fragments thereof, S. aureus protein A, and the like. Preferably the specific
binding
agent binds the binding member species when that species is present as part of
a
complex.

In preferred embodiments, the specific binding agent is labelled. However,
when the
diagnostic system includes a specific binding agent that is not labelled, the
agent is
typically used as an amplifying means or reagent. In these embodiments, the Ia-

belled specific binding agent is capable of specifically binding the
amplifying means
when the amplifying means is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an "ELISA" format
to
detect the quantity of a preselected ligand in a fluid sample. "ELISA" refers
to an
enzyme-linked immunosorbent assay that employs an antibody or antigen bound to

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
43
a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and
quantify the amount of an antigen present in a sample and is readily
applicable to
the present methods.

Thus, in some embodiments, a binding member of the present invention can be af-

fixed to a solid matrix to form a solid support that comprises a package in
the sub-
ject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from an aqueous
me-
dium although other modes of affixation applicable to proteins and
polypeptides can
be used that are well known to those skilled in the art. Exemplary adsorption
meth-
ods are described herein.

Useful solid matrices are also well known in the art. Such materials are water
in-
soluble and include the cross-linked dextran available under the trademark
SEPHA-
DEX from Pharmacia Fine Chemicals (Piscataway, N.J.); agarose; beads of
polysty-
rene beads about 1 micron to about 5 millimeters in diameter available from
Abbott
Laboratories of North Chicago, Ill.; polyvinyl chloride, polystyrene, cross-
linked poly-
acrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or
paddles; or
tubes, plates or the wells of a microtiter plate such as those made from
polystyrene
or polyvinylchloride.

The binding member species, labelled specific binding agent or amplifying
reagent
of any diagnostic system described herein can be provided in solution, as a
liquid
dispersion or as a substantially dry power, e.g., in lyophilized form. Where
the indi-
cating means is an enzyme, the enzyme's substrate can also be provided in a
sepa-
rate package of a system. A solid support such as the before-described
microtiter
plate and one or more buffers can also be included as separately packaged ele-
ments in this diagnostic assay system.
Diagnostic methods

The present invention also contemplates various assay methods for determining
the
presence, and preferably amount, of a Streptococcus, in particular
Streptococcus
pneumoniae, typically present in a biological sample.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
44
Accordingly, the present invention relates to a method of detecting or
diagnosing a
disease or disorder associated with Pneumococcus in an individual comprising

- providing a biological sample from said individual
- adding at least one binding member as defined above to said biological
sample,
- detecting binding members bound to said biological sample, thereby detecting
or
diagnosing the disease or disorder.

The bound binding members may be detected either directly or indirectly, to
the
amount of the Streptococcus in the sample.

Those skilled in the art will understand that there are numerous well known
clinical
diagnostic chemistry procedures in which a binding reagent of this invention
can be
used to form an binding reaction product whose amount relates to the amount of
the
ligand in a sample. Thus, while exemplary assay methods are described herein,
the
invention is not so limited.

Various heterogenous and hom*ogeneous protocols, either competitive or noncom-
petitive, can be employed in performing an assay method of this invention.

Binding conditions are those that maintain the ligand-binding activity of the
receptor.
Those conditions include a temperature range of about 4 to 50 degrees
Centigrade,
a pH value range of about 5 to 9 and an ionic strength varying from about that
of
distilled water to that of about one molar sodium chloride.

The detecting step can be directed, as is well known in the immunological
arts, to
either the complex or the binding reagent (the receptor component of the
complex).
Thus, a secondary binding reagent such as an antibody specific for the
receptor
may be utilized.

Alternatively, the complex may be detectable by virtue of having used a
labelled
receptor molecule, thereby making the complex labelled. Detection in this case
comprises detecting the label present in the complex.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
A further diagnostic method may utilize the multivalency of a binding member
com-
position of one embodiment of this invention to cross-link ligand, thereby
forming an
aggregation of multiple ligands and polypeptides, producing a precipitable
aggre-
5 gate. This embodiment is comparable to the well-known methods of immune
precipi-
tation. This embodiment comprises the steps of admixing a sample with a
binding
member composition of this invention to -form a binding admixture under
binding
conditions, followed by a separation step to isolate the formed binding
complexes.
Typically, isolation is accomplished by centrifugation or filtration to remove
the ag-
10 gregate from the admixture. The presence of binding complexes indicates the
pres-
ence of the preselected ligand to be detected.

Pharmaceutical compositions

15 In a preferred aspect the present invention contemplates pharmaceutical
composi-
tions useful for practising the therapeutic methods described herein.
Pharmaceutical
compositions of the present invention contain a physiologically tolerable
carrier to-
gether with at least one species of binding member as described herein,
dissolved
or dispersed therein as an active ingredient. In a preferred embodiment, the
phar-
20 maceutical composition is not immunogenic when administered to a human
individ-
ual for therapeutic purposes, unless that purpose is to induce an immune
response.
In one aspect the invention relates to a pharmaceutical composition comprising
at
least one binding member as defined above. In a preferred embodiment the phar-
25 maceutical composition comprises at least two different binding members as
defined
above in order to increase the effect of the treatment.

As used herein, the terms "pharmaceutically acceptable", "physiologically
tolerable"
and grammatical variations thereof, as they refer to compositions, carriers,
diluents
30 and reagents, are used interchangeably and represent that the materials are
capa-
ble of administration to or upon a human without the production of undesirable
physiological effects such as nausea, dizziness, gastric upset and the like.

The preparation of a pharmacological composition that contains active
ingredients
35 dissolved or dispersed therein is well understood in the art. Typically
such composi-

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
46
tions are prepared as sterile injectables either as liquid solutions or
suspensions,
aqueous or non-aqueous; however, solid forms suitable for solution, or
suspensions,
in liquid prior to use can also be prepared. The preparation can also be
emulsified.

The active ingredient can be mixed with excipients which are pharmaceutically
ac-
ceptable and compatible with the active ingredient and in amounts suitable for
use in
the therapeutic methods described herein. Suitable excipients are, for
example, wa-
ter, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
In addi-
tion, if desired, the composition can contain minor amounts of auxiliary
substances
such as wetting or emulsifying agents, pH buffering agents and the like, which
en-
hance the effectiveness of the active ingredient.

The pharmaceutical composition of the present invention can include
pharmaceuti-
cally acceptable salts of the components therein. Pharmaceutically acceptable
salts
include the acid addition salts (formed with the free amino groups of the
polypeptide)
that are formed with inorganic acids such as, for example, hydrochloric or
phospho-
ric acids, or such organic acids as acetic, tartaric, mandelic and the like.
Salts
formed with the free carboxyl groups can also be derived from inorganic bases
such
as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides,
and
such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,
his-
tidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplary of
liquid carri-
ers are sterile aqueous solutions that contain no materials in addition to the
active
ingredients and water, or contain a buffer such as sodium phosphate at
physiologi-
cal pH value, physiological saline or both, such as phosphate-buffered saline.
Still
further, aqueous carriers can contain more than one buffer salt, as well as
salts such
as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene
glycol
and other solutes.
Liquid compositions can also contain liquid phases in addition to and to the
exclu-
sion of water. Exemplary of such additional liquid phases are glycerin,
vegetable oils
such as cottonseed oil, organic esters such as ethyl oleate, and water-oil
emulsions.

A pharmaceutical composition contains a binding member of the present
invention,

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
47
typically an amount of at least 0.1 weight percent of antibody per weight of
total
pharmaceutical composition. A weight percent is a ratio by weight of antibody
to
total composition. Thus, for example, 0.1 weight percent is 0.1 grams of
antibody
per 100 grams of total composition.
The invention also relates to a method for preparing a medicament or
pharmaceuti-
cal composition comprising an antibody of the invention, the medicament being
used
for immunotherapy of a disease or disorder associated with Streptococcus, in
par-
ticular Streptococcus pneumoniae, such as pneumonia, meningitis and sepsis,
com-
prising admixing at least one binding member as defined above with a
physiologi-
cally acceptable carrier.

Furthermore, the invention relates to the use of a binding member as defined
above
for the production of a pharmaceutical composition for the treatment of a
disease or
disorder associated with Streptococcus, in particular Streptococcus
pneumoniae,
such as pneumonia, meningitis and sepsis.

The pharmaceutical composition may also be a kit-in-part further including an
antibi-
otic agent, such as antibiotics selected from P-lactams, cephalosporins,
penicilins
and aminoglycosides, and/or include an immunostimulating agent, such as cyto-
kines, interferons, growth factors, for example GCSF or GM-CSF. The kit-in-
part
may be used for simultaneous, sequential or separate administration.

Furthermore, the pharmaceutical composition may include the binding member ac-
cording to the invention in combination with the Streptococcus protein
Pneumolysin,
in particular as a vaccine. It has been found that by combining the binding
member
according to the invention with the protein Pneumolysin, the immunising
properties
of the combination product is better than for the protein Pneumolysin alone.
This
may be due to the fact that the protein Pneumolysin is presented to the immune
system by the binding member.

In another embodiment, the antibody according to the invention is combined
with
another antibody against Streptococcus pneumoniae, such as another anti-Pneumo-

lysin antibody, for example a non-haemolytic anti-Pneumolysin antibody.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
48
The antibody according to the invention may also be an anti-PsaA antibody as
de-
scribed in International patent application no. PCT/DK2004/000492.

Therapeutic methods
The binding members according to the present invention are particular useful
in
therapeutic methods due to their high affinity and specificity. Accordingly,
the bind-
ing members can be used immunotherapeutically towards a disease or disorder
associated with Streptococcus, in particular Streptococcus pneumoniae, such as
pneumonia, meningitis and sepsis.

The term "immunotherapeutically" or "immunotherapy" as used herein in
conjunction
with the binding members of the invention denotes both prophylactic as well as
therapeutic administration. Thus, the binding members can be administered to
high-
risk patients in order to lessen the likelihood and/or severity of disease,
administered
to patients already evidencing active infection, or administered to patients
at risk of
infection.

The dosage ranges for the administration of the binding members of the
invention
are those large enough to produce the desired effect in which the symptoms of
the
disease are ameliorated or the likelihood of infection decreased. Generally,
the dos-
age will vary with the age, condition, sex and extent of the disease in the
patient and
can be determined by one of skill in the art. The dosage can be adjusted by
the indi-
vidual physician in the event of any complication.
A therapeutically effective amount of an binding member of this invention is
typically
an amount of antibody such that when administered in a physiologically
tolerable
composition is sufficient to achieve a plasma concentration of from about 0.1
micro-
gram ( g) per milliliter (ml) to about 100 g/ml, preferably from about 1
g/ml to
about 5 g/ml, and usually about 5 g/ml. Stated differently, the dosage can
vary
from about 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg to
about
200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or
more
dose administrations daily, for one or several days.

The binding members of the invention can be administered parenterally by
injection

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
49
or by gradual infusion over time. Although the infection may be systemic and
there-
fore most often treated by intravenous administration of pharmaceutical
composi-
tions, other tissues and delivery means are contemplated where there is a
likelihood
that targeting a tissue will result in a lessening of the disease. Thus,
antibodies of
the invention can be administered parenterally, such as intravenously,
intraperito-
neally, intramuscularly, subcutaneously, intracavity, transdermally, and can
be de-
livered by peristaltic means.

The pharmaceutical compositions containing a binding member of this invention
are
conventionally administered intravenously, as by injection of a unit dose, for
exam-
ple. The term "unit dose" when used in reference to a pharmaceutical
composition of
the present invention refers to physically discrete units suitable as unitary
dosage for
the subject, each unit containing a predetermined quantity of active material
calcu-
lated to produce the desired therapeutic effect in association with the
required dilu-
ent; i.e., carrier, or vehicle.

The therapeutic method may further include the use of a kit-in-part as defined
above.

Passive immune protection
The binding members may be particular useful for passive immune protection,
whereby the binding member neutralise the action of Pneumolysin. The binding
member may be evaluated in an assay as described in Example 1. The result of
the
assay demonstrates that administration of a binding member towards Pneumolysin
may prolong survival upon S. pneumoniae infection in mice and thus induction
of
passive immune protection.

Active immune protection
The antigenic epitopes of the invention can be used as vaccines to stimulate
an im-
munological response in a mammal directed against Pneumolysin, a mammal for
example being a mouse, dog, cat, swine, horse, bovine etc. and preferably a
human
being. Such an response may include induction of Pneumolysin specific
antibodies.
Antibodies directed against the antigenic epitopes of the invention can
inhibit Pneu-
molysin function as described above, and immunisation may further be used for
prophylactic treatment and infection caused by S. pneumoniae.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
Pneumolysin peptide
In an aspect the invention relates to a Pneumolysin peptide comprising an
epitope
recognised by a binding member according to the invention. Preferably the
Pneumo-
5 lysin peptide, fragment or variants preferably comprise an amino acid
sequence
identified by SEQ ID NO 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36. A
Pneumolysin
peptide according to the invention may be a peptide consisting of amino acid 1-
436
of SEQ ID NO 11. Further included are fragments and variants of the
Pneumolysin
peptide consisting of amino acid 1-436 of SEQ ID NO 11, this includes
fragments
10 comprising amino acid 50-436, or more preferably amino acid 100-436 of
Pneumo-
lysin as identified by SEQ ID NO 11. In specific embodiments the Pnemolysin
pep-
tide comprise amino acid 200-436 or 300-436 of Pneumolysin as identified by
SEQ
ID NO 11. Variants or hom*ologues of Pneumolysin peptides may be defined as
hom*ologues in relation to binding members as described above.
The Pneumolysin peptide, fragment or variants preferably comprise an amino
acid
sequence identified by SEQ ID NO 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36. It
is pre-
ferred that the Pneumolysin peptide is constituted by at the most 100, such as
80,
60, 40, 30, 25, 20, 15 or such as 12 amino acids. It may further be preferred
that the
Pneumolysin peptide is constituted by at the least 12, such as 15, 20, 25, 30,
40, 60,
80, or such as at least 100 amino acids.

In specific embodiments the Pneumolysin peptide fragment s are identified by
SEQ
ID NO 27, 29, 30, 31 or 32.
The Pneumolysin peptides may be used as antigenic epitopes capable of stimulat-

ing the immune system.

Vaccine composition
A vaccine composition according to the invention can be formulated according
to
known methods such as by the admixture of one or more pharmaceutically
acceptable excipients or carriers with the active agent, preferably acceptable
for
administration to humans. Examples of such excipients, carriers and methods of
formulation may be found e.g. in Remington's Pharmaceutical Sciences (Maack
Publishing Co, Easton, PA). To formulate a pharmaceutically acceptable

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
51
composition suitable for effective administration, such compositions will
according to
the invention contain an effective amount of a Pneumolysin polypeptide or an
analog
there of.

Vaccine compositions according to the invention may be administered to an
individual in therapeutically effective amounts. The effective amount may vary
according to a variety of factors such as the individual's condition, weight,
sex and
age. Other factors include the mode of administration.

In the following vaccine compositions are meant to encompass compositions
useful
for therapeutic use, including stimulating an immune response.

To obtain vaccines or immunogenic compositions it may be required to combine
the
Pneumolysin peptide or analog molecules with various materials such as
adjuvants,
immunostimulatory components and/or carriers. Adjuvants are included in the
vaccine composition to enhance the specific immune response.

Such adjuvants may be any compound comprising an adjuvant effect known to the
person skilled in the art. For example such adjuvants could be of mineral,
bacterial,
plant, synthetic or host origin or they could be oil in water emulsions.

Adjuvants could be selected from the group consisting of: AIK(S04)2,
AINa(SO4)2,
AINH4 (SO4), silica, alum, AI(OH)3, Ca3 (P04)2, kaolin, carbon, aluminum
hydroxide,
muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-
acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-
MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-
sn -
glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-

PE), RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80® emulsion, lipopoly-
saccharides and its various derivatives, including lipid A, Freund's Complete
Adju-
vant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides
(for
example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis,
sub-
stances found in Corynebacterium parvum, Bordetella pertussis, and members of
the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil
A,
ALUN (see US 58767 and 5,554,372), Lipid A derivatives, choleratoxin
derivatives,

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
52
HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP,
Interleukin 1
and Interleukin 2.

A large number of adjuvants have been described and used for the generation of
antibodies in laboratory animals, such as mouse, rats and rabbits. In such
setting
the tolerance of side effect is rather high as the main aim is to obtain a
strong anti-
body response.

For use and for approval for use in pharmaceuticals, and especially for use in
hu-
mans it is required that the components of the vaccine composition, including
the
adjuvant, are well characterised. It is further required that the composition
has mini-
mal risk of any adverse reaction, such as granuloma, abscesses or fever.

In a preferred embodiment the vaccine composition is suitable for
administration to a
human subject, thus a preferred adjuvant are suitable for administration to a
human
subject.

Adjuvants useful in therapeutic vaccines may be mineral salts, such as
aluminium
hydroxide and aluminium or calcium phosphates gels, oil emulsions and
surfactant
based formulations such as MF59 (microfluidised detergent stabilised oil in
water
emulsion), QS21 (purified saponin), AS02 (SBAS2, oil-in-water emulsion + mono-
phosphoryl lipid A (MPL) + QS21), Montanide ISA 51 and ISA-720 (stabilised
water
in oil emulsion), Adjuvant 65 (containing peanut oil, mannide monooleate and
alumi-
num monostearate), RIBI ImmunoChem Research Inc., Hamilton, Utah), particulate
adjuvants, such as virosomes (unilamellar liposomal cehicies incorporating
influenza
haemagglutinin), ASO4 (Al salt with MPL), ISCOMS (structured complex of
saponins
and lipids (such as cholesterol), polyactide co-glycolide (PLG), microbial
derivatives
(natural and synthetic) such as monophosphoryl lipid A (MPL), Detox (MPL + M.
Phlei cell wall skeleton), AGP (RC-529 (synthetic acylated monosaccharide)),
DC_chol (lipoidal immunostimulators able to self organise into liposomes), OM-
174
(lipid A derivative), CpG motifs (synthetic oligonucleotides containing
immunostimu-
latory CpG motifs), modified bacterial toxins, LT and CT, with non-toxic
adjuvant
effects, Endogenous human immunomodulators, e.g., hGM-CSF or hIL-12 or Immu-
daptin (C3d tandem array), inert vehicles such as gold particles.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
53
In some embodiments, the vaccine composition may further comprise one or more
additional immunostimulatory components. These include, without limitation,
mura-
myldipeptide (MDP); e.g. N-acetyl-muramyl-L-alanyl-D-isoglutamine (ala-MDP), N-

acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-
alanyl-D-isoglutamine (CGP 11637, nor-MDP) and N-acetyl-muramyl-L-alanyl-D-
isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-
hydroxyphosphoryloxy)-
ethylamine (CGP 19835A, MTP-PE), dimethylglycine, tuftsin, and trehalose
dimyco-
late. monophosphoryl-lipid A (MPL), and formyl-methionine containing tri-
peptides
such as N-formyl-Met-Leu-Phe. Such compounds are commercially available from
Sigma Chemical Co. (St. Louis, MO) and RIBI ImmunoChem Research, Inc. (Hamil-
ton, MT), for example.

A carrier may be present independently of an adjuvant. The function of a
carrier can
for example be to increase the molecular weight of in particular survivin
fragments in
order to increase their activity or immunogenicity, to confer stability, to
increase the
biological activity, or to increase serum half-life. The carrier may be any
suitable
carrier known to the person skilled in the art. A carrier protein could be but
is not
limited to keyhole limpet haemocyanin, serum proteins such as transferrin,
bovine
serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobu-
lins, or hormones, such as insulin or paimitic acid. For immunization of
humans, the
carrier must be a physiologically acceptable carrier acceptable to humans and
safe.
However, tetanus toxoid and/or diptheria toxoid are suitable carriers in one
embodi-
ment of the invention. Alternatively, the carrier may be dextrans for example
sepha-
rose.
In an embodiment the vaccine composition comprise a Pneumolysin peptide com-
prising an amino acid sequence identified by SEQ ID NO 27, 28, 29, 30, 31 or
32.
Vaccines comprising peptides comprising an amino acid sequence identified by
SEQ ID NO 29, 30 or 31 are preferred. Especially preferred are peptides
comprising
the amino acid sequence of 400-436, 422-436 or 425- 436 of pneumolysin as
identi-
fied by SEQ ID NO 11.

It is preferred that the Pneumolysin peptide is constituted by at the most
100, such
as 80, 60, 40, 20, 15, 12, 10 8 or such as 6 amino acids. It may further be
preferred
that the Pneumolysin peptide is constituted by at the least 6, such as 8, 10,
12, 15,
20, 25, 30, 40, 60, 80, or such as at least 100 amino acids. In an embodiment
the

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
54
vaccine composition comprise at least one Pneumolysin peptide identified by
SEQ
ID NO 27, 28, 27, 30, 31, 32, 33, 34, 35 or 36. Vaccines comprising peptides
identi-
fied by SEQ ID NO 28, 29, 30 or 31 are preferred. Especially preferred are
peptides
comprising the amino acid sequences identified as AA 423-438, 424-437, 425-436
or 426-436 of pneumolysin as identified by SEQ ID NO 11.

A vaccine composition capable of stimulating an immune response is preferred.
It is
particularly relevant that the vaccine composition is capable of inducing an
antibody
response upon administration. Mostly preferred are vaccines capable of
inducing a
Pneumolysin inhibiting response, by inducing the production of antibodies
capable
of inhibition the lytic activities of Pneumolysin. Other preferred embodiments
include
antibodies capable of enhancing phagocytosis of Pneumolysin. Such antibodies
may be characterised by comprising a variable region as the binding member de-
scribed here in.
Detailed description of drawings

Figure 1. Schematic drawing of a Fab fragment.
The antigen pocket composed of VL, CDR1, CDR2, CDR3 and VH, CDRI, CDR2,
CDR3 is shown.

Figure 2. Pneumolysin amino acid sequence having SEQ ID NO 11.
The amino acid sequence of Pneumolysin corresponding to the sequence of Gene-
bank no. X52474 is shown.
Figure 3. Anti-Pneumolysin light chain and heavy chain variable segments.
Figure 3A includes the consensus sequences of the variable light and heavy
chain
and the complementarity determining regions of antibody 26-5F12.1. Figure 3B
in-
cludes the consensus sequences of the variable light and heavy chain and the
com-
plementarity determining regions of antibody 26-23C 2.2. Figure 3C includes
the
consensus sequences of the variable light and heavy chain and the
complementarity
determining regions of antibody 22-1 C11. The sequences are obtained as
described
in example 6.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
Figure 4. Survival diagram for mice inoculated with Pneumococcus and antibody.
The survival of mice injected with Pneumococcus D39 alone or in combination
with
penicillin and/or Pneumolysin antibody (26-5F12) evaluated 24 hours after
inocula-
tion as described in example 1.
5
Figure 5. Antihaemolytic activity of Pneumolysin antibodies.
The anti-haemolytic activity of Pneumolysin antibodies analysed by evaluating
the
inhibitory effect on Pneumolysin mediated lysis of erythrocytes as described
in ex-
ample 3. Three antibodies (26-5F1 2, 26-23C 2 and 22-6E6) are particular
effective.
Figure 6 Peptides for epitope mapping.
An overview of the amino acid sequence 419-446 of Pneumnolysin and various pep-

tide sequences for epitope mapping.

Figure 7 Pneumolysin antibody epitopes.
Figure 7A and Figure 7B are graphic illustrations of the results obtained as
de-
scribed in example 7 related to identification of the antibody epitope.

Figure 8 Isolation of 26-5F12 clones
Figure 8A shows the total RNA isolated from the 26-5F12 hybridoma cells. The
RNA
was used for cDNA synthesis of heavy chain and light chain variable regions.
The
PCR products are shown in figure 8B. After cloning the positive transformants
were
identified using colony PCR (figure 8C).

Figure 9 Isolation of 26-23C2 clones
Figure 9A shows the total RNA isolated from the 26-23 C2 hybridoma cells. The
RNA was used for cDNA synthesis of heavy chain and light chain variable
regions.
The PCR products are shown in figure 9B. After cloning the positive
transformants
were identified using colony PCR (figure 9C).
Figure 10 Isolation of 22 1 C11 clones
Total RNA isolated from 22 1 C11 hybridoma cells was used for cDNA synthesis
of
heavy chain and light chain variable regions. The PCR products are shown in
figure
10BA. After cloning the positive transformants were identified using colony
PCR
(figure 10B).

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
56
Figure 11 CDR sequences of 26-5F12, 26-23C2 and 22 1 C11.
The sequences of the light and heavy chain CDR's of 26-5F12, 26-23 C2 and 22
1 C11 are aligned. The heavy chain of 26-5F1 2 and 26-23C2 is almost identical
whereas CDR 2 and CDR3 of 22 1 C11 heavy chain diverge from the sequence of
the 6-5F12 and 26-23C2.

Examples
The invention is further explained through the examples below; the examples
are
not to be construed as limiting to the invention.

Example 1
Study of the effect of antibodies and penicillin on survival of transgenic
female mice
inoculated with Pneumococcus D39 (type 2)

Materials
= 82 transgenic female mice (M-B project no. #249, project name CD64, about 8-
12 weeks old)
= 0.9% saline (AAS)
= PBS qH 7.4
= Syringes
= Needles
= 5% blood plates
= Filtered bovine broth
= Solvent ad penicillin
= Penicillin 1 million IU (Loven D6726), 10 mg/mouse -40 mg/ml
Strains: Pneumococcus D39 (type 2) (F1/S1/4E2)

Antibodies:
PdB26-5F12.1, 1.0 mg/mI 040520
OmpA6-4B6.1, 1.38 mg/mI

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
57
Method:

Hours -24: The Pneumococcus strain is seeded onto 3 x 5% blood plate and incu-
bated overnight at 35 C/CO2.

Hours 0: The Pneumococcus strain is slurried in filtered broth to 108 CFU/ml
(cf.
MU/F074-01) and diluted to 2 x 105 CFU/ml (120,u1 10$ CFU/ml in 59.88 ml of
PBS).
The antibody is diluted to 200,ug/ml:
3.00 ml of PdB26-5F12.1 + 12.00 ml of PBS
2.17 ml of OmpA6-4B6.1 + 12.83 ml of PBS
The mice are treated with bacteria (0.5 ml i.p.) and antibody (0.5 ml i.p.).

Hours 18: Penicillin: 1 ampoule is diluted in 3 ml solvent ad pen. -200 mg/mI;
further
dilution: 3 ml "200 mg/mi" + 12.00 ml of saline -40 mg/mi.
The antibodies are diluted to 200,ug/mI:
3.00 ml of PdB26-5F12.1 + 12.00 ml of PBS
2.17 ml of OmpA6-4B6.1 + 12.83 ml of PBS
The mice are treated with penicillin (0.25 mi s.c.) and antibody (0.5 ml
i.p.).
Hours 48: Penicillin: 1 ampoule is diluted in 3 ml solvent ad pen. -200 mg/mI;
further
dilution: 3 ml "200 mg/mI" + 12.00 ml of saline -40 mg/ml.
The mice are treated with penicillin (0.25 ml s.c.).

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
58
Cage No. of mice 0 hours 18 hours 48 hours
no.
Grp 1 5 Bacteria + 5F12.1 5F12.1 + PEN PEN
1 2 5
3 4
Grp 4 5 Bacteria + 5F12.1 5F12.1
2 5 5
6 4
Grp 7 5 Bacteria + 6-4B6 6-4B6 + PEN PEN
3 8 5
9 4
Grp 10 5 Bacteria + 6-4B6 6-4B6
4 11 5
12 4
Grp 13 5 Bacteria PEN PEN
14 5
3
Grp 16 5 Bacteria
6 17 5
18 3

Morning and afternoon the following days for the duration of the experiment:
The
mice are scored according to scale 1-4.

inoculate undiluted 10-1 10-2 10" 10" 10" CFU/ml
Pn. D39 ~ oo i.t. 12/20 4/2 8.0 x 10
5
Results
The survival of the mice is evaluated at 24 hours.
The results of the experiments performed using 26-5F12.1 is summarised in
figure
4, showing an increase survival rate at 24 hours.

Example 2

Detection of anti-haemolytic properties in antibodies from culture
supernatant.

Description:
Antibodies against Pneumolysin can inhibit the lytic effect of Pneumolysin.
The lytic
effect is abolished in the presence of serum, thereby rendering it necessary
to bind
the antibodies and remove the serum by washing before performing an anti-haemo-

lytic assay.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
59
Devices:
Incubator 37 C
Pipettes
Centrifuge
ELISA reader, BIO-TEK EL 800
Digital Camera, Canon Powershot S20
Materials:
Tips
Reagent tray
Plate cover
96-well microwell plate (Nunc 260836 - flat bottom)
Reacti-Bind Protein G coated microwell strips, Pierce no. 15133
Reagents:
Rec. PdB, diluted in PBS w.10 mM DTT to 4,ug/ml
Dithiothreitol (DTT)
PBS, pH 7.4
Dem. H20
Sheep erythrocytes 50% in Alsever's Fluid, SSI no. 29431
Buffers:
PBS pH 7.4
PBS pH 7.4 with 0.05%Tween20
Controls:
Catching:
Negative: PBS pH 7.4 with 0.05 IoTween20
Haemolysis: PBS pH 7.4 with 0.05%Tween20
Positive High: PdB22-6E6 diluted to 10 yg/ml in PBS
Positive Low: PdB22-6E6 diluted to 2,ug/mI in PBS
Samples:
Samples are undiluted culture supernatants with antibody concentrations
expected
to be 1-5,ug/ml.

Procedure:
Strips are washed three times in PBS/0.05%Tween
Add 50,u1/well of PBS/0.05%Tw20 followed by 50,u1/well of undiluted culture
super-
natant or 50,u1/well of controls.
Incubate I h at room temperature.
Wash x4 with PBS (without Tween20)
50,uI PBS is added to each well, A1-B1 are added 100,u1/well.
Recombinant PdB is diluted to 4pg/ml in pre-heated PBS and activated with 10
mM
DTT (final concentration) for 15 min at 37 C
Add 50,u1/well of activated PdB, except for A1-B1.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
Incubate for 30 min at 37 C.
Sheep erythrocytes are washed thrice in PBS and resuspended to 2% vol/vol in
PBS.
Add 50,uI to each well and incubate for 30 min at 37 C.
5 Centrifuge plates 5 min at 1000 xg.
A digital image of the plate is obtained.
Carefully transfer 100 /jI of supernatant to flat-bottomed microwells and read
OD at
405 nm.

STRIP NO. 1 2 3 4
A Negative Sample 1 Sample 5 Sample 9
B Negative Sample I Sample 5 Sample 9
C Haemolysis Sample 2 Sample 6 Sample 10
D Haemolysis Sample 2 Sample 6 Sample 10
E Positive High Sample 3 Sample 7 Sample 11
F Positive High Sample 3 Sample 7 Sample 11
G Positive Low Sample 4 Sample 8 Sample 12
H Positive Low Sample 4 Sample 8 Sample 12

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
61
Example 3

Determination of ability of antibody to inhibit the haemolytic activity of
Pneumolysin
Description:
Purified antibodies against Pneumolysin can inhibit the lytic effect seen on
erythro-
cytes, representing a functional assay for the screening of antibodies.

Devices:
Incubator 37 C
Pipettes
Centrifuge
ELISA reader, BIO-TEK EL 800
Digital Camera, Canon Powershot S20
Materials:
Tips
Reagent tray
Plate cover
96-well microwell plate (Nunc 260170 - U-shaped)
96-well microwell plate (Nunc 260836 - flat bottom)
Reagents:
Rec. Pneumolysin (PLY) or Rec. Pneumolysoid (PdB)
PdB Lot #P01103 0.2 mg/ml in PBS diluted to 10,ug/mI
Dithiothreitol (DTT)
PBS, pH 7.4
Dem. H20
Sheep erythrocytes 50% in Alsever's Fluid, SSI no.29431
Buffer:
PBS pH 7.4
PBS with 10 mM DTT
Samples:
Purified antibody samples are diluted in PBS.
Procedure:
Determining Haemolytic endpoint:
This is determined for each new batch of PLY or PdB. All samples are done in
tripli-
cates. Controls are:
Blank: 100 ,ul Buffer (0% Haemolysis)
Total: 100,u1 Dem. H20 (100% Haemolysis)
A dilution series of PLY/PdB is prepared in PBS w. 10 mM DTT: 40-20-10-5-2,5-
1,25-0,625-0,3125,ug/ml. Add 100,u1 to each well and incubate 15 min at 37 C.
Sheep erythrocytes (50 %) are washed three times in PBS and restored to 2%

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
62
vol/vol. Add 50 ,ul to each well and incubate for 30 min at 37 C. Centrifuge 5
min at
1000 xg.
A digital image of the plate is obtained.
100,u1 supernatant is transferred to a flat bottom microwell plate and read at
405
nm. Twice the concentration of Pneumolysin giving 90% haemolysis is used as
standard concentration in the inhibition assay.

Inhibition assay:
All tests are done in duplicates round-bottom microwell plates.
Controls are:
Blank= 100 pI PBS
Total Haemolysis = 100,u1 dem. H20
Negative = 50,u1 Pneumolysin + 50,u1 PBS

Pneumolysin: Pool PdB 031201, 0.5 mg/mI diluted to 20 /ug/mI = 1,ug/well
PLY/PdB is diluted in pre-heated PBS and activated with 10 mM DTT (final
concen-
tration) for 15 min at 37 C.
50,u1 antibody dilution is added to each well followed by 50 ,uI activated
PLY/PdB.
The plate is incubated for 30 min at 37 C.
Sheep blood is washed thrice in PBS and restored to 2% vol/vol.
Add 50,u1 to each well and incubate plate for 30 min at 37 C.
Centrifuge 5 min at 1000 xg.
A digital image of the plate is obtained.
100,u1 of supernatant is transferred to a second flat bottom microwell plate
(plate 2)
and read at 405 nm (an example is shown in table 1). The titer is determined
as the
dilution of antibody which inhibits 50 % haemolysis and is included in table 4
below.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
63
Samples:
All purified antibodies are diluted to 500,ug/mI in PBS.
S1= Ra-a-Pneumolysin
S2= OmpA17-10C7 031024
S3= 22-6E6.5 040224
S4= 26-51=12.1040520
S5= 26-23C2.2 040319
S6= 26-18G8.2 040319
S7= 26-30H10.2 040319
S8= 28-10E7.2 040514
S9= 26-14G4 040305
S10= 13-2E12.1 031105
S11= 22-1C11.1031211
Plate Setup:
PLATE 1:

1 2 3 4 5 6 7 8 9 10 11 12
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Sil
A Blank 500 pg/mI
B Blank 100 Ng/mI
C Total 20 pg/mI
D Total 4 Ng/mI
E Negative 800 ng/ml
F Negative 160 ng/ml
G 32 ng/ml
H 6 ng/ml
PLATE 2:

1 2 3 4 5 6 7 8 9 10 11 12
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11
A Blank 500 pg/mI
B Blank 100 Ng/mI
C Total 20 Ng/mI
p Total 4 Ng/mI
E Negative 800 ng/ml
F Negative 160 ng/ml
G 32 ng/ml
H 6 ng/ml

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
64
The data relating to sample I to 11 are shown in the tables here below.

Blank 0,05
Total 1,15
Negative 1,18
Sample 1 2 3 4 5 6 7 8 9 10 11
no.
Antibody, N
ng/ml >, ~ N N ~ N ~-
~ ~ ~ N (N ~ CV
~ QU w u c~i o~0 0 0 d w U
~c CO L? N M r ~ N
O~ N N N N N N N ~ N
500000 1,15 1,23 0,05 0,05 0,05 0,05 0,05 0,05 0,06 1,17 1,23
100000 1,15 1,16 0,08 0,05 0,05 0,05 0,04 0,05 0,05 1,11 1,18
20000 1,14 1,17 0,06 0,05 0,04 0,04 0,05 0,07 0,05 1,13 1,16
4000 1,14 1,16 0,04 0,06 0,04 0,14 0,04 0,68 0,12 1,12 1,15
800 1,14 1,17 0,09 0,09 0,06 0,92 0,11 1,03 0,70 1,13 1,15
160 1,11 1,17 0,13 0,09 0,06 1,09 1,06 1,11 1,15 1,12 1,14
32 1,11 1,13 1,09 1,13 1,08 1,15 1,12 1,14 1,14 1,13 1,16
6 1,14 1,16 1,15 1,15 1,13 1,14 1,12 1,17 1,19 1,14 1,16
Table 1. OD at 405 nm.

The % of haemolysis is calculated from the obtained data (table 1) and shown
in
table 2 here below.

N
N
-p LO
ni N 00
E o O O W
~ U
~ E W LL M ~ o CO L? N - C? r
:3
(N (O (O (O cfl o0 Cfl c~ ) N
~ 0 N N N N N N (N r N
6 96 98 97 97 96 96 95 99 100 96 98
32 94 96 92 95 91 97 95 96 96 96 98
160 94 99 11 8 5 92 90 94 97 95 96
800 96 99 7 8 5 78 9 87 59 95 98
4000 97 98 4 5 3 12 4 58 10 95 97
20000 96 99 5 4 4 3 5 6 4 95 98
100000 98 98 7 4 4 4 4 4 5 94 99
500000 97 104 4 5 4 4 5 4 5 99 104
Tabel 2. Haemolysis in %.

The % of inhibition is calculated from the obtained data (table 2) and shown
in table
3 here below.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536

m c- N N N
N r- ~
LO o
00 ~ -~ d' N
a E W u. U o~o 0 o d~- W U
E (O U') N M ~ r N
:3 N CO CO CO (O 00 (O c~ N
Q-' 0 N N N N N N N r- N
6 3,9 1,7 2,8 3,0 4,1 3,7 5,2 1,3 -0,4 3,7 1,9
32 5,8 4,2 7,6 4,8 8,9 2,7 5,1 4,0 3,6 4,4 2,1
160 5,9 1,3 89,1 92,5 95,1 7,7 10,1 5,8 3,0 5,4 3,6
800 3,6 1,3 92,5 92,1 94,7 21,9 90,9 13,0 40,9 4,6 2,5
4000 3,3 2,0 96,3 95,1 96,8 88,0 96,4 42,3 90,0 5,2 2,6
20000 4,1 0,7 94,7 96,1 96,4 96,6 95,5 94,3 95,9 4,6 1,9
100000 2,5 1,7 92,9 96,1 96,1 96,2 96,2 95,7 95,4 6,3 0,7
500000 3,0 -3,7 95,5 95,4 95,5 95,7 95,4 95,5 95,2 0,9 -4,0
Tabel 3. % of inhibition of haemolysis.

Graphic illustrations of the results are depicted in Figure 5.
5
The titer of the antibodies was determined based on the data described above
and
summarized in table 4 here below.

Anti haemolytic actitity
Mab 0,5 N/ml ED50, ng/ml

17-10C7.1 >500
22-6E6.5 < 0,100
26-5F12.1 < 0,100
26-23C2.2 < 0,100
13-2E12.1 >500
22-1 C11.1 >500
27-11 A8 >500
28-10E7.2 >500
10 Tabel 4. The titter of selected antibodies.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
66
Example 4

Affinity Characterization of anti-Pneumolysin HuMabs

Avidity measurements were made by flowing one concentration of mAbs on antigen
coated surface.

Methods & Materials:
Material coated on chip: Protein-G Chip type: CM5. Chip prepared on: Sep 16,
2003
Coating density: FC1 & 3 = blank, FC2= 6286 RUs, FC4 = 6700RUs
Coating conditions: Conc. of protein = 5 g/mL, dilution buffer = sodium
acetate, pH
= 4.5
Running Buffer: HBS-EP.
Reagents:
Antibodies (purified):
1. 4E8 0.94 mg/mL
2. 22-6E6 2.50 mg/mL
3. 26-23C2 3.40 mg/mL
4. 26-5F12 1.26 mg/mL
5. 22-1C11 5.80 mg/mL
6. 13-2E12 1.03 mg/mL

7. 10-3G7.2 1.10 mg/mL
8. 10-5G3.3 0.82 mg/mL
9. 10-14A5.2 0.91 mg/mL
10. 10-5G3.2 1.14 mg/mL

Antigen: 0.6 mg/mL, 57kDa (full length w/ His-tag)
Experimental Conditions:

Capture (Ab) Conc: 20ug/mL concentration, 200 uL @ 50 uL/min flow rate
Association time: 4min.
Dissociation time: 20 min.
Regeneration of chip: one pulse of 17uL of 50mM NaOH + 75 NaCl @ 75uL/min
flow rate

Results:
The estimate affinity and rate constants from this experiment are listed in
Table 1.
here below. The first few seconds of association and dissociation have been
fit to a
1:1 Langmuir model to obtain the affinity and rate constants.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
67
Sample ID KD x 10-9 (M) Ko~ x 105 Koff x 10-4
(1/Ms) (1/s)
4E8 0.22 11.6 2.5
22-6E6 0.31 13 4.0
26-23C2 0.69 5.2 3.6
26-5F12 0.82 5.3 4.3
22-1 C11 11.7 0.62 0.7
13-2E12 24.7 1.24 3.1
10-3G7.2 0.66 0.95 30.6
10-5G3.3 1.1 0.39 0.44

10-14A5.2 28.7 0.71 20.3
10-5G3.2 0.66 0.41 0.27
Table 1. Affinity and rate constants of Pneumolysin antibodies.
Example 5

Generation of anti-CD64 x anti-Pneumolysin 5-9A7 Bispecific Antibody

F(ab')2 fragments of each of the HuMAbs, anti-CD64 (88.53), and anti-
Pneumolysin
are generated by pepsin digestion and purified to hom*ogeneity by Superdex 200
gel
filtration chromatography. Size exclusion HPLC is performed, and by this type
of
analysis both of the F(ab') 2 fragments are >95% pure.

A Fab' fragment of the 88.53 is generated by mild reduction of the inter-heavy
chain
disulfide bonds of the F(ab') 2 fragment with mercaptoethanolamine (MEA). The
exact reducing conditions are determined prior to conjugation in small-scale
experi-
ments. Size exclusion HPLC is performed, and by this type of analysis the
88.53
Fab' is >90% pure.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
68
The Fab' fragment of the 88.53 is separated from free MEA by G-25 column chro-
matography. The Fab' fragment is incubated with dinitrothiobenzoate (DTNB) to
generate a Fab-TNB conjugate.

A Fab' fragment of the anti-Pneumolysin antibody is generated by mild
reduction of
the inter-heavy chain disulfide bonds of the F(ab') 2 fragment with
mercaptoethanol-
amine (MEA). The exact reducing conditions are determined prior to conjugation
in
small-scale experiments. Size exclusion HPLC is performed, and by this type of
analysis the Fab' is >90% pure.
The Fab' fragment is separated from free MEA by G-25 column chromatography
and mixed with 88.53 Fab-TNB at a 1:1 molar ratio overnight at room
temperature.
The bispecifc antibody is purified from contaminating Fab' molecules by
Superdex
200 size exclusion chromatography, and the purified molecule is analyzed by
HPLC.
For control anti-CD64 x anti-CD89 Bispecific Antibody are generated. F(ab') 2
frag-
ments of each of the HuMAbs, anti-CD64 (88.53), and anti-CD89 (14A8) are gene-
rated by pepsin digestion and purified to hom*ogeneity by Superdex 200 gel
filtration
chromatography. Size exclusion HPLC is performed, and by this type of analysis
both of the F(ab') 2 fragments are >95% pure.

A Fab' fragment of the 88.53 is generated by mild reduction of the inter-heavy
chain
disulfide bonds of the F(ab')2 fragment with mercaptoethanolamine (MEA). The
ex-
act reducing conditions are determined prior to conjugation in small-scale
experi-
ments. Size exclusion HPLC is performed, and by this type of analysis the
88.53
Fab' is >90% pure.

The Fab' fragment of the 88.53 is separated from free MEA by G-25 column chro-
matography. The Fab' fragment is incubated with dinitrothiobenzoate (DTNB) 16a
and 16b to generate a Fab-TNB conjugate.

A Fab' fragment of the 14A8 is generated by mild reduction of the inter-heavy
chain
disulfide bonds of the F(ab')2 fragment with mercaptoethanolamine (MEA). The
ex-
act reducing conditions are determined prior to conjugation in small-scale
experi-

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
69
ments. Size exclusion HPLC is performed, and by this type of analysis the 14A8
Fab' is >95% pure.

The Fab' fragment of the 14A8 is separated from free MEA by G-25 column chroma-

tography and mixed with 88.53 Fab-TNB at a 1:1 molar ratio overnight at room
tem-
perature.

The bispecific antibody is purified from contaminating Fab' molecules by
Superdex
200 size exclusion chromatography, and the purified molecule is analyzed by
HPLC.
The 88.53 x 14A8 bispecific antibody is purified to near hom*ogeneity.

Characterization of the Binding Specificity of the anti-CD64 x anti-
Pneumolysin Bis-
pecific Antibody - Bispecific ELISA

1. ELISA plates are coated with recombinant Pneumolysin, 50 l/well, 5 g/ml
and incubated overnight at 4 C.
2. The plates are blocked with 5% BSA in PBS.
3. Titrations of the bispecific antibody are added to the plate. Controls
include
the anti-CD64 x anti-CD89 bispecific (control bispecific) and the F(ab')2 frag-

ments of the anti-CD64 Ab, 88.53 or of the anti-Pneumolysin Ab.
4. The plates are then incubated with a supernatant containing a fusion
protein
consisting of soluble CD64 linked to the Fc portion of human IgM.
5. The plates are finally incubated with an alkaline phosphatase labelled goat
anti-human IgM antibody. Positive wells are detected with the alkaline phos-
phatase substrate.

Characterization of the BindincSpecificity of the anti-CD64 x anti-Pneumolysin
Bis-
pecific Antibody - Binding to CD64 on Human CD64-transgenic Mice

Blood is taken from CD64 transgenic mice or from non-transgenic littermates,
and
incubated with the 88.53 x anti-Pneumolysin bispecific antibody at a
concentration of
30 g/ml for 30 minutes at room temperature.

The blood is washed and then incubated with an FITC-labelled anti-human IgG
anti-
body for 30 minutes at room temperature. The red blood cells are lysed and the
re-

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
maining leukocytes are analyzed for staining by flow cytometry. Regions corre-
sponding to the lymphocyte, monocyte, and neutrophil populations are gated and
analyzed separately.

5 Human CD64 is expressed on monocytes and, to a lesser extent, neutrophils of
CD64 transgenic mice. As in humans, CD64 is not expressed by lymphocytes of
the
transgenic mice. The bispecific antibody binds to CD64 transgenic monocytes
and
neutrophils, but not to any cell populations derived from non-transgenic mice.

Example 6

Sequencing of monoclonal antibody

The DNA encoding antibodies according to the present invention are sequenced
as
described below for the antibody 26-5F12.1.

Total RNA was isolated from hybridoma cells using STAT60 reagent (BioGenesis)
and converted into cDNA for use as a template in PCR. Agarose gel analysis
showed a high yield of the extracted RNA from the pellet (figure (8A)

cDNA was created from the RNA. Heavy chain and light chain variable regions
were
amplified using Heavy Primers and Light Primer Mix from Amersham Biosciences.
The PCR products were analysed on a Tris-acetate-EDTA agarose gel. The PCR
using these primers on the cDNA gave the bands shown in figure 8B.

Direct cloning of the PCR products gave poor transformation efficiency, so the
PCR
products were gel purified and cloned. Samples positive in the PCRs were
cloned
into the pCR4-TOPO vector in the TOPO TA Cloning Kit (Invitrogen).

The purified VL and VH PCR products were cloned into a sequencing vector and
positive transformants were determined by colony PCR (figure 8C).

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
71
All positive clones were picked (normally 3) for each chain and sequenced with
both
forward and reverse sequencing primers. The clones were sequenced by the dide-
oxy method with the BigDye V3.1 DNA sequencing kit (Applied Biosystems).

Sequencing analysis identified five correct clones for the variable heavy
chain and
seven for the variable light chain of monoclonal antibody 26-5F 12.1. The DNA
and
protein sequences for each of these clones are shown below.

Monoclonal Antibody 26-5F 12.1 sequencing results
26-5F 12.1 VH Clone 4 DNA Sequence
AGGTGCAGCTGCAGGAGTCAGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTTTCCTGCACGGCTTCTGGATACATCTTCACTAGCTATGCTATACATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
GGCTATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCAGCATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGTTGAGCAGCCTGAGATCT
GAAGACACGGCTGTGTATTACTGTGCGAGAAGGGGGCAGCAGCTGGCCTTTG
ACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

26-5F 12.1 VH Clone 4 Protein Sequence

V Q L Q E S G A E V K K P G A S V K V S C T A S G Y I F T S Y A I H W V R Q A
PGQRLEWMetGWINAGYGNTKYSQKFQGRVSITRDKSAST
A Y Met E L S S L R S E D T A V Y Y C A R R G Q Q L A F D Y W G Q G T T V T V
SS

26-5F 12.1 VH Clone 3 DNA Sequence
AGGTGAAGCTGCAGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTTTCCTGCACGGCTTCTGGATACATCTTCACTAGCTATGCTATACATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
G G CTATG GTAACACAAAATATTCACAGAAGTTC CAG G G CAGAGTCAG CATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGTTGAGCAGCCTGAGATCT
GAAGACACGGCTGTGTATTACTGTGCGAGAAGGGGGCAGCAGCTGGCCTTTG
ACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

26-5F 12.1 VH Clone 3 Protein Sequence

V K L Q E S G A E V K K P G A S V K V S C T A S G Y I F T S Y A I H W V R Q A
PGQRLEWMetGWINAGYGNTKYSQKFQGRVSITRDKSAST
AYMetELSSLRSEDTAVYYCARRGQQLAFDYWGQGTTVTV
S S

26-5F 12.1 VH Clone 6 DNA Sequence

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
72
AGGTGAAGCTGCAGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTTTCCTGCACGGCTTCTGGATACATCTTCACTAGCTATGCTATACATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
GGCTATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCAGCATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGTTGAGCAGCCTGAGATCT
GAAGACACG G CTGTGTATTACTGTG CGAGAAG G G G G CAG CAG CTG G C CTTT G
ACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTC

26-5F 12.1 VH Clone 6 Protein Sequence

V K L Q Q S G A E V K K P G A S V K V S C T A S G Y I F T S Y A I H W V R Q A
P G Q R L E W MetG W I N A G Y G N T K Y S Q K F Q G RV S I T R D KSAST
AYMetELSSLRSEDTAVYYCARRGQQLAFDYWGQGTTVTV
S

26-5F 12.1 VH Clone 15 DNA Sequence
AGGTGAAGCTGCAGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTTTCCTGCACGGCTTCTGGATACATCTTCACTAGCTATGCTATACATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
GGCTATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCAGCATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGTTGAGCAGCCTGAGATCT
GAAGACACGGCTGTGTATTACTGTGCGAGAAGGGGGCAGCAGCTGGCCTTTG
ACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTC

26-5F 12.1 VH Clone 15 Protein Sequence
VKLQQSGAEVKKPGASVKVSCTASGYI FTSYAIHWVRQA
PGQRLEWMetGWINAGYGNTKYSQKFQGRVSITRDKSAST
AYMetELSSLRSEDTAVYYCARRGQQLAFDYWGQGTTVTV
S

26-5F 12.1 VH Clone 10 DNA Sequence

AGGTGAAGCTGCAGGAGTCAGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTTTCCTGCACGGCTTCTGGATACATCTTCACTAGCTATGCTATACATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
G G CTATG GTAACACAAAATATTCACAGAAGTTC CAG G G CAGAGTCAG CATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGTTGAGCAGCCTGAGATCT
GAAGACACGGCTGTGTATTACTGTGCGAGAAGGGGGCAGCAGCTGGCCTTTG
ACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
26-5F 12.1 VH Clone 10 Protein Sequence
V K L Q E S G A E V K K P G A S V K V S C T A S G Y I F T S Y A I H W V R Q A
PGQRLEWMetGWINAGYGNTKYSQKFQGRVSITRDKSAST

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
73
AYMetELSSLRSEDTAVYYCARRGQQLAFDYWGQGTTVTV
SS

26-5F 12.1 VL Clone 2 DNA Sequence
GACATCCAGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCT
GGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC
AGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAG
ACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACT
GTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGCACCAAGCTGGAA
ATCAAACGG

26-5F 12.1 VL Clone 2 Protein Sequence

D I Q Met T Q S P G T L S L S P G E R A T L S C R A S Q S V S S S Y L A W Y Q
Q K P G Q A P R L L I Y G A S S R A T G I P D R F S G S G S G T D F T L T I S
R
L E P E D F A V Y Y C Q Q Y G S S P F T F G P G T K L E I K R

26-5F 12.1 VL Clone 3 DNA Sequence
GACATCCAGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCT
GGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC
AGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAG
ACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACT
GTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGCACCAAGCTGGAA
ATCAAACGG

26-5F 12.1 VL Clone 3 Protein Sequence

D I Q Met T Q S P G T L S L S P G E R A T L S C R A S Q S V S S S Y L A W Y Q
QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR
L E P E D F A V Y Y C Q Q Y G S S P F T F G P G T K L E I K R

26-5F 12.1 VL Clone 4 DNA Sequence

GACATCCAGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCT
GGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC
AGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAG
ACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACT
GTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGCACCAAGCTGGAA
ATCAAACGG

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
74
26-5F 12.1 VL Clone 4 Protein Sequence

D I Q Met T Q S P G T L S L S P G E R A T L S C R A S Q S V S S S Y L A W Y Q
QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR
L E P E D F A V Y Y C Q Q Y G S S P F T F G P G T K L E I K R

26-5F 12.1 VL Clone 5 DNA Sequence
GACATCCAGATGACTCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCT
GGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC
AGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAG
ACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACT
GTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGCACCAAGCTGGAA
ATCAAACGG

26-5F 12.1 VL Clone 5 Protein Sequence

D I Q Met T Q S P G T L S L S P G E R A T L S C R A S Q S V S S S Y L A W Y Q
QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR
L E P E D F A V Y Y C Q Q Y G S S P F T F G P G T K L E I K R

26-5F 12.1 VL Clone 6 DNA Sequence
GACATCCAGATGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCT
GGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC
AGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAG
ACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACT
GTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGCACCAAGCTGGAA
ATCAAACGG

26-5F 12.1 VL Clone 6 Protein Sequence

D I Q Met T Q S P G T L S L S P G E R A T L S C R A S Q S V S S S Y L A W Y Q
QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR
L E P E D F A V Y Y C Q Q Y G S S P F T F G P G T K L E I K R

26-5F 12.1 VL Clone 10 DNA Sequence
GACATCCAGATGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCT
GGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC
AGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAG
ACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACT

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
GTCAG CAGTATG GTAG CTCACCATTCACTTTC G G CCCTG G CAC CAAG CTG GAA
ATCAAACGG

5 26-5F 12.1 VL Clone 10 Protein Sequence

D IQMetTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ
QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR
LEPEDFAVYYCQQYGSSPFTFGPGTKLEIKR

26-5F 12.1 VL Clone 12 DNA Sequence
GACATCCAGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCT
GGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC
AGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAG
ACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACT
GTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGCACCAAGCTGGAA
ATCAAACGG

26-5F 12.1 VL Clone 12 Protein Sequence

D I Q Met T Q S P G T L S L S P G E R A T L S C R A S Q S V S S S Y L A W Y Q
QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR
LEPEDFAVYYCQQYGSSPFTFGPGTKLEIKR

Monoclonal antibody 26-5F 12.1 consensus seguence
VH consensus protein sequence

V K L Q E S G A E V K K P G A S V K V S C T A S G Y I F T S Y A I H W V R Q A
P
GQRLEWMetGWINAGYGNTKYSQKFQGRVSITRDKSAST
AYMetELSSLRSEDTAVYYCARRGQQLAFDYWGQGTTVT
VSS

VL consensus Protein Sequence

D IQMetTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQ
KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEP
E D F A V Y Y C Q Q Y G S S P F T F G P G T K L E I K R
The sequences of the variable light and heavy chain of 26-5F 12.1 are shown in
figure 3A, where the sequence of the CDRs is also included.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
76
Monoclonal Antibody 26-23 C2.2 Sequencing Analysis

RNA is extracted as described above showing a high yield (Figure 9A).

cDNA was created from the RNA. The initial PCR reactions prepared to amplify
the
VL region were unsuccessful. New primers were ordered to amplify VHand VL in
separate reactions. The PCR using these primers on the original cDNA gave the
VH
and VL bands shown in figure 9B.

The purified VH and VL PCR products were cloned into a sequencing vector and
positive transformants were determined by colony PCR (figure 9C).

VH and VL clones were picked and sequenced. The sequence of 5 VH clones and 3
VL clones is shown here below.
Monoclonal Antibody 26-23 C2.2 sequencing results
26-23 C2.2 VH Clone 1 DNA sequence

AGGTGAAGCTGCAGGAGTCAGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTTTCCTG CACGGCTTCTGGATACATCTTCACTAGCTATGCTATGCATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
GGCTATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCAGCATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGCTGACCAGCCTGAGATCT
GAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGCAGCAGCTGGCCTTTG
ACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
26-23 C2.2 VH Clone 1 amino acid sequence

VKLQESGAEVKKPGASVKVSCTASGYI FTSYAMHWVRQAPGQRLEW MGW I NAGY
GNTKYSQKFQGRVSITRDKSASTAYMELTSLRSEDTAVYYCARRGQQLAFDYW-
GQGTTVTVSS

26-23 C2.2 VH Clone 2 DNA sequence
AGGTGAAACTGCAGCTGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTTTCCTGCACG GCTTCTGGATACATCTTCACTAGCTATGCTATGCATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
GGCTATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCAGCATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGCTGACCAGCCTGAGATCT
GAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGCAGCAGCTGGCCTTTG
ACTACTGGGGCCAAGGGACCACGGTCAACGTCTCCTCA

26-23 C2.2 VH Clone 2 amino acid sequence

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
77
VKLQLSGAEVKKPGASVKVSCTASGYIFTSYAMHWVRQAPGQRLEWMGWINAGY
GNTKYSQKFQGRVSITRDKSASTAYMELTSLRSEDTAVYYCARRGQQLAFDYW-
GQGTTVNVSS

26-23 C2.2 VH Clone 3 DNA sequence
AGGTCAAACTGCAGGAGTCAGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAG GTTTC CTG CACG G CTTCTG GATACATCTTCACTAG CTATG CTATG CATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
GGCTATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCAGCATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGCTGACCAGCCTGAGATCT
GAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGCAGCAGCTGGCCTTTG
ACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

26-23 C2.2 Clone VH3 amino acid sequence
VKLQESGAEVKKPGASVKVSCTASGYI FTSYAMHWVRQAPGQRLEWMGWINAGY
GNTKYSQKFQGRVSITRDKSASTAYMELTSLRSEDTAVYYCARRGQQLAFDYW-
GQGTTVTVSS

26-23 C2.2 VH Clone 4 DNA sequence

AGCTCAAGCTGCAGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTTTCCTGCACGGCTTCTGGATACATCTTCACTAGCTATGCTATGCATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
GGCTATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCAGCATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGCTGACCAGCCTGAGATCT
GAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGCAGCAGCTGGCCTTTG
ACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
26-23 C2.2 VH Clone 4 amino acid sequence
LKLQESGAEVKKPGASVKVSCTASGYI FTSYAMHWVRQAPGQRLEW MGW I NAGY
GNTKYSQKFQGRVSITRDKSASTAYMELTSLRSEDTAVYYCARRGQQLAFDYW-
GQGTTVTVSS

26-23 C2.2 VH Clone 5 DNA sequence
AGGTGCAGCTGCAGGAGTCAGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAG GTTTCCTG CACG G CTTCTG GATACATCTTCACTAG CTATG CTATG CATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
GGCTATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCAGCATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGCTGACCAGCCTGAGATCT
GAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGCAGCAGCTGGCCTTTG
ACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

26-23 C2.2 VH Clone 5 amino acid sequence

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
78
VQLQESGAEVKKPGASVKVSCTASGYI FTSYAMHW VRQAPGQRLEW MGW I NAG
YGNTKYSQKFQG RVSITRDKSASTAYMELTSLRSEDTAVYYCARRGQQLAFDYW
GQGTTVTVSS

26-23C 2.2 VL clone 2 DNA sequnece
GACATCCGGGTGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAG
GGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTT
ATACGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTAT
CTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAA
CCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGG
TGGAAATCAAAA

26-23C 2.2 VL clone 2 Protein Sequence
DIRVTQSPASLAVSLGQRATISYRASKSVSTSGYSYTHWNQQKPGQPPRLLIYLVS
NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPRWKSK
26-23C 2.2 VL clone 3 DNA Sequence

GACATCCAGATGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAG
GGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTT
ATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTAT
CTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAA
CCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGC
TGGAGATCAAAA

26-23C 2.2 VL clone 3 Protein Sequence
DIQMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLV
SNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWRSK
26-23C 2.2 VL clone 4 DNA Sequence

GACATCCAGTTGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAG
GGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTT
ATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTAT
CTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAA
CCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGG
TGGAAATCAAAA

26-23C2.2 VL clone 4 Protein Sequence

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
79
DIQLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVS
NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPRWKSK

Monoclonal antibody 26-23C2.2 consensus sequences
VH consensus DNA sequence

AGGTGAAGCTGCAGGAGTCAGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAG GTTTC CTG CACG G CTTCTG GATACATCTTCACTAG CTATG CTATG CATTG
GGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCT
GGCTATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCAGCATTAC
CAGGGACAAATCCGCGAGCACAGCCTACATGGAGCTGACCAGCCTGAGATCT
GAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGCAGCAGCTGGCCTTTG
ACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
VH consensus amino acid sequence
VKLQESGAEVKKPGASVKVSCTASGYI FTSYAMHW VRQAPGQRLEW MGW I NAGY
GNTKYSQKFQGRVSITRDKSASTAYMELTSLRSEDTAVYYCARRGQQLAFDYW-
GQGTTVTVSS

VL consensus DNA Sequence
GACATCCAGDTGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAG
GGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTT
ATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTAT
CTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAA
CCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGG
TGGAAATCAAAA

VL consensus Protein Sequence
DIQLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVS
NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPRWKSK

The sequences of the variable light and heavy chain of 26-23C2 are shown in
figure
3B, where the sequence of the CDRs is also included.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
Monoclonal Antibody 22 1C 11 Sequencing Analysis

cDNA was created from mRNA. PCR reactions to amplify the VH and VL regions of
the monoclonal antibody DNA gave the bands shown in figure 10A.

5

The purified VH and VL PCR products were cloned into a sequencing vector and
positive transformants were determined by colony PCR (figure 10 B):

Seven VH and six VL clones were picked for each chain and sequenced with both
10 forward and reverse sequencing primers. Sequencing analysis identified 5
correct
clones for the VH chain of monoclonal antibody 22-1 C11.

The VL sequencing was of poorer quality. A further six clones were picked and
se-
quenced to obtain a consensus sequence from a total of six clones.
The DNA and protein sequences for the positive VH and VL clones are shown
below
Monoclonal Antibody 22-1C 11 Sequencing results
22-1 C 11 VH Clone 1 DNA sequence
AGGTGCAACTGCAGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCC
TAAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACT
GGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTA
TGATGGAAGTAATAAATACTATGCAGACTTCGTGAAGGGCCGATTCACCATCTC
CAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCG
AGGACACGGCTGTGTATTACTGTGCGAGAAGGGGAAATTACTATGGTTTGGGG
AGCTTCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCG-
TCTCCTCA

22-1 C 11 VH Clone I Amino acid sequence:
VQLQESGGGWQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWYD
GSNKYYADFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGNYYGLGSFY
YYGMDVWGQGTTVTVSS

22-1 C 11 VH Clone 2 DNA sequence
AGGTGAAGCTGCAGGAGTCTGGGGGAGGCGTGGCCCAGCCTGGGAGGTCCC
TAAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACT
GGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTA
TGATGGAAGTAATAAATACTATGCAGACTTCGTGAAGGGCCGATTCACCATCTC
CAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCG

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
81
AG GACAC G G CTGTGTATTACTGTG C GAGAAG G G GAAATTACTATG GTTTG G G G
AGCTTCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCG-
TCTCCTCA

22-1 C 11 VH Clone 2 Amino Acid sequence:
VKLQESGGGVAQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWYD
GSNKYYADFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGNYYGLGSFY
YYGMDVWGQGTTVTVSS

22-1 C 11 VH Clone 3 DNA sequence

AGGTCCAACTGCAGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCT
AAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACTG
GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTAT
GATGGAAGTAATAAATACTATGCAGACTTCGTGAAGGGCCGATTCACCATCTCC
AGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGA
GGACACGGCTGTGTATTACTGTGCGAGAAGGGGAAATTACTATGGTTTGGGGA
GCTTCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCG-
TCTCCTC

22-1 C 11 VH Clone 3 Amino acid sequence
VQLQESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWYD
GSNKYYADFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGNYYGLGSFY
YYGMDVWGQGTTVTVS

22-1 C 11 VH Clone 4 DNA Sequence
AGGTCAAACTGCAGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCT
AAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACTG
GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTAT
GATGGAAGTAATAAATACTATGCAGACTTCGTGAAGGGCCGATTCACCATCTCC
AGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGA
GGACACGGCTGTGTATTACTGTGCGAGAAGGGGAAATTACTATGGTTTGGGGA
GCTTCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCG-
TCTCCTCA

22-1 C 11 VH Clone 4 Amino Acid Sequence
VKLQESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWYD
GSNKYYADFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGNYYGLGSFY
YYGMDVWGQGTTVTVSS

22-1 C 11 VH Clone 8 DNA sequence

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
82
AGGTGAAGCTGCAGGAGTCAGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCC
TAAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACT
GGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTA
TGATGGAAGTAATAAATACTATGCAGACTTCGTGAAGGGCCGATTCACCATCTC
CAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCG
AGGACACGGCTGTGTATTACTGTGCGAGAAGGGGAAATTACTATGGTTTGGGG
AGCTTCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCG-
TCTCCTCA

22-1 C 11 VH Clone 8 Amino Acid Sequence
VKLQESGGGWQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWYD
GSNKYYADFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGNYYGLGSFY
YYGMDVWGQGTTVTVSS

22-1C 11 VL Clone 3 DNA Sequence
GACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGT
ACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAAC
AGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGCCTGGGACAGACT
TCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTC
AGCAGCGTAGCAACTGGCATCCGACGTTCGGCCAAGGCACCAAGCTG-
GAAATCAAACGG

22-1 C 11 VL Clone 3 Amino Acid Sequence

DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWHPTFGQ
GTKLXNQT

22-1 C 11 VL Clone 6 DNA Sequence
ACACAGTNTCCNGCCNCCCTGTNTTNGTCTNCAGNGGAAAGANCCACCCTNTC
CNGCAGGNCCAGTCANAGTGTTNGCAGCTANTTAGCCTGGTACCAACAGAAAN
NTGGNCAGGCTCCCAGGCTCCTCATCTATGANGCATCCAACNGGGCCACTGG
CATCCCAGCCAGGTTCAGNGGCAGTGGGTNTGGGACAGACTTCACTCTCACCA
TCAGCAGCNTAGAGCCTGAAGATTTNGCAGTTTATTACTGTCAGCAGTGTAGCA
ACTGGCATCCGACATTCGGCCAAGGCACCAAGCTGGAAATCAAANGN
Sequence of bad quality.

22-1 C 11 VL Clone 7 DNA sequence
GACATCCAGATGACCCAGTTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGA
GCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTA
CCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACA
GGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCA

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
83
GCAGTGTAGCAACTGGCATCCGACGTTCGGCCAAGGCACCAAGCTGGAAAT-
CAAACGG

22-1 C 11 VL Clone 7 Amino Acid sequence
DIQMTQFQPPCLCLQGKEPPSPAGPVRVLAAT*PGTNRNLARLPGSSSMM
HPTGPLASQPGSVAVGLGQTSLSPSAA*SLKILQFITVSSVATGIRRSAK
APSWKSN

22-1 C 11 VL Clone 11 DNA sequence
GACATCCAGATGACACAGTCTCCAGCCACCCTGTCTTTGTCTNCAGGGGAAAG
AGCCACCCTCTCCNGCAGGNCCAGTCAGAGTGTTAGCAGNTANTTAGCCTGGT
ACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAAC
AGGGCCACTGGCATCCCANNCAGGTTCAGTGGCAGTGGGTCTGGGACAGACT
TCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTNGCAGTTTATTACTGTC
AGCAGTGTAGCAACTGGCATCNGACATTCGGCCAAGGCACCAAGCTGGAAATC
AAACGG

Sequence of bad quality.

22-1 C 11 VL Clone 12 DNA sequence

GACATCCAGATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGT
ACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAAC
AGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACT
TCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTC
AGCAGTGTAGCAACTGGCATCCGACTTCGGCCAAGGCACCAAGCTGGAAATCA
AACGG

22-1 C 11 VL Clone 12 Amino Acid sequence
D IQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQCSNWHPTSAK
APSWKSN

22-1 C 11 VL Clone 14 DNA sequence
GACATCCAGATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGT
ACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAAC
AGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACT
TCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTC
AGCAGTGTAGCAACTGGCATCNGACATTCGGCCAAGGCACCAAGNTGGAAAN
CAAACGG

22-1 C 11 VL Clone 14 Amino Acid sequence

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
84
DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQCSNWHLTFGQ
GTK

Monoclonal antibody 22-1 C 11 consensus sequences
VH consensus DNA sequence

AGGTGAAACTGCAGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCT
AAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACTG
GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTAT
GATGGAAGTAATAAATACTATGCAGACTTCGTGAAGGGCCGATTCACCATCTCC
AGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGA
GGACACGGCTGTGTATTACTGTGCGAGAAGGGGAAATTACTATGGTTTGGGGA
GCTTCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGT
CTCCTCA

VH consensus Amino Acid sequence
VKLQESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWYD
GSNKYYADFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGNYYGLGSFY
YYGMDVWGQGTTVTVSS

VL consensus DNA sequence
GACATCCAGATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGT
ACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAAC
AGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACT
TCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTC
AGCAGTGTAGCAACTGGCATCCGACATTCGGCCAAGGCACCAAGCTGGAAATC
AAACGG

VL consensus Amino Acid sequence

DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRA
TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQCSNWHPTFGQGTKLEIKR
The sequences of the variable light and heavy chain of 22-1 C11 are shown in
figure
3C, where the sequence of the CDRs is also included.

An alignment of the CDR sequences of 26-5F12, 26-23 C2 and 22 1 C11 is shown
in
figure 11.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
Example 7

Identification of localisation of epitopes

5 Synthetic peptide fragments of Pneumolysin of 12 amino acids, representing
28
amino acid residues of Pneumolysin are produced. The peptides overlap with
neighbouring fragments with at least 8 amino acid residues. The peptides are
shown
in figure 6. Antibody binding to the fragments is tested in a standard ELISA
assay as
described here below. All peptides used are biotinylated peptides.
Devices:
Incubator at 37
Pipettes
Elisa reader
Material:
Tips
Reagent tray
Plate cover
Reacti-Bind Streptavidin HBC Coated 96-well micro-well plates (Pierce)
Reagents:
Rabbit-a-Human IgG HRP (DAKO P0214)
OPD (o-Phenylenediamine)
Buffers:
Wash and dilution buffer: PBS with 0.05% Tween20
Blocking buffer: wash buffer added 2 % SMP (skimmed milk powder)
Controls
Negative: blank
Negative: PsaA Peptide 9144 Biotin-KDPNNKEFYEKNLKEYTDKLDKLDK-NH2, I
mg/mI 040630
Positive: PLY Peptide 10146 Biotin-ECTGLAWEWWRT-OH, 5 mg/mI
Peptides:
Peptide "GNT-01" Biotin-RECTGLAWEWWR-OH, 5 mg/mi
Peptide "GNT-02" Biotin-IRECTGLAWEWW-OH, 5 mg/mI
Peptide "GNT-03" Biotin-KIRECTGLAWEW-OH, 50,ug/ml
Peptide "GNT-04" Biotin-VKIRECTGLAWE-OH, 50,ug/mI
Peptide "GNT-05" Biotin-SVKIRECTGLAW-OH, 50 /ug/mI
Peptide "GNT-06" Biotin-LSVKIRECTGLA-OH, 50 1-ig/mI
Peptide "GNT-061" Biotin-NLSVKIRECTGL-OH, 50,ug/mI
Peptide "GNT-062" Biotin-RNLSVKIRECTG-OH, 50,ug/mI
Peptide "GNT-07" Biotin-CTGLAWEWWRTV-OH, 50,ug/mI
Peptide "GNT-08" Biotin-TGLAWEWWRTVY-OH, 50,ug/mi
Peptide "GNT-09" Biotin-GLAWEWWRTVYE-OH, 50,ug/mi
Peptide "GNT-10" Biotin-LAWEWWRTVYEK-OH, 50,ug/mi

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
86
Peptide "GNT-1 3" Biotin-EWWRTVYEKTDL-OH, 50,ug/ml
Peptide "GNT-14" Biotin-WWRTVYEKTDLP-OH, 50,ug/mi
Procedure
The coated plates is rinsed well with 3 x 3001ul of wash buffer per well.
All peptides are diluted in PBS to 2,5,ug/mI. 100,u1 is added per well and the
plated
is incubated for 1 hour at room temperature. The set up is shown here below.
The plate is flowingly rinsed with 3 x 200,u1 of wash buffer per well and
blocked for
30 min at RT with wash buffer including 2 % SMP. Subsequently each well is
rinsed
with 3 x 200,u1 of wash buffer. All Mabs are diluted to 0.5,ug/mI and 100,u1
is added
per well and the plate is incubated for 1 h at 37C. The antibody is applied as
shown
below.
The plated is rinsed using 2 x 200pi of wash buffer per well
The secondary antibody Rabbit-a-Human IgG HRP (DAKO P0214) is diluted 1:2000
in blocking buffer, 1001u1 is added per well and the plate incubated 30 min at
37C.
Each well is rinsed with 3 x 200,u1 of wash buffer and developed with OPD for
30
minutes.
Three independent experiments are performed and the result summarised here be-
low. An overview of the results of plate I is shown in figure 7A and the
results of
plate 2 is shown in figure 7B.

Peptide set up (plate 1)

1 2 3 4 5 6 7 8 9
A
Blank P9144 P10146 GNT-01 GNT-02 GNT-07 GNT-08 GNT-13 GNT-14
B
Blank P9144 P10146 GNT-01 GNT-02 GNT-07 GNT-08 GNT-13 GNT-14
C
Blank P9144 P10146 GNT-01 GNT-02 GNT-07 GNT-08 GNT-13 GNT-14
D
Blank P9144 P10146 GNT-01 GNT-02 GNT-07 GNT-08 GNT-13 GNT-14
E
Blank P9144 P10146 GNT-01 GNT-02 GNT-07 GNT-08 GNT-13 GNT-14
F
Blank P9144 P10146 GNT-01 GNT-02 GNT-07 GNT-08 GNT-13 GNT-14
G
Blank P9144 P10146 GNT-01 GNT-02 GNT-07 GNT-08 GNT-13 GNT-14
H
Blank P9144 P10146 GNT-01 GNT-02 GNT-07 GNT-08 GNT-13 GNT-14

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
87
Peptide set up (plate 2)

1 2 3 4 5 6 7 8 9 10 11 12
A GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT-
Blank P9144 P10146 01 02 03 04 05 06 061 062 07
B GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT-
Blank P9144 P10146 01 02 03 04 05 06 061 062 07
C GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT-
Blank P9144 P10146 01 02 03 04 05 06 061 062 07
D GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT-
Blank P9144 P10146 01 02 03 04 05 06 061 062 07
E
GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT-
Blank P9144 P10146 01 02 03 04 05 06 061 062 07
F
GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT-
Blank P9144 P10146 01 02 03 04 05 06 061 062 07
G GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT-
Blank P9144 P10146 01 02 03 04 05 06 061 062 07
H GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT-
Blank P9144 P10146 01 02 03 04 05 06 061 062 07

Peptide set up (plate 2 continued)

13 14 15 16 17
A
GNT-08 GNT-09 GNT-10 GNT-13 GNT-14
B
GNT-08 GNT-09 GNT-10 GNT-13 GNT-14
C
GNT-08 GNT-09 GNT-10 GNT-13 GNT-14
D
GNT-08 GNT-09 GNT-10 GNT-13 GNT-14
E
GNT-08 GNT-09 GNT-10 GNT-13 GNT-14
F
GNT-08 GNT-09 GNT-10 GNT-13 GNT-14
G
GNT-08 GNT-09 GNT-10 GNT-13 GNT-14
H
GNT-08 GNT-09 GNT-10 GNT-13 GNT-14

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
88
Antibody set up for both plates
A 17-10C7.1
B 22-6E6.5
C 26-5F12.1
D 26-23C2.2
E 13-2E12.1
F 22-1 C11.1
G 27-11 A8
H 28-10E7.2
i
Elisa readings (plate 1):
PsaA PLY GNT- GNT- GNT- GNT- GNT- GNT-
Mab Blank pept. Pept. 01 02 07 08 13 14
17-10C7.1 0,11 0,08 0,21 0,24 0,35 0,15 0,28 0,08 0,09
17-10C7.1 0,08 0,08 0,30 0,37 0,42 0,19 0,35 0,08 0,09
17-10C7.1 0,10 0,06 0,22 0,27 0,32 0,12 0,24 0,07 0,07
22-6E6.5 0,10 0,09 2,17 1,56 3,00 0,17 0,63 0,09 0,10
22-6E6.5 0,09 0,08 3,00 2,25 3,00 0,36 0,64 0,09 0,08
22-6E6.5 0,08 0,07 3,00 2,83 3,00 0,53 0,80 0,08 0,07
26-5F12.1 0,09 0,09 2,56 1,68 3,00 0,21 0,89 0,09 0,09
26-5F12.1 0,08 0,08 3,00 2,98 3,00 0,47 1,09 0,09 0,09
26-5F12.1 0,08 0,07 3,00 3,00 3,00 0,60 1,23 0,09 0,08
26-23C2.2 0,08 0,08 0,30 0,29 2,80 0,14 0,29 0,07 0,08
26-23C2.2 0,08 0,08 0,52 0,49 3,00 0,23 0,38 0,08 0,08
26-23C2.2 0,09 0,07 0,55 0,50 3,00 0,18 0,26 0,07 0,07
13-2E12.1 0,08 0,09 0,20 0,23 0,35 0,13 0,27 0,08 0,08
13-2E12.1 0,08 0,08 0,29 0,37 0,41 0,18 0,36 0,08 0,08
13-2E12.1 0,07 0,06 0,20 0,26 0,33 0,14 0,22 0,07 0,07
22-1C11.1 0,19 0,16 1,09 0,72 2,05 0,24 0,71 0,11 0,10
22-1011.1 0,11 0,10 1,71 1,16 2,51 0,58 0,91 0,12 0,10
22-1 C 11.1 0,22 0,19 2,24 1,70 3,00 1,01 1,52 0,25 0,22
27-11A8 0,09 0,08 1,99 0,92 3,00 0,15 0,34 0,08 0,09
27-11 A8 0,08 0,08 3,00 1,53 3,00 0,29 0,41 0,09 0,08
27-11 A8 0,08 0,07 3,00 1,84 3,00 0,28 0,32 0,07 0,07
28-10E7.2 0,08 0,08 0,23 0,27 0,39 0,14 0,30 0,08 0,09
28-10E7.2 0,08 0,08 0,35 0,42 0,47 0,21 0,37 0,09 0,08
28-10E7.2 0,07 0,06 0,26 0,32 0,38 0,16 0,26 0,07 0,08

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
89
Overview of results from Elias readings of plate 1:

z > o
~o W w ~ ~ w Y
W Q w 3:
z ~ w ~
pz W I~- U W
~ Y W ~ w
Sequence
Mab 0,5 PsaA PLY GNT- GNT- GNT- GNT- GNT- GNT-
,u /ml Blank pept. Pept. 01 02 07 08 13 14
17-10C7.1 0,11 0,08 0,21 0,24 0,35 0,15 0,28 0,08 0,09
22-6E6.5 0,10 0,09 2,17 1,56 >3 0,17 0,63 0,09 0,10
26-5F12.1 0,09 0,09 2,56 1,68 >3 0,21 0,89 0,09 0,09
26-23C2.2 0,08 0,08 0,30 0,29 2,80 0,14 0,29 0,07 0,08
13-2E12.1 0,08 0,09 0,20 0,23 0,35 0,13 0,27 0,08 0,08
22-1C11.1 0,19 0,16 1,09 0,72 2,05 0,24 0,71 0,11 0,10
27-11A8 0,09 0,08 1,99 0,92 >3 0,15 0,34 0,08 0,09
28-10E7.2 0,08 0,08 0,23 0,27 0,39 0,14 0,30 0,08 0,09
Elisa readings plate 2:
PsaA PLY GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT-
Mab Blank e t. Pe t. 01 02 03 04 05 06 061 062 07
17-10C7.1 0,062 0,077 0,119 0,117 0,185 0,081 0,077 0,08 0,072 0,077 0,088
0,071
22-6E6.5 0,072 0,074 2,25 2,175 ****** 2,111 0,235 0,133 0,096 0,151 0,109
0,108
26-5F12.1 0,071 0,069 OUT OUT ****** 2,814 0,262 0,154 0,104 0,152 0,107 0,132
26-23C2.2 0,068 0,061 1,112 0,764 ****** 0,711 0,133 0,108 0,089 0,112 0,096
0,094
13-2E12.1 0,067 0,065 0,128 0,118 0,171 0,078 0,074 0,075 0,069 0,072 0,077
0,08
22-1C11.1 0,567 0,539 1,577 1,274 OUT 1,103 0,748 0,816 0,56 0,61 0,844 0,681
27-11A8 0,082 0,073 OUT 1,699 ****** 0,938 0,15 0,148 0,088 0,158 0,112 0,113
28-10E7.2 0,074 0,08 0,174 0,166 0,251 0,127 0,098 0,097 0,086 0,098 0,107
0,089
Elisa readings plate 2 continued:
GNT-
Mab GNT-08 GNT-09 GNT-10 GNT-13 14
17-1007.1 0,083 0,065 0,07 0,062 0,068
22-6E6.5 0,323 0,322 0,09 0,089 0,088
26-5F12.1 0,761 0,293 0,09 0,09 0,092
26-23C2.2 0,16 0,105 0,076 0,076 0,086
13-2E12.1 0,085 0,067 0,066 0,066 0,074
22-1C11.1 0,73 0,475 0,403 0,264 0,257
27-11A8 0,175 0,095 0,076 0,071 0,076
28-10E7.2 0,106 0,081 0,071 0,072 0,072
Overview of results from Elisa readings of plate 2:

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
Sequence

W~ ~ w W (D v
Y w w
z o ~ c~ ~ ~- v W a! Y > g
Z~ ~ U ~ ~ ~ > J
~Y Y W w x > > J Z za~ U
Mab 0,5 PsaA PLY GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT- GNT-
,ug/ml Blank pept. Pept. 01 02 03 04 05 06 061 062 07
17-10C7.1
0,06 0,08 0,12 0,12 0,19 0,08 0,08 0,08 0,07 0,08 0,09 0,07
22-6E6.5
0,07 0,07 2,25 2,18 >3 2,11 0,24 0,13 0,10 0,15 0,11 0,11
26-5F12.1
0,07 0,07 >3 >3 >3 2,81 0,26 0,15 0,10 0,15 0,11 0,13
26-23C2.2
0,07 0,06 1,11 0,76 >3 0,71 0,13 0,11 0,09 0,11 0,10 0,09
13-2E12.1
0,07 0,07 0,13 0,12 0,17 0,08 0,07 0,08 0,07 0,07 0,08 0,08
22-1 C11.1
0,57 0,54 1,58 1,27 >3 1,10 0,75 0,82 0,56 0,61 0,84 0,68
27-11 A8
0,08 0,07 >3 1,70 >3 0,94 0,15 0,15 0,09 0,16 0,11 0,11
28-10E7.2
0,07 0,07 >3 >3 >3 2,81 0,26 0,15 0,10 0,15 0,11 0,13
plate 2 continued
Sequence
w
p ~
w

w
W
w
w III>~---
~ (D
Mab 0,5
,ug/ml GNT-08 GNT-09 GNT-10 GNT-13 GNT-14
17-10C7.1
0,08 0,07 0,07 0,06 0,07
22-6E6.5
0,32 0,32 0,09 0,09 0,09
26-5F 12.1
0,76 0,29 0,09 0,09 0,09
26-23C2.2
0,16 0,11 0,08 0,08 0,09
13-2E12.1
0,09 0,07 0,07 0,07 0,07
22-1 C11.1
0,73 0,48 0,40 0,26 0,26
27-11 A8
0,18 0,10 0,08 0,07 0,08
28-
10E7.2 0,11 0,08 0,07 0,07 0,07

A graphic illustration of the results is shown in figure 7A and 7B.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
91
References

Alexander, J.E., R.A. Lock, C.A.M. Peeter, J.T. Poolman, P.W. Andrew, T.J.
Mitchell, D.
Hansman, and J.C. Paton. 1994. Immunization of mice with pneumolysin
toxoid confers a significant degree of protection against at least nine sero-
types of Streptococcus pneumoniae. Infect.immun. 62:5683

AlonsoDeVelasco, E. Streptococcus pneumoniae: virulence factors, pathogenesis,
and
vaccines. Microbiol.Rev.1995.Dec. 59:591-603.

Anonymous. 1985. Acute respiratory infections in under fives: 15 million
deaths a year.
Lancet 2:699-701.

Barry, A.M., R.A. Lock, D. Hansman, and J.C. Paton. 1989. Contribution of
autolysin to
virulence of Streptococcus pneumoniae. Infect.lmmun. 57:2324-2330.
Benton et al. 1997. Differences in virulence for mice among Streptococcus
pneumoniae
strains of capsular types 2, 3, 4, 5 and 6 are not attributable to differences
in
Pneumolysin production. Infect. Immun. 65:1237-1244.

Bonev, B.B., Gilbert, R.J.C., Andrew, P.W. et al. 2001. Structural analysis of
the pro-
tein/lipid complexes associated with pore formation by the bacterial toxin
pneumolysin. J. Biol. Chem. 276(8):5714-5719.Camara, M., G.J. Boulnois,
P.W. Andrew, and T.J. Mitchell. 1994. A neuraminidase from Streptococ-
cus pneumoniae has the features of a surface protein. Infect.lmmun.
62:3688-3695.

Crain MJ, et al. Pneumococcal surface protein A (PspA) is serologically highly
variable and
is expressed by all clinically important capsular serotypes of Streptococcus
pneumoniae. Infect.Immun.1990.Oct. 58:3293-3299.

de los Toyos JR, et al. Functional analysis of pneumolysin by use of
monoclonal antibod-
ies. Infect.Immun.1996.Feb. 64:480-484.

Kuo, J., M. Douglas, H.K. Ree, and A.A. Lindberg. 1995. Characterization of a
recombi-
nant pneumolysin and its use as a protein carrier for pneumococcal type
18C conjugate vaccines. Infect.Immun. 63:2706-2713.

Lee CJ, et al. Immunologic epitope, gene, and immunity involved in
pneumococcal glyco-
conjugate. Crit.Rev.Microbiol.1997. 23:121-142.

Lock, R.A., J.C. Paton, and D. Hansman. 1988. Comparative efficacy of
pneumococcal
neuraminidase and pneumolysin as immunogens protective against Strepto-
coccus pneumoniae. Microb.Pathog. 5:461-467.

Lomholdt, H. 1995. Evidence of recombination and an antigenically diverse
immunoglobulin
Al protease among strains of Streptococcus pneumoniae. Infect.lmmun.
63:4238-4243.

McDaniel LS, et al. Monoclonal antibodies against protease-sensitive
pneumococcal anti-
gens can protect mice from fatal infection with Streptococcus pneumoniae.
J.Exp.Med.1984.Aug.1. 160:386-397.

McDaniel LS, et al. PspA, a surface protein of Streptococcus pneumoniae, is
capable of
eliciting protection against pneumococci of more than one capsular type. In-
fect.Immun.1991.Jan. 59:222-228.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
92
Mitchell, T.J., F. Mendez, J.C. Paton, P.W. Andrew, and G.J. Boulnois. 1990.
Compari-
son of pneumolysin genes and proteins from Streptococcus pneumoniae
type I and 2. Nuclei.Acids.Res. 18:4010

Musher, D.M., Mediwala, R., Phan, H.M. et al. 2001. Nonspecificity of assaying
for IgG
antibody to pneumolysin in circulating immune complexes as a means to di-
agnose pneumococcal pneumonia. Clin. Infect. Dis. 32:534-538.

Paton, J.C., R.A. Lock, and D. Hansman. 1983. Effect of immunization with
pneumolysin
on survival time of mice challenged with Streptococcus pneumoniae. in-
fect.immun. 40:548

Paton, J.C., R.A. Lock, C.J. Lee, J.P. Li, A.M. Barry, T.J. Mitchell, P.W.
Andrew, D.
Hansman, and G.J. Boulnois. 1991. Purification and immunogenicity of
genetically obtained pneumolysin toxoids and their conjugation to Strepto-
coccus pneumoniae type 19F polysaccharide. Infect.lmmun. 59:2297-2304.

Rapola, S., Jannti,V., Haikala,R., Carlone,G.M., Sampson,J.S., Briles,D.E.,
Paton,J.C.,
Takala,A.K., Kilpi,T.M., and Kayhty,H. 2000. Natural development of anti-
bodies to pneumococcal surface protein A, pneumococcal surface adhesin
A, and pneumolysin in relation to pneumococcal carriage and acute otitis
media. J.Infect.Dis. 182:1146-1152.

Simell, B., Korkeila,M., Pursiainen,H., Kilpi,T.M., and Kayhty,H. 2001.
Pneumococcal
carriage and otitis media induce salivary antibodies to pneumococcal sur-
face adhesin A, pneumolysin, and pneumococcal surface protein A in chil-
dren. J.Infect.Dis. 183:887-896.

Sorensen, U.B. Pneumococcal polysaccharide antigens: capsules and C-
polysaccharide.
An immunochemical study. Dan.Med.Bu11.1995.Feb. 42:47-53.

Swiatlo, E., M.J. Crain, L.S. McDaniel, A. Brooks-Walter, T.J. Coffey, B.G.
Spratt, D.A.
Morrison, and D.E. Briles. 1996. DNA polymorphisms and variants penicil-
lin-binding proteins as evidence that relatively penicillin-resistant pneumo-
cocci in Western Canada are clonally related. J.Infect.Dis. 174:884-888.

Tai, S.S., T.R. Wang, and C.J. Lee. 1997. Characterization of hemin binding
activity of
Streptococcus pneumoniae. Infect.lmmun. 65:1083

Talkington DF, et al. A 43-kilodalton pneumococcal surface protein, PspA:
isolation, protec-
tive abilities, and structural analysis of the amino-terminal sequence. In-
fect.Immun.1991.Apr. 59:1285-1289.

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
SEQUENCE LISTING

<110> Genesto A/S

<120> Binding member towards Pneumolysin
<130> P885PC00

<160> 26

<170> Patentln version 3.1
<210> 1
<211> 12
<212> PRT
<213> hom*o sapiens
<400> 1

Ile Arg Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp
1 5 10
<210> 2
<211> 16
<212> PRT
<213> Nomo sapiens
<400> 2
Val Lys Ile Arg Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr
1 5 10 15
<210> 3
<211> 109
<212> PRT
<213> artificial sequence
<220>
<223> variable light chain 26-5F12.1
Page 1

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
<400> 3
Asp ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gin Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe ser
50 55 60
Gly ser Gly ser Gly Thr Asp Phe Thr Leu Thr ile ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95
Phe Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 4
<211> 117
<212> PRT
<213> artificial sequence
<220>
<223> variable light chain 26-5F12.1
<400> 4
val Lys Leu Gln Glu Ser Gly Ala Glu val Lys Lys Pro Gly Ala ser
1 5 10 15
Val Lys val ser Cys Thr Ala Ser Gly Tyr ile Phe Thr Ser Tyr Ala
20 25 30
Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly
35 40 45

Trp ile Asn Ala Gly Tyr Gly Asn Thr Lys Tyr ser Gln Lys Phe Gln
50 55 60
Gly Arg val ser Ile Thr Arg Asp Lys ser Ala ser Thr Ala Tyr Met
65 70 75 80
Glu Leu ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Page 2

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
Arg Arg Gly Gln Gln Leu Ala Phe Asp Tyr Trp G1y.Gln Gly Thr Thr
100 105 110
Val Thr val Ser Ser
115
<210> 5
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> CDR1 light chain 26-5F12.1
<400> 5
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala
1 5 10
<210> 6
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> CDR2 light chain 26-5F12.1
<400> 6

Gly Ala ser ser Arg Ala Thr
1 5
<210> 7
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> CDR3 light chain 26-5F12.1
<400> 7

Gin Gln Tyr Gly Ser Ser
1 5

Page 3

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
<210> 8
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> CDR 1 heavy chain 26-5F12.1
<400> 8

5er Tyr Ala Ile His
1 5
<210> 9
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> CDR2 heavy chain 26-5F12.1
<400> 9
Trp ile Asn Ala Gly Tyr Gly Asn Thr Lys Tyr Ser Gln Lys
1 5 10
<210> 10
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> CDR3 heavy chain 26-5F12.1
<400> 10
Arg Gly Gln Gln Leu Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val
1 5 10 15
Thr

<210> 11
<211> 471

Page 4

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
<212> PRT
<213> hom*o sapiens
<400> 11

Met Ala Asn Lys Ala Val Asn Asp Phe Ile Leu Ala Met Asn Tyr Asp
1 5 10 15
Lys Lys Lys Leu Leu Thr His Gln Gly Glu ser Ile Glu Asn Arg Phe
20 25 30
ile Lys Glu Gly Asn Gln Leu Pro Asp Glu Phe val Val Ile Glu Arg
35 40 45

Lys Lys Arg Ser Leu Ser Thr Asn Thr Ser Asp ile Ser Val Thr Ala
50 55 60
Thr Asn Asp Ser Arg Leu Tyr Pro Gly Ala Leu Leu val Val Asp Glu
65 70 75 80
Thr Leu Leu Glu Asn Asn Pro Thr LeU Leu Ala Val Asp Arg Ala Pro
85 90 95
Met Thr Tyr Ser Ile Asp Leu Pro Gly Leu Ala Ser Ser Asp Ser Phe
100 105 110

Leu Gln Val Glu Asp Pro Ser Asn Ser Ser Val Arg Gly Ala Val Asn
115 120 125
Asp Leu Leu Ala Lys Trp His Gln Asp Tyr Gly Gln Val Asn Asn Val
130 135 140
Pro Ala Arg Met Gln Tyr Glu Lys Ile Thr Ala His Ser Met Glu Gln
145 150 155 160
Leu Lys Val Lys Phe Gly ser Asp Phe Glu Lys Thr Gly Asn ser Leu
165 170 175

Asp ile Asp Phe Asn Ser val His ser Gly Glu Lys Gln Ile Gln Ile
180 185 190
val Asn Phe Lys Gln Ile Tyr Tyr Thr Val Ser val Asp Ala val Lys
195 200 205
Asn Pro Gly Asp val Phe Gln Asp Thr val Thr val Glu Asp Leu Lys
210 215 220

Gln Arg Gly Ile ser Ala Glu Arg Pro Leu Val Tyr ile ser ser val
225 230 235 240
Ala Tyr Gly Arg Gln Val Tyr Leu Lys Leu Glu Thr Thr Ser Lys ser
Page 5

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
245 250 255
Asp Glu Val Glu Ala Ala Phe Glu Ala Leu ile Lys Gly Val Lys val
260 265 270
Ala Pro Gln Thr Glu Trp Lys Gln zle LeU Asp Asn Thr Glu Val Lys
275 280 285

Ala val Ile LeU Gly Gly Asp Pro Ser Ser Gly Ala Arg Val Val Thr
290 295 300
Gly Lys Val Asp Met val Glu Asp Leu ile Gln Glu Gly Ser Arg Phe
305 310 315 320
Thr Ala Asp His Pro Gly Leu Pro Ile Ser Tyr Thr Thr Ser Phe Leu
325 330 335
Arg Asp Asn val val Ala Thr Phe Gin Asn ser Thr Asp Tyr Val Glu
340 345 350

Thr Lys val Thr Ala Tyr Arg Asn Gly Asp Leu Leu Leu Asp His Ser
355 360 365
Gly Ala Tyr Val Ala Gln Tyr Tyr ile Thr Trp Asp Glu Leu Ser Tyr
370 375 380
Asp His Gln Gly Lys Glu Val Leu Thr Pro Lys Ala Trp Asp Arg Asn
385 390 395 400
Gly Gln Asp Leu Thr Ala His Phe Thr Thr ser Ile Pro Leu Lys Gly
405 410 415

Asn val Arg Asn Leu Ser val Lys Ile Arg Glu cys Thr Gly LeU Ala
420 425 430
Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp Leu Pro Leu Val
435 440 445
Arg Lys Arg Thr Ile Ser Ile Trp Gly Thr Thr Leu Tyr Pro Gln Val
450 455 460
Glu Asp Lys Val Glu Asn Asp
465 470
<210> 12
<211> 110
<212> PRT
<213> artificial sequence

Page 6

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
<220>
<223> variable light chain 26-23C2.2
<400> 12

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile ser Tyr Arg Ala Ser Lys ser Val ser Thr ser
20 25 30
Gly Tyr ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45

Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe ser Gly ser Gly Ser Gly Thr Asp Phe Thr Leu Asn 71e His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg
85 90 95
Glu Leu Thr Arg Ser Glu Gly Gly Pro Arg Trp Lys Ser Lys
100 105 110
<210> 13
<211> 117
<212> PRT
<213> artificial sequence
<220>
<223> variable heavy chain 26-23C2.2
<400> 13
Val Lys Leu Gln Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala ser
1 5 10 15
Val Lys Val Ser Cys Thr Ala ser Gly Tyr Ile Phe Thr Ser Tyr Ala
20 25 30
Met His Trp Val Arg Gln Ala Pro Gly Gln Arg LeU Glu Trp Met Gly
35 40 45

Trp Ile Asn Ala Gly Tyr Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln
50 55 60
Gly Arg Val Ser Ile Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr Met
65 70 75 80

Page 7

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
G1u Leu Thr Ser Leu Arg Ser Glu Asp Thr Ala val Tyr Tyr CyS Ala
85 90 95
Arg Arg Gly Gln Gln Leu Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val ser ser
115
<210> 14
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> CDR1 light chain 26-23c2.2
<400> 14

Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser
1 5 10
<210> 15
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> CDR2 light chain 26-23C2.2
<400> 15

Leu Val Ser Asn Leu Glu Ser
1 5
<210> 16
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> CDR3 light chain 26-23C2.2
<400> 16

Page 8

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
Gln His Ile Arg G1u Leu
1 5
<210> 17
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> CDR1 heavy chain 26-23c2.2
<400> 17

Ser Tyr Ala Met His
1 5
<210> 18
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> CDR 2 heavy chain 26-23C2.2
<400> 18

Trp Ile Asn Ala Gly Tyr Gly Asn Thr Lys Tyr ser Gln Lys
1 5 10
<210> 19
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> variable light chain 22-lcll
<400> 19
Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
Page 9

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln cys Ser Asn Trp His Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys LeU Glu Ile Lys Arg
100 105
<210> 20
<211> 125
<212> PRT
<213> artificial sequence
<220>
<223> variable heavy chain 22-1C11
<400> 20

Val Lys Leu Gln Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser
1 5 10 15
Leu Arg Leu Ser cys Ala Ala Ser Gly Phe Thr Phe ser Asn Tyr Gly
20 25 30
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
35 40 45

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Phe Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn ser Leu Arg Ala Glu ASP Thr Ala Val Tyr Tyr cys Ala
85 90 95
Arg Arg Gly Asn Tyr Tyr Gly Leu Gly Ser Phe Tyr Tyr Tyr Gly Met
100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 21

Page 10

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> CDR1 light chain 22-1C11
<400> 21

Arg Ala Ser Gin Ser Val Ser Ser Tyr Leu Ala
1 5 10
<210> 22
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> CDR2 light chain 22-1C11
<400> 22

Asp Ala ser Asn Arg Ala Thr
1 5
<210> 23
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> CDR3 light chain 22-1C11
<400> 23

Gln Gln cys Ser Asn Trp
1 5
<210> 24
<211> 6
<212> PRT
<213> artificial sequence
<220>
Page 11

CA 02578361 2007-02-22
WO 2006/021210 PCT/DK2005/000536
<223> CDR1 heavy chain 22-1C11
<400> 24

Ser Asn Tyr Gly Met His
1 5
<210> 25
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> CDR2 heavy chain 22-1C11
<400> 25

Val Ile Trp Tyr Asp Gly Ser Asn LYS Tyr Tyr Ala Asp Phe
1 5 10
<210> 26
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> cDR3 heavy chain 22-1C11
<400> 26
Ser Phe Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val
1 5 10 15
Thr

Page 12