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Search Report and Written Opinion as issued by Intellectual Property Communication as issued by European Patent Office for European
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consideration by the Appeal Board, and a signed Declaration by

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in European Patent Application No. 01945896.7. Jan. 15, 2010. * cited by examiner

U.S. Patent Sep. 27, 2011 Sheet 1 of 16 US 8,025,878 B2

Figure 1

IgG immunoglobulin

4-CDR1

-- CDR2

Finge region

IgG4/CHy2 does NOT

bind complement

factor C1q.

IgG1/CHy3 bind high

affinity Fc receptors on

microglial cells

U.S. Patent Sep. 27, 2011 Sheet 2 of 16 US 8,025,878 B2

Figure 2: Characterization of a high affinity protofibril selective monoclonal antibody.

s-Os Profibri

-O-Fb.

2
as Moorer
wr Abeta 1-18

rr Abeta 17-40

00001 OO O odoo

Conc. of peptide nM

O,O1 O,1 O 100 OOO 100 E

Conc. of AB peptide nM

U.S. Patent Sep. 27, 2011 Sheet 3 of 16 US 8,025,878 B2

Figure 3. Therapeutic efficacy of a high affinity protofibril selective antibody in

transgenic mouse model (APPswe)

Figure 4
Human AS protofibrils are measured at pM levels by proximity ligation technique,

30

32

-- roome

s
34
-O-protofibril

36

38

40
O 100 100 10000 OOOOO

AB piv

U.S. Patent Sep. 27, 2011 Sheet 4 of 16 US 8,025,878 B2

Figure 5

A mAb158 B Positive controls

2 4-8 1632 64 1259 8 16 32 64 125 ing

digi

Medin fibril pAb179

APP fibril pAA110 APPri

ot-synuclein fibril Ab211 o-synuclein fibril

Mouse brain HEK-ce Wouse brain HEK-cell

C homogenate culture media homogenate culture media Positive control

s g
c e
2

x. 5. 9 & sa f.: g5 ?5
. . . f.

5 9
S < a. 2O O
S Dil o
gg

~ 100 kDa 3: : i.

i.p. with mAb158 i.p. with 6E10 ..W p with

A 1586E

Western blot with 6E10

U.S. Patent Sep. 27, 2011 Sheet 5 of 16 US 8,025,878 B2

Figure 6

A. B C

20 10 0.8

E 1.5
3. 1 --Asprotothri
E O6

Q
1.0 ro,998 r
8
--AArc protofibri)
-o-LMWAf O
0.4 -armAb158sandwich ESA
-A-6E10 sandwich ELSA
0.5 o AB protofibril --AB-18 2

o.O.
ApArc protofibrit 0.1
0.0

O 100
Asplm
2do 3o -12. -11 -10 -9 -8
og ABM
7 -8 -5 -4 to to to its
A 1-16. Approtofibris
city

U.S. Patent Sep. 27, 2011 Sheet 6 of 16 US 8,025,878 B2

Figure 7

A HEK-Cell Culture media

40

3O

2O

1O

Mock APPswe APFArc-swe

B Mouse brain homogenates

600

5OO

400

300

200

1OO

Non-tg APPswe APPArc-swe

U.S. Patent Sep. 27, 2011 Sheet 7 of 16 US 8,025,878 B2

Fig. 8. AB protofibril levels in APPswear transgenic

mouse brain TBS extracts after 4 months treatment

with either mAb158 or placebo.

Placebo mAb158

Fig. 9. Total AB levels in APPswear transgenic

mouse brain formic acid extracts after 4

months

15000

10000

5000

Placebo mAb158

U.S. Patent Sep. 27, 2011 Sheet 8 of 16 US 8,025,878 B2

Fig. 10. pKN100 vector

U.S. Patent Sep. 27, 2011 Sheet 9 of 16 US 8,025,878 B2

Fig.11. pG1D200 vector

PGD2OOSEQ

7268 Bp

U.S. Patent Sep. 27, 2011 Sheet 10 of 16 US 8,025,878 B2

Fig. 12. Ab monomer binding by chimeric and mouse 158 antibodies

EC

g -- mouse 158 //

10

IgG ng/ml

Legend: Direct ELISA coated with Af 1-40 (217 ng?well) and using serial dilutions of mAb.

Amount of FCS at highest concentration (1 g/ml) is 2.7%. Detection with anti-mouse k or anti

humank light chain conjugates,

U.S. Patent Sep. 27, 2011 Sheet 11 of 16 US 8,025,878 B2

Fig. 13. Competition of monomeric or protofibrillar A8 for binding to chimeric 158 or mouse

158 antibody

E
O
l
w
O
-- Abeta1-40/chimeric158
-O-PF/chimeric158
- A - Abeta1-40/Thouse 158
r-A-PF/mouse158

10 1000
Abeta (nW)
Legend: Monomeric AB 1-40 (OA) or protofibrils (PF) (OA) were incubated in solution with
chimeric 158 (OO) or mouse 158 (AA). The final concentration of FCS was 0.3%. After an
incubation for 1 h, the mixture was added to a plate coated with Amonomers. The binding of
antibody to the plate was detected by anti-mouse k or anti-human klight chain conjugates.
U.S. Patent Sep. 27, 2011 Sheet 12 of 16 US 8,025,878 B2

Fig. 14. Competition of monomeric or protofibrillar AB for binding to chimeric 158, mouse 158
antibody and humanized 158 antibody (BAN2401),

egend: Monomeric AB 1-40, or protofib

antibody (chim), mouse 158 antibody (mAb158) or humanized 158 antibody BAN2401 (Hum).
The final concentration of FCS was 0.3%. After an incubation for h, the mixture was added to
a plate coated with Amonomers, The binding of antibody to the plate was detected by anti
mouse k or anti-human klight chain conjugates.
U.S. Patent Sep. 27, 2011 Sheet 13 of 16 US 8,025,878 B2

Signal P-NN prediction ceuk networks) : Sequence

i
2. army-T"

Position

Figure 15
U.S. Patent Sep. 27, 2011 Sheet 14 of 16 US 8,025,878 B2

Signal P-NN prediction ceuk networks) ; Secuence

2. i v + 1,

3 5 12 15 22 25 32
Position

Figure 16
U.S. Patent Sep. 27, 2011 Sheet 15 of 16 US 8,025,878 B2

Signal P-NN prediction ceuk networks) Secuence

Position

Figure 17
U.S. Patent Sep. 27, 2011 Sheet 16 of 16 US 8,025,878 B2

Signal P-NN prediction ( euk networks) : Secuence

C score
1. S. arg -------.
score

Position

Figure 18
 US 8,025,878 B2
1.
PROTOFIBRIL SELECTIVE ANTIBODES
AND THE USE THEREOF
CROSS-REFERENCE TO RELATED
APPLICATIONS
This application is the U.S. National Stage of International
Application No. PCT/SE2007/000292, filed Mar. 23, 2007,
which claims the benefit of SE 0600662-1, filed Mar. 23,
2006, and SE 0602591-0, filed Nov.30, 2006.
BACKGROUND
Field of Invention
This invention pertains to the prevention, treatment and
diagnosis of neurodegenerative diseases, in particular Alzhe
imer's disease, and other similar disease. More precisely, to
high affinity 107M, preferably 10M, even less than 10M
or less than 10' M or 10' M antibodies, selective for
amyloid beta protein (AB) in its protofibril conformation and
of IgG class and IgG1 or IgG4 Subclass or combinations
thereof or mutations thereof, retaining high Fc receptor bind
ing and low C1 (C1q) binding, effective in clearance of AB
protofibrils and with reduce risk of inflammation.
Alzheimer's disease (AD) is a progressive and irreversible
neurodegenerative disorder causing cognitive, memory and
behavioural impairments. It is the most common cause of
dementia in the elderly population affecting roughly 5% of
the population above 65 years and 20% above 80years of age.
AD is characterized by an insidious onset and progressive
deterioration in multiple cognitive functions. The neuropa
thology involves both extracellular and intracellular argyro
phillic proteineous deposits. The extracellular deposits,
referred to as neuritic plaques, mainly consist of amyloid beta
protein (AB) surrounded by dystrophic neurites (swollen,
distorted neuronal processes). A? within these extracellular
deposits are fibrillar in its character with a B-pleated sheet
structure. AB in these deposits can be stained with certain
dyes, e.g. Congo Red, and display a fibrillar ultra structure.
These characteristics, adopted by AB in its fibrillar structure
in neuritic plaques, are the definition of the generic term
amyloid. The classic intracellular AD pathologic lesion is the
neurofibrillary tangle (NFT) which consists of filamentous
structures called paired helical filaments (PHFs), composed
of twisted strands of hyperphosphorylated microtubule-asso
ciated protein tau. Frequent neuritic plaques and neurofibril
lary tangle deposits in the brain are diagnostic criteria for AD,
as carried out post mortem. AD brains also display macro
scopic brain atrophy, nerve cell loss, local inflammation (mi
crogliosis and astrocytosis) and often cerebral amyloid angi
opathy (CAA) in cerebral vessel walls.
Two forms of AB peptides, AB40 and AB42, are the domi
nant species in AD neuritic plaques while AB40 is the promi
nent species in cerebrovascular amyloid associated with AD.
Enzymatic activities allow AB to be continuously formed
from a larger protein called the amyloid precursor protein
(APP) in both healthy and AD afflicted subjects in all cells of
the body. Two major APP processing events through B- and
Y-secretase activities enables AB production, while a third
enzyme called C-Secretase, prevents AB generation by cleav
age inside the AB sequence (Selkoe, 1994: Ester 2001; U.S.
Pat. No. 5,604,102). The AB42 is a forty two amino acid long
peptide, i.e. two amino acids longer at the C-terminus, as
compared to AB40. AB42 is more hydrophobic, and does
more easily aggregate into larger structures of A peptides
(Jarret 1993) such as AB dimers, AB trimers, AB tetramers, AB
2
oligomers, AB protofibrils or AB fibrils. A? fibrils are hydro
phobic and insoluble, while the other structures are all less
hydrophobic and soluble. All these higher molecular struc
tures of AB peptides are individually defined based on their
biophysical and structural appearance e.g. in electron micros
copy, and their biochemical characteristics e.g. by analysis
with size-exclusion chromatography/western blot. These AB
peptides, particularly AB42, will gradually assemble into a
various higher molecular structures of AB during the life
10 span. AD, which is a strongly age-dependent disorder, will
occur earlier in life if this assembly process occurs more
rapidly. This is the core of the "amyloid cascade hypothesis”
of AD which claims that APP processing, the A342 levels and
their assembly into higher molecular structures is a central
15 cause of AD. All other neuropathology of AD brain and the
symptoms of AD Such as dementia are somehow caused by
A? or assembled forms thereof.
A? can exist in different lengths i.e. 1-39, 1-40. 1-42 and
1-43 and fragments sizes i.e. 1-28 and 25-35. Truncations
might occur at the N-terminus of the peptide. All these pep
tides can aggregate and form soluble intermediates and
insoluble fibrils, each molecular form having a unique struc
tural conformation and biophysical property. Monomeric
AB1-42 for example, is a 42 amino acid long Soluble and non
25 toxic peptide, that is Suggested to be involved in normal
synapse functions. Under certain conditions, the AB1-42 can
aggregate into dimers, trimers, tetramers, pentamers up to
12-mer and higher oligomeric forms, all with its distinct
physicochemical property Such as molecular size, EM struc
30 ture and AFM (atomic force microscopy) molecular shape.
An example of a higher molecular weight soluble oligomeric
AB form is the protofibril (Walsh 1997), which has an appar
ent molecular weight >100 kDa and a curvelinear structure of
4-11 nm in diameter and <200 nm in length. It has recently
35 been demonstrated that soluble oligomeric A? peptides Such
as AB protofibrils impair long-term potentiation (LTP) a mea
Sure of synaptic plasticity that is thought to reflect memory
formation in the hippocampus (Walsh 2002). Furthermore,
oligomeric Arctic AB peptides display much more profound
40 inhibitory effect than witAB on LTP in the brain, likely due to
their strong propensity to form AB protofibrils (Klyubin
2003).
There are also other soluble oligomeric forms described in
the literature that are distinctly different from protofibrils.
45 One such oligomeric form is ADDL (Amyloid Derived Dif
fusible Ligand) (Lambert 1998). AFM analysis of ADDL
revealed predominantly small globular species of 4.7-6.2 mm
along the Z-axis with molecular weights of 17-42 kDa (Stine
1996). Another form is called ASPD (Amyloidspheroids)
50 (Hoshi 2003). ASPD are spherical oligomers of AB1-40. Tox
icity studies showed that spherical ASPD-10 nm were more
toxic than lower molecular forms (Hoshi 2003). This idea has
gained support from recent discovery of the Arctic (E693)
APP mutation, which causes early-onset AD (US 2002/
55 0162129 A1; Nilsberth et al., 2001). The mutation is located
inside the AB peptide sequence. Mutation carriers will
thereby generate variants of AB peptides e.g. Arctic A?40 and
Arctic AB42. Both Arctic A?40 and Arctic A?42 will much
more easily assemble into higher molecular structures i.e.
60 protofibrils. Thus, the pathogenic mechanism of the Arctic
mutation Suggests that the Soluble higher molecular
protofibrils are causing AD and contains a specific unique
epitope i.e. “the AD disease epitope'.
In the Alzheimer's disease (AD) brain, extracellular amy
65 loid plaques are typically found in parenchyma and vessel
walls. The plaques are composed of amyloid (A338-43 amino
acid long hydrophobic and self-aggregating peptides, which
 US 8,025,878 B2
3 4
gradually polymerize prior to plaque deposition. The soluble
A? oligomeric species have been proposed to be better dis
ease correlates than the amyloid plaques themselves
(McLean et al., 1999; Näslund et al., 2000). Among these
pre-fibrillar intermediate A3 species, oligomeric forms have
been shown to elicit adverse biological effects both in vitro
and in vivo (Walsh et al., 2002) and may thus play a central
role in disease pathogenesis. Several oligomeric AB species
of various molecular sizes are known. Importantly, the con
formation of monomeric, oligomeric and fibrillar forms of AB
are different and can be targeted by conformational selective
antibodies. The identity of the main AB pathogen is unclear,
although some evidence Suggests high-molecular weight AB
oligomers to be especially neurotoxic (Hoshi et al., 2003).
Pathogenic mutations in the amyloid precursor protein
(APP) gene, causing early onset AD have been described.
One of them, the Swedish APP mutation (Mullanet al., 1992),
causes increased levels of AB. The other the Arctic APP
mutation (E693G) located within the AB domain, was found
to enhance the formation of protofibrils, large AB oligomers,
Suggesting these AB intermediates to be particularly patho
genic (US 2002/0162129 A1: Nilsberth et al., 2001). The
identification of the Arctic APP mutation and the elucidation
of toxic effects for AB protofibrils have increased the focus on
AB oligomers in AD pathogenesis.
Active immunization as a therapeutic strategy for Alzhe
imer's disease was first reported by (Schenket al. 1999). The
target for the immunization strategy was the fibrillar form of
AB found in Alzheimer plaques. A recent clinical phase I/II
trial of active A3 vaccination using fibrillized AB as a vaccine
(AN-1792) had to be halted because of the development of
meningoencephalitis in a small number of patients (Bayer et
al., 2005). The side effects seen in this study were likely
caused by anti-Afantibodies reacting against fibrillar amy
loid in vessel walls. The fibrillary amyloid in CAA is in close
proximity to the blood-brain-barrier (BBB) and the antigen
antibody reaction could thus generate damage to the BBB
leading to infiltration of T-lymphocytes into the CNS (Pfeifer
et al., 2002; Racke et al., 2005). Moreover, only a minority of
the participating patients displayed an immune response to
the AB vaccine. Although the study ended prematurely, it
seems to imply that active A? immunization may be benefi
cial only to a subset of AD patients.
Monoclonal antibodies selective for human AB protofibrils
have been described (US 2002/0162129 A1). The method to
generate highly pure and stable human AB protofibrils,
involves the use synthetic AB42 peptides with the Arctic
mutation (Glu22Gly). The mutation facilities immunization
and hybridoma screening for AB protofibril selective antibod
ies. Importantly, these antibodies bind both wild-type AB
protofibrils and AB-Arc protofibrils (PCT/SE 2005/000993).
Antibodies that are selective towards other conformations
of AB such as AB fibrils (O'Nuallain 2002), micellar AB
(Kayed 2003), ADDL (Lambert 2001), have been described.
However, non of these are AB protofibril selective.
SUMMARY OF THE INVENTION
The present invention pertains to improved antibodies i.e.
high affinity (less than 107M) A? protofibril selective anti
bodies of class IgG and Subclass IgG1 or IgG4 or combination
thereof or mutations thereof, with reduced risk of inflamma
tion, for improved prevention, treatment and diagnosis of
Alzheimer's disease, Downs syndrome or other neurodegen
erative disorders. Said antibodies have been developed by
classical hybridoma techniques and antibody engineering.
The invention discloses the consensus amino acid
sequence of the CDR1-3 regions on the VL and VH chains
from antibodies that selectively bind oligomeric A? forms,
i.e. A? protofibrils constituting the Alzheimer disease
epitope', combined with modifications of the Fc region to
reduce complement factor C1q binding, reducing the risk for
complement activation and inflammation.
The constant region of an antibody has many important
functions notably binding Fc-receptors and complement fac
10 tor C1q. The latter function has been inactivated to avoid
inflammatory reactions.
In summary, this type of high affinity protofibril selective
antibodies have the following distinct advantages as com
pared to other known immunotherapeutic treatment modali
15 ties:
1) targets disease causing AB protofibrils with high affinity
2) reduces the risk for inflammatory side-effects i.e. menin
gioencephalitis, by low or no binding to complement factor
C1q.
3) high affinity antibody reduces the clinical dose needed for
an effective treatment
4) provides a modality of accurate dosing
5) less binding to AB fibrils in the blood vessel wall i.e. CAA,
reducing the risk for inflammatory side-effects.
25 6) Less antibody is bound in the periphery, thus more will
cross the blood brain barrier and be available for binding
and elimination of AB oligomeric forms in the brain.
One aspect of the invention is the discovery of the antibody
consensus amino acid sequence of the CDR regions that bind
30 human wild type AB protofibrils (Example 1). This discovery
defines the binding sites (CDR regions) that confer high affin
ity and high selectivity for wild-type human AB protofibrils
for use as therapeutics or diagnostics. The basic structure of
an immunoglobulin (IgG) molecule comprises two identical
35 light chains and two identical heavy chains linked together by
disulphide bridges (FIG. 1). The light chain, which is either
lambda or kappa, has a variable region (VL) and a constant
region (CL) of approximately 110 amino acid residues each.
The heavy chain has a variable region (VH) of about 110
40 amino acid residues, but a much larger constant region (CH)
of 300-400 amino acid residues, comprising CHy1, CHy2 and
CHY3 regions or domains.
The constant region (Fc) activates the complement system
and binds to a Fc receptor on macrophages, microglia and
45 neutrophiles, which ingest and destroys infecting microor
ganisms or foreign/non-self antigens. This function is par
ticular important since it is part of the therapeutic principle of
the antibody, i.e. Fc receptor mediated microglial phagocy
tosis and clearance of AB protofibrils. Other antibody medi
50 ated clearance mechanisms are also operating, i.e. anti-aggre
gation properties of AB antibodies and clearance of AB
protofibrils in the periphery, according to the sinkhypothesis.
The variable region of the heavy and light chains contains 3
hyper variable regions called complementary determining
55 regions or CDRs. The CDR regions are short stretches of
about 13-23 amino acid long, located in the VL and VH
regions. The six CDRs regions on one "arm' of the antibody
forms the “pocket' that binds the antigen. FIG. 1 shows the
basic structure of an IgG immunoglobulin and its subdo
60 mains.
Another aspect of the invention pertains to protofibril
selective antibodies of high affinity. Affinities in the range of
107M preferably 10 M, even less than 10 M, less than
10' M, or less than 10' M for protofibrils are described
65 (Example 2). These antibodies have the advantage that they
can be administered at lower doses compared to antibodies
with affinities in the 10M range. This has significant clini
 US 8,025,878 B2
5 6
cal advantage in that these high affinity antibodies, which are
administered by injection, can be given Subcutaneously since
only a low amount of the antibody is needed to achieve
efficacy. Administration modalities are not limited to Subcu
taneous injections. Furthermore, the lower doses needed for
efficacy will reduce cost of goods for production of the anti
body.
Another aspect of the invention is that the antibodies are of
IgG class, Suitable for therapeutic use since it can pass over
the blood brain barrier. Clearance of AB protofibrils in the
brain parenchyma is achieved by Fc receptor mediated
phagocytosis by microglia cells. Other anti-A clearance
mechanisms are likely to operate as well. This clearance of
soluble AB protofibrils is a central mechanism of the treat
ment. A? protofibrils are considered highly neurotoxic, initi
ating and driving the disease process. Clearance of AB
protofibrils in the brain is of significant clinical value. In
addition to clearance of AB protofibrils, other AB oligomeric
forms including AB fibrils, will be reduced indirectly via
removal of AB protofibrils since different AB aggregated
forms, i.e. dimers, trimers, tetramers and higher oligomeric
forms including protofibrils and fibrils, are in equilibrium.
Example of reduction of plaques, which contain AB fibrils, is
shown in a Alzheimer transgenic mouse model (APPSwe)
after 72 hour treatment with a high affinity protofibril selec
tive antibody (mAb 158) (Example 3). Hence, clearance of
A? protofibrils by said antibody will also have the advantage
to indirectly reduce other AB aggregated or oligomeric forms.
Yet another aspect of the invention is a high affinity human
A? protofibril selective antibody of subclass IgG1, which has
a high affinity for human FcyRI receptors present on micro
glial cells in the brain. A high affinity antibody will lead to
efficient clearance of AB protofibrils which will be of signifi
cant therapeutic value. Hence, the antibodies will exhibit
clearance of AB protofibrils, both in CNS and periphery as
compared to other immunotherapeutic strategies such as
active vaccination or monoclonal antibody treatments with
other monoclonal antibodies of IgG1 Subclass targeting other
A? forms. Importantly, the treatment will be efficient early in
the disease process when toxic soluble AB spices such as AB
protofibrils are present at elevated levels but also later in the
disease process. Elevated levels of oligomeric AB forms have
been described in a transgenic mouse model exhibiting the
Swedish and Arctic mutations APP swearc (Lord A. et al.
2006).
Yet another aspect of the invention is that the high affinity
A? protofibril selective antibodies can reduce or inhibit AB
aggregation thereby reducing levels of soluble oligomeric AP
forms in the brain.
Yet, another aspect of the invention is that the high affinity
A? protofibril selective antibodies can bind oligomeric forms
of AB, i.e. A? protofibrils outside CNS as well, thereby shift
ing the equilibrium of said AB forms over the blood brain
barrier in such a way as to lower CNS levels of said AB forms
(drainage).
As discussed above, the Elan clinical study using an AB
vaccine (AN-1792) selective for AB fibrils to treat Alzheimer
patients resulted in a side-effect, i.e. meningioencephalitis, in
6% of the cases. The strategy to target AB fibrils, that are the
core of amyloid plaques present in the brain parenchyma but
importantly also in the blood vessel walls, resulted in severe
side-effects. The side-effects was most likely caused by the
binding of the antibodies to CAA (Cerebral Amyloid Angi
opathy) in the blood vessel walls of the brain, starting an
inflammatory process. This significant clinical problem is
avoided by the improved high affinity protofibril selective
antibodies with reduced complement activation activity.
These antibodies will retain high clearance efficacy of AB
protofibrils reduced risk of side-effects, i.e. meningioen
cephalitis.
Another aspect of the invention is that the high affinity
protofibril selective antibodies have low AB fibril binding
(See example 2), reducing the risk for side effects, by less
binding to AB fibrils present in CAA.
Yet another aspect of the invention is that the high affinity
A? protofibril selective IgG antibodies are engineered to
10 reduce complement factor C1q binding to the CH2 domain of
IgG1 and reduce complement activation and risk of inflam
mation. This modification can be done in several different
ways. One way is to make a chimeric antibody where the
CHY2 domain of the IgG1 constant region has been deleted
15 and exchanged for the corresponding domain from IgG4 or
part of the domain that confers C1q binding. It is well estab
lished that IgG4 does not bind C1q and hence does not acti
vate the complement cascade. To achieve this the constant
region of the heavy chain (CH) is engineered is such a way as
to combine the high affinity Fc-receptor domain (CHY3) on
IgG1 with the IgG4 domain (CHY2) which has no binding for
the complement factor C1q. This new antibody containing the
chimeric constant heavy chain (IgG1 :CHy1, CHY2:IgG4,
CHY3:IgG1) will have the important properties of both effi
25 cient clearance of A? protofibrils through Fc-receptor medi
ated phagocytosis and reduced risk for side-effects, i.e
inflammation Such as meningioencephalitis.
Yet another way of reducing the risk of inflammation is to
alter the oligosaccharides structure of the antibody which will
30 reduce complement factor C1q binding and complement acti
vation. 30 different structures of the complex biantennary
oligosaccharides at Asn-297 in human IgG1 has been
described. The absence of CH2 associated carbohydrates is
believed to cause a conformational change in the “hinge’
35 region of the antibody, reducing interaction efficacies with
effector molecules and loss of complement activation func
tion and C1q binding.
The modification of a high affinity human AB protofibril
selective antibody by site-directed mutagenesis of Asn-297 to
40 any other amino acid will generate an antibody of retained
Fc-receptor binding with less C1q binding and hence reduced
risk of inflammation in particular at the blood brain barrier.
An alternative to modify the glycosylation on the antibody is
to expressing the antibody in a cell type where the enzyme
45 N-acteylglucosaminyl-transferase I has been inactivated.
This will yield an antibody with altered carbohydrate struc
ture at Asn-297. A structure of Man-GlcNAc, but not limited
to this structure, is formed. This carbohydrate modification
will reduce complement factor C1q binding and inhibit
50 inflammation (Wright at al. 1998). Alternatively, glycosy
lated protofibril selective antibodies can be achieved by cul
turing cells expressing antibodies in the presence of tunica
mycin, which inhibits glycosylation. These antibodies will
have altered complement activating activity as well as altered
55 Fc-receptor function (Leatherbarrow el al. 1985). Screening
of clones expressing antibodies with low complement activa
tion and high Fc-receptor binding will generate protofibril
selective antibodies that exhibit high Fc-mediated clearance
of AB protofibrils and low C1q binding.
60 Yet another aspect of the invention is a high affinity human
A? protofibril selective antibody, of IgG1 subclass, where the
complement factor C1q binding site has been modified, i.e.
Pro331>Ser331 (Xuetal. 1994), in such away as to reduce or
inhibit binding of complement factor C1q, for the treatment
65 or prevention of AD. The proline residue at position 331 in
human IgG1 can also be changed to a threonine or glycine or
any other polaramino acid. This modification can be achieved
 US 8,025,878 B2
7 8
by standard molecular biology techniques such as site-di
rected mutagenesis or DNA deletions.
Yet another aspect of the invention is the use of high affinity
human AB protofibril selective IgG antibodies to specifically
determine protofibril levels in human tissues, in particular in
cerebrospinal fluid, blood, urine or saliva as a diagnostic tool
or biomarker for Alzheimer's disease. Levels of human AB
protofibrils in CSF or blood are likely to be different as
compared to a matched elderly control group not having
Alzheimer's disease. A person who is developing Alzhe
imer's disease is likely to have increased levels of AB
protofibril levels in CSF or blood. Hence, by determination of
A? protofibril levels in CSF or blood an early diagnosis of the
disease can be made. This is possible to achieve with the new
high affinity A?8 protofibril selective antibodies in combina
tion with a sandwich ELISA method (Example 2A), where
A? protofibrils have been determined down to 10 pM level.
Interference of other AB forms such as AB fibrils, AB mono
mers and AB fragments (1-16; 17-40) in the assay, is 10% or
less.
The invention further pertains to the use of a high affinity
protofibril specific antibodies for determinations of AB
protofibrils in human and animal tissues, for example, cere
brospinal fluid, blood, serum, urine and brain tissue but not
limited to these tissues, providing for a possible diagnostic
method for Alzheimer's disease. Suitable methods for assay
ing AB protofibrils in these tissues as well as in cell cultures
using an anti-A?protofibrilantibody are immunoassays Such
as ELISA, RIA, Western blotting or dot blotting. The method
would be suitable to follow treatment efficacy (protofibril
reduction) in clinical trials and suitable as a diagnostic test for
Alzheimer's disease or Down's syndrome.
Since AB protofibrils levels are very low in CSF and blood,
a high affinity A?8 protofibril selective antibody is needed in a
diagnostic test based on an ELISA method, to be able to
measure low levels of AB protofibrils. Other supersensitive
methods such as proximity ligation (Example 4) (Gullberg
2004) or similar amplification systems or Biacore or similar
techniques, can be used to increase sensitivity. The proximity
ligation technique is based on the discovery that different
antibodies, raised against different epitopes on an analyte (in
this case a protein), may bind near each other on said analyte.
If said different antibodies are conjugated to oligonucle
otides, the distance between said oligonucleotides will be
short enough for a connector oligonucleotide, with the aid of
ligation components, to form a bridge between the oligo
nucleotides. Amplification components are also added, upon
which RT-PCR may be performed. By this principle, an
amplifiable DNA sequence, reflecting the identity and
amount of the target protein, is generated. This technique
makes it possible to obtain an enhanced signal response and
thus to detect lower concentrations of analyte.
The present inventors Surprisingly discovered that a modi
fied proximity ligation technique may also be used with their
A? protofibril-specific antibodies, to detect low concentra
tions of larger AB peptide structures, i.e. A? protofibrils but
not AB monomers. They discovered that the AB peptides, in
the protofibril conformation, exhibits a structure (repetitive
units) that makes it possible for two antibodies, according to
the present invention, to bind sufficientitly near each other on
the protofibril. If said antibodies are conjugated to oligo
nucleotides, said oligonucleotides may be bridged using a
connector oligonucleotide. PCR is performed using amplifi
cation components. By this principle, an amplifiable DNA
sequence, reflecting the identity and amount of the target
protofibril, is generated (see FIG. 4).
Proximity ligation or a version of the technique called
“rolling circle', is a highly sensitive technique and particu
larly well suited for detection of polymeric structures with
repeated sequences, such as AB protofibrils to be used for
5 diagnosis of Alzheimer's disease and other neurodegenera
tive disorders.
The invention further pertains to the use of high affinity
protofibril specific antibodies in imaging for detection, local
ization and quantitation of AB protofibrils in human and ani
10 mal tissues. The antibody could be label with a radioactive
ligand such as I'', C''', H or Gallium, but not limited to
these radioisotopes, for detection purposes. The method will
be suitable as a diagnostic tool for Alzheimer's disease or
Down's syndrome.
15 Yet another aspect of the invention is to make the antibody
spices specific for use in veterinary medicine. The diagnostic
methods outlined are also suitable for veterinary use.
Another aspect of the invention is the humanization of said
antibodies to avoid side-effect, i.e. to avoid an immunore
sponse against said antibodies in humans when used as a
therapeutic or diagnostic agent.
Yet another aspect is a formulation of the antibody in a
physiological buffer, for example PBS but not limited to PBS,
Suitable for administration to humans and animals. The anti
25 body product can be freeze dried for better stability. The
freeze dried formulation can contain an excipient Such as
manitol but not limited to manitol to stabilize the product after
freeze drying.
The antibody product can contain an antibacterial agent.
30 The antibodies or fragments according to the inventions
may exhibit amino aciddeletions, Substitutions and insertions
within said CDR regions and/or its framework. Inserted or
Substituted amino acids may also be amino acid derivatives,
with the proviso that the affinity and specificity of the anti
35 body is still intact.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the basic structure of an IgG immunoglobulin
40 and its subdomains.
FIG. 2A shows the characterization of a high affinity
protofibril selective monoclonal antibody as measured by
sandwich ELISA.
FIG. 2B shows the characterization of a high affinity
45 protofibril selective monoclonal antibody as measured by
competitive ELISA.
FIG. 3 shows the therapeutic efficacy of a high affinity
protofibril selective antibody in transgenic mouse model
(APPs).
50 FIG. 4 shows human AB protofibrils measured at pM levels
by proximity ligation technique.
FIG. 5A shows the reactivity of mAb158 with AB
protofibril, AB fibril, medin fibril, iset amyloid polypeptide
(IAPP) fibril, and C-synuclein fibril in a dot blot assay.
55 FIG. 5B shows the reactivity of 6E10 (AB), p.Ab179 (me
din), pAbA110 (IAPP), and mAb211 (O-synuclein) with AB
protofibril, AB fibril, medin fibril, IAPP fibril, and C.-sy
nuclein fibril in a dot blot assay.
FIG. 5C shows immunoprecipitation data from HEK-cell
60 culture media (mock, APPs and APPs) and mouse
brain homogenates (non-transgenic, APPs, and
APPs). mAb158 and 6E10 were used for immunopre
cipitation and 6E19 was used as the detecting antibody.
FIG. 6A shows sandwich ELISA data using mAb158 as
65 both the capturing and the detecting antibody.
FIG. 6B shows ELISA data validating the conformation
specificity of mAb158.
 US 8,025,878 B2
9 10
FIG. 6C shows data from a mAB158 ELISA and a 6E10 affinity and high specificity binding to human wild-type AB
6E10 sandwich ELISA performed by adding an increasing protofibrils. Where a polar amino acid is present in a particu
excess of AB1-16 to a fixed concentration of AB protofibrils. lar position in a CDR region that particular amino acid can be
FIG. 7A shows the AB protofibril concentration in substituted by another polar amino acid, with retained or
APPs HEK-cell culture media and APPs HEK-cell 5 improved high affinity and specificity binding to AB
culture media. protofibrils. Likewise, if a non-polar or negatively or posi
FIG. 7B shows the AB rotofibril levels in APP re-Swe a ind tively charged amino acids is presentata certain position, that
APPs transgenic mouse brains. amino acid can be substituted for by a similaramino acid from
FIG. 8 shows the AB protofibril levels in APPs trans the same group.
genic mouse brain TBS extracts after four months treatment 10 Also, a particular amino acid oramino acids are exchanged
with either mab158 or placebo. in any position in the CDR regions by functional equivalents
FIG. 9 shows the total AB levels in is transgenic that confers a similar function and structure to the antibody.
mouse brain formic acid extracts after four months.
FIG. 10 shows the vector map of pKN100. EXAMPLE 2
FIG. 11 shows the vector map of pG1D200. 15
FIG. 12 shows AB monomer binding by chimeric and Characterization of an High-affinity Human AB
mouse 158 antibodies. Wild-type Profibril Selective Monoclonal Antibody
FIG. 13 shows competition of monomeric and protofibril by ELISA
lar AB for binding to chimeric 158 or mouse 158 antibody.
FIG. 14 shows competition of monomeric and protofibril- 20 Example 2 shows a high affinity protofibril selective anti
lar AB for binding to chimeric 158, mouse 158 antibody and body that cross-reacts a 200-1000-fold less with A? mono
humanized 158 antibody (BAN2401). mers and less than 40-fold with Affibrils, as measured by a
sandwich ELISA (FIG. 2A). From competitive ELISA
DESCRIPTION experiments, the antibody has a strong affinity for human
25 Af342 wild-type protofibrils, but only very weak affinity for
The following examples are provided for illustration and the N-terminal part of the AB peptide and AB monomers. No
are not intended to limit the invention to these specific binding was observed to the C-terminal fragment of AB (FIG.
examples. 2B). Furthermore, the antibody does not cross-react with
other types of amyloids, like medin or transthyretin. Further
EXAMPLE1 30 more the antibody does not recognize human APP, the abun
dant precursor of AB.
Human wild-type AB protofibril selective monoclonal anti In FIG.2A a sandwich ELISA is shown. Antibody 158 was
bodies were cloned and sequenced. The amino acid sequence coated in the wells and different AB forms subsequently
of the variable heavy chain region (VH) and the variable light added to the well in increasing concentrations. Measurement
chain region (VL) are shown in Table 1. The positions of the 35 of bound AB forms was made by adding biotinylated mAb
CDR regions 1-3 are underlined and shown as well in Table 2 158 and HRP labelled Streptavidine. Colour development
and 3. The amino acid sequences of the CDR regions form the was measured according to the procedure recommended by
structural basis for binding human wild-type AB protofibrils the manufacturer.
constituting the Alzheimer disease epitope'. In FIG. 2B a competitive ELISA is shown. An ELISA plate
The amino acid sequence of the CDR regions 1-3 of the VL 40 was coated with human AB protofibrils. Antibody 158 was
and VH chains for a high affinity protofibril specific antibody Subsequently incubated with increasing amounts of different
BA9/158 is shown in Table 1, 2 and 3. Sequencing data of A? forms (competition). The incubation mix was added to the
other protofibril selective antibodies (BA2, BA3, BA4 and microtiter plate wells and free antibody was allowed to bind
BA7) provide alternative amino acids sequences of the CDR to immobilized protofibrils in the wells. Bound 158 antibody
regions but not limited to these. The combined amino acid 45 was measured by a second antibody using standard proce
sequences of the CDR1-3 regions of the VH and VL chains dures.
create the molecular “pocket' which binds human AB wild
type protofibrils with high affinity and specificity. This EXAMPLE 3
"pocket' forms the structural basis of the Alzheimer's dis
ease epitope'. Variations in the CDR amino acid sequence 50 The efficacy of high affinity A? protofibril selective anti
length are observed in both the VH chain and the VL is body was determined in an Alzheimer transgenic mouse
compatible binding to human AB protofibrils (Table 2 and 3). model (APPSwe) by an acute intracranial injection. Trans
A shorter CDR region provides a more restricted three dimen genic mice used for efficacy evaluation express human APP,
sional structure of the binding pocket of the antibody, with the Swedish mutation (APPs). In this paradigm, anti
whereas a longer is more flexible. 55 bodies are injected directly into plaque-rich regions of the
We claim the CDR sequences as shown in Tables 1, 2 and brain parenchyma and effects on neuropathology areassessed
3 as well as amino acid sequences in the “mouse framework after 72 hours (Wilcock et al., 2003). Other studies have
regions of the VHandVL chains, i.e. outside the CDR regions shown that the direct application of anti-Afantibodies results
as well as the human VL and VH framework regions for in a rapid clearance of amyloid deposits in vivo (Bacskaietal,
protofibril specific antibodies as shown in Table 4 and 5, but 60 2001; Brendza et al., 2005). The injection of high affinity AB
not limited to those. protofibril selective antibody leads to a significant plaque
The amino acid sequence of the framework region of VL reduction in the APPs mouse model (FIG. 3).
and VH regions 1-3 of the VL and VH chains from a high In FIG. 3 the therapeutic efficacy of a high affinity
affinity protofibril specific antibody BA9/158 is shown in protofibril selective antibody in transgenic mouse model
Table 4 and 5. 65 (APPswe) was tested. A: A 14 months old APPSwe transgenic
Other amino acid substitution in the CDR regions than mouse was intracranially injected with PBS and B: high affin
what is shown in Table 1, 2 and 3 are compatible with high ity protofibril selective antibody (158) at 1 lug/ul and exam
 US 8,025,878 B2
11
ined 72 hours following injection. Marked clearance of AB
burden is noticeable in the Subiculum close to the injection
site (B: arrow) as compared to the control side (A; arrow).
EXAMPLE 4
Proximity ligation in combination with high affinity
protofibril selective antibody for measurement of AB
protofibrils. Human wild-type AB protofibrils were detected
down to 10 pM-range whereas the AB monomer preparation
were not detected at all. The combination of the hypersensi
tive proximity ligation method and a high affinity antibody is
particularly advantageous since it provides a system to deter
mine only oligomeric forms of the analyte, which is particu
larly Suitable when diagnosing Alzheimer's disease and other
protein “aggregation' diseases such as prion disease,
Creutzfelt-Jacob, amyloidosis and Parkinson's disease.
In FIG. 4 human AB protofibrils are measured at pM levels
by the proximity ligation technique. ProXimity ligation assay:
Method description (from Gullberg et al., 2004): Step 1.
incubation of sample with proximity probe pair (s1 h); Step 2.
addition of all components required for ligation and detection
by quantitative PCR (s.5 min ligation time). A high affinity
protofibril selective monoclonal antibody was used in the
assay; step 3, quantitative PCR (s2 h). Synthetic A? mono
mer and AB protofibril preparations were diluted and tested
for their reactivity in proximity ligation assay described
above.
EXAMPLE 5
mAb 158 does not Recognize a Generic Amyloid Epitope.
Previously reported AB conformation dependent antibod
ies have been shown to bind oligomers and fibrils of other
amyloidogenic proteins, Suggesting a common epitope
present on all amyloid aggregates. Due to technical difficul
ties in generating protofibrils from other amyloidogenic pro
teins than Af, mAb158 was instead tested against different
amyloid fibrils. The dot blot assay was used for these experi
ments since inhibition ELISA, where the antibody-antigen
reactions take place in Solution, is not suitable for insoluble
antigens like fibrils. The dot blotassay is however not suitable
for evaluation of antibody specificity for various AB forms,
i.e. for measuring differences in selectivity for profibrils and
fibrils. Fibrils of medin, islet amyloid polypeptide (IAPP) and
C-synuclein were immobilized on a nitrocellulose membrane
to maintain their native conformations. mAb158 did not
exhibit reactivity with any amyloid other the AB fibril (FIG.
5A). The binding of mAb 158 to AB fibrils suggests that part
of the AB protofibril epitope is present also in the AB fibril
structure. As positive controls the antibodies 6E10 (AB),
pAb179 (medin), p.AbA110 (IAPP) and mAb211 (C-sy
nuclein) were used (FIG. 5B). Representative blots from
repeated experiments (n=3).
mAb158 does not Bind APP
Levels of APP and soluble APP fragments commonly
exceed the levels of Afin biological samples such as CSF and
brain homogenate, and therefore an AB-antibody's cross
reactivity to APP could inhibit a treatment by binding to APP.
resulting in less free antibody for binding and elimination of
A? protofibrils and/or AB oligomers. Also, it could disturb
measurements of AB protofibrils in biological samples by a
sandwich ELISA assay of AB. To elucidate whether mab 158
binds to native APP, immunoprecipitation experiments were
performed. HEK-cell culture media (mock, APPs and
APPArc-Save) and mouse brain homogenates (non-transgenic,
APPShe and APPs) were immunoprecipitated with
12
mAb158 or 6E10, followed by a denaturing Western blot with
6E10 as detecting antibody (FIG.5C). As seen in FIG. 5C,
mAb158 did not immunoprecipitate CAPPs from cell culture
media or full length APP from mouse brain homogenates,
whereas, as expected, 6E10 did. The synthetic AB protofibrils
used as control were immunoprecipitated equally well by
both antibodies (FIG. 5C). Representative blots from
repeated experiments (n=3).
10 EXAMPLE 6
Establishment of an AB protofibril specific sandwich
ELISA. To enable measurements of AB protofibrils in bio
logical samples a sandwich ELISA with mAb158 as both
15 capturing and detecting antibody was established. This assay
measures AB protofibrils with a detection limit of 1 pM and
with a linear range up to 250 pM (FIG. 6A, lines indicate
linear regression of the standard curves). Due to uncertainties
concerning the size of the AB protofibrils used in the standard
curve, the concentration 1 pM is based on the molecular
weight of one AB monomer (451.4 g/mol), Though, since the
molecular weight of a protofibril has been estimated to be at
least 100 kDa, the limit of detection calculated as molar AB
protofibrils could be as low as 50 fM. A standard curve of
25 A? Arc protofibrils gave a lower signal than wild type AB
protofibrils, possibly due to differences in A? protofibril size
(FIGS. 6A, 6B). Titrated synthetic LMW-AB (Low Molecular
Weight AB). By the term “Low Molecular Weight AB, it is
meant monomers, dimers and trimers of AB having a molecu
30 lar weight of approximately 4-12 kDa. A? protofibrils and
Af31-16 were used to validate the conformation specificity of
the ELISA (FIG. 6B), where the hydrophilic AB1-16 peptide
was used since it is not expected to aggregate. An ELISA
composed of two identical antibodies requires at least a dimer
35 of a protein to produce a signal and as predicted, AB1-16 was
not detected with the mAb 158 sandwich-ELISA even at
uM-concentrations (FIG. 6B). When pre-treating the LMW
A? and AB protofibrils with 70% formic acid (FA), known to
dissociate aggregated AB into monomers, the sandwich
40 ELISA the signal was lost (data not shown). Hence, the detec
tion of LMW-AB at high nM concentrations (FIG. 6B) is
probably due to a small aggregate content of the peptide
preparation.
A large excess of monomeric Af, holoAPP and APP-frag
45 ments, naturally occurring in biological samples, could inter
fere with the AB protofibril analysis by occupying binding
sites of the capture antibody coat, thus inhibiting the
protofibrils from binding. This problem was investigated by
adding an increasing excess of AB1-16 to a fixed concentra
50 tion of AB protofibrils (50 pM, expressed as monomer units)
and analyzing it with both the mab 158 ELISA and a 6E10
6E10 sandwich ELISA (FIG. 6C). A 500 000-fold molar
excess of AB1-16, as compared to AB protofibrils, did not
disturb the measurements with the mAb158 sandwich
55 ELISA, as expected since AB1-16 binds poorly to the capture
antibody. In contrast, a 500 fold excess of AB1-16 was enough
to decrease the signal in the 6E10-6E10 ELISA, where AB1
16 binds with high affinity to the capture antibody (FIG. 6C).
Moreover, when synthetic A? protofibrils was added to mock
60 HEK cell culture media or non-transgenic mouse brainhomo
genates, 90% of the signal was recovered (data not shown).
EXAMPLE 7
65 Measurement of AB Protofibrils in Biological Samples.
The presence of A? protofibrils in cell and mouse models
carrying the Arctic mutation have been Suggested, though
 US 8,025,878 B2
13 14
until now there has been no method for direct assaying of AB levels in APPswearc transgenic mouse brain formic acid
protofibrils in biological samples. The mab158 sandwich extracts after 4 months treatment with either mAb158 or
ELISA therefore provides the first opportunity to measure AB placebo.
protofibril levels in such cell and mouse models and to com
pare them to models without this intra-A mutation. Samples EXAMPLES 9-11
from cells and mice carrying only the Swedish mutation were
compared to the wild type AB protofibril standard curve,
whereas samples from cells and mice expressing AB with the
Arctic mutation were compared to ABArc protofibril standard 10 Abbreviations
curve (FIG. 6A). To ensure that all AB measured in this assay
was in a soluble state, and to exclude any possible interfer A. Adenine
ence from AB fibrils, all samples were centrifuged for 5 min Ab protocol AERES biomedical protocol
at 17900xg before analysis. Groups of cell media from tran BHK baby hamster kidney
bp base pairs
siently transfected APPs and APPs HEK-cells were 15 C Centrigrade
analyzed and compared to mock HEK-cell culture media. AB C Cytosine
protofibril levels were calculated from the standard curves CHO Chinese Hamster Ovary
CMF Calcium and Magnesium Free
(FIG. 6A) as the mean value of triplicates and were then COS 7 African green monkey kidney fibroblast cell line
normalized to APP levels to compensate for differences in dhfr Dihydrofolate-reductase
transfection levels (according to Stenh et al.). The AB DMEM Dulbecco's Modified Eagles Medium
DMSO Dimethylsulphoxide
protofibril concentration in APPs HEK-cell culture DNA Deoxyribonucleic acid
media was 28 pM (+2), significantly higher (p<0.0001) than ELISA Enzyme linked immuno-adsorbent assay
the 8.2 pM (+0.3) seen in APPs (FIG. 7A). No AB FCS
9.
Foetal Calf Serum
grams
protofibrils could be detected in mock media. Levels of AB G Guanine
protofibrils were also measured in brains from 10 months old 25 hir hour
APPs and APPs transgenic mice with both plaques HRP Horseradish peroxidase
and intraneuronal A? pathology (according to Lord et al.). IgG Immunoglobulin
Brains were homogenized in TBS and centrifuged prior to K G or T (IUPAC convention)
LSAP Large Soluble Amyloid Product
analysis in order to recover the soluble AB fraction. Similar to mAb monoclonal antibody
the analysis using cell culture media, A? protofibril levels 30 SeC. Second
differed significantly (p=0.005) between the groups, with 397 min minute
pM (+59) in APPs and 108 pM (+14) in APPs trans M A or C (IUPAC convention)
MTX Methotrexate
genic mouse brains (FIG. 7B). NIMR National Institute for Medical Research (UK)
In the above-mentioned figures (FIGS. 6 and 7) the number OD
ill nanometer
optical density
of samples were; mock cells (n=3) and transiently transfected 35
PBS Phosphate Buffered Saline
with APPsSave (n=8) and APPs (n=11). Levels of AB PCR Polymerase chain reaction
protofibrils in APP is media were approximately 9 fold R A or G (IUPAC convention)
higher than in APP media, whereas mock media gave no RT Room Temperature
signal (A). Measurements of AB protofibril levels in the TBS S C or G (IUPAC convention)
soluble fraction of non-transgenic mouse brain homogenates 40 T Thymine
UV UltraViolet
(n-6) were compared to transgenic mice (APPs, n=3, and V variable
APPs, n=6) (B). Similar to the cell culture media, AB V A or C or G (IUPAC convention)
protofibril levels of APP is mice were 7 fold higher than VH Immunoglobulin heavy chain variable region
in APPShe mice. Error bars show SEM. WK Immunoglobulin kappa light chain variable region
45
W A or T (IUPAC convention)
Y C or T (IUPAC convention)
EXAMPLE 8

mAb158 Significantly Lowers AB Protofibrils and total A? in Materials

APPswearc transgenic mice. After i.p. Administration
mAb158 (12 mg/kg) was injected i.p. once weekly for 18 50
weeks in 9-10 months old APPSwearc mice. After the study, Equipment
brains were isolated and homogenised in TBS and subse
quently centrifuged to sediment insoluble material. The Equipment UK Supplier Catalog Number
insoluble material was solubilised informic acid. Hence, two
fractions were obtained from mouse brains i.e. a TBS fraction 55 DNA thermal cycler: PerkinElmer N801-0177
GeneAmp 9600
and a formic acid fraction. A? protofibril levels in the TBS A designated tissue culture Walker Safety Cabinets N/a
fractions were determined by an ELISA. A significant reduc aboratory containing a class Ltd.
tion of AB protofibrils was found in the mab 158 treatment I microbiological safety
group compared to the placebo group (FIG. 8). FIG. 8 shows cabinet fitted with a UV-lamp
the AB protofibril levels in APPswearc transgenic mouse innova (R) bench top
60 incubator shaker
New Brunswick Scientific 4000
brain TBS extracts after 4 months treatment with either Bench top centrifuge Fisher Scientific CEK-126-01ON
mAb158 or placebo. CO2-gassed 37 incubator RossLab plc HSO-SO1 TVBB
Total A? in the formic acid fraction was determined by an Microbiological incubator Kendro Heraeus B6060
Electroporator Model: Bio-Rad Laboratories Ltd. 341BR-3092
ELISA (the formic acid was used to solubilise all AB forms, Gene Pulser II
in order to make all A3 forms detectable). A significant reduc 65 ELISA reader: Microplater Bio-Rad Laboratories Ltd. 3550
tion of total AB was observed in the treatment group com Reader 3550
pared to the placebo group (FIG.9). FIG.9 shows the total AB
 US 8,025,878 B2
15 16
-continued -continued
Equipment Immunology and molecular biology reagents
Equipment UK Supplier Catalog Number Article UK Supplier Catalog No. Lot No.
5
Microplate Manager (R) 2.2 Bio-Rad Laboratories Ltd. Na QIAquick PCR purification kit Qiagen 28106 G10.112
data analysis Software Red Stop Solution (For K Blue) SkyBio Ltd, 301.475 O6O104
package for Macintosh Qiagen 74.106 10916587
computer Shrimp alkaline phosphatase USB 70092Y 107635
96-Well GeneAmp PCR ABI Subcloning Efficiency TM Invitrogen 44 OO98 1164658
System 9700 10 DH5CTM Chemically
ABIPRISM 310 Genetic Applied Biosystems 310-00-100,120 Competent E. coli
Analyzer T4 DNA Ligase Promega M1801 16708O
T100 surface plasmon Biacore TMB One-Step substrate for SkyBio Ltd, KB176
HRP
resonance detector
TOPO-TA Cloning (R) kit Invitrogen 45-0641 1350.772
15 X-Gal Sigma B-9146 20965701

Plastic consumables
Solutions from National Institute of Medical Research
Catalog
Article UK Supplier Number Solution name: Components Amount
75 cm2 tissue culture flask Sarstedt Ltd 83.1812.002
25 cm2 tissue culture flask Corning Costar 3056
PBS A Dulbeccos (Ca & NaCl 8g
30 ml universal container Sterilin 128C
Mg Free) KC 0.2 g
75 cm2 tissue culture flask Sarstedt Ltd 83.1813.002 25
NaHPO. 1.15g
Electroporation cuvettes Bio-Rad Laboratories Ltd. 16S-2088 KH2PO 0.2 g
Water 1 L
ELISA plates: Nunc MaxiSorp Invitrogen Life 43945A
LB Bacto Tryptone 10 g
Technologies Yeast Extract 5 g
GeneAmp TM PCR reaction PerkinElmer N801-018O NaCl 10 g
tubes Water 1 L
Glasstic (R) disposable Bio-statDiagnostic 887144 30 LBagar LB 1 L
cell-counting slide Agar (Difico) 15 g
Nunc inoculating needles Life Technologies
issue culture petri 100 x 20 Helena Biosciences
mm, multi-vent
issue culture plate: 6-well + lid Corning
issue culture plate: 24-well + lid Corning 35
Culture Reagents
UK Catalog Lot Expiry
Article Supplier Number Numbers date
Immunology and molecular biology reagents 40 DMEM (1X) Invitrogen 41966-047 9206 July 2007
Dulbecco's Modified
Article UK Supplier Catalog No. Lot No. Eagle Medium (High
glucose) with
1st strand synthesis kit Amersham 27-9261-01 3375.313 GlutaMAX TM I,
Biosciences 4500 mg/L D
Advantage (R)-HF 2 PCR Kit Clontech 639.123 604O151 Glucose, Sodium
Agarose (UltraPure TM) Invitrogen 1551O-O27 3019491 45
Puruvate
Albumin bovine (BSA) Calbiochem 126575 B65755 DMSO (Dimethyl Sigma D26SO 12SK2409 December
Ampicillin Sigma A-9518 63HO992 Sulfoxide) 2007
Apa I Promega R636 16007OO3 Penicillin & Invitrogen 15070-063 12984.01
Themoprime + DNA Abgene ABO3O1 O14f0103.11 Streptomycin
Polymerase O19,0607.13 Serum: Fetal Clone I Perbio SH30080 AMM17779 December
O2O1808:13 50 Science 2007
Bam HI Promega R602 15851606
SOC Invitrogen 15544-034 1306051
BigDye (R) Terminator v3.0 ABI 4390242 O6OS143 Trypan Blue Sigma T8154 19H2388
Cycle Sequencing Ready 0608154 Trypsin-EDTA Sigma T4049 48K2342 April 2008
Reaction Kit
Ethidium Bromide (10 mg/ml) Sigma E-1510 43H9414 Solution, cell culture
Goat anti-human IgG (Fe Stratech 109-005-098 68215 tested, 0.25%
55
ragment specific) antibody Scientific
Goat anti-human kappa chain Sigma A7164 O32K9157
horseradish peroxidase EXAMPLE 9
conjugate
HindIII Promega R604 168348O3
Human IgG1/kappa antibody. The Binding BP078
Site
223.729 60 DNA Sequence of 158 Antibody
K-Blue HRP Substrate SkyBio 3O8176 060823
Oligonucleotides Sigma Ila. 9.1-RNA Preparation
PBS Tablets Sigma P4417 11K8204 Snap-frozen cell pellets of the mouse hybridoma 158, (la
QIAGEN Plasmid Maxi Kit Qiagen 12162 1241 14870
(25) belled vials 060824#1585x10° cells) were received by TAG
QIAprep Spin Miniprep Kit Qiagen 27106 1241.17906 65 on Oct. 3, 2006. These cells were stored frozen until process
QIAquick gel purification kit Qiagen 28704 1154974O ing using the Qiagen RNeasy midikit to isolate RNA follow
ing the manufacturers protocol.
 US 8,025,878 B2
17 18
9.2–1' Strand Synthesis 9.6–158 VHDNA Sequence
About 5 micrograms of 158 RNA was subjected to reverse The consensus sequence (158VH) of VH clones generated
transcription to produce 158 clNA using the Amersham Bio using PCR primers MHV5 and MHCG1/2a/2b/3 mixture on
Sciences 1st Strand synthesis kit following the manufacturers 1 strand cDNAs rounds 1-3 is shown in Table 15. As with
protocol This was repeated to generate 3 independent 5 158 VK, there are some differences from the FW1 sequence
cDNA products (rounds 1, 2 and 3) in order to obviate DNA shown in Tables 1, 4 and 5. The most identical mouse VH
mutations due to the RT reaction. leader sequence to the fragment of leader, not encoded by our
9.3 Cloning of the 158 Immunoglobulin cDNA primers, was NL-1 (Table 16).
Hybridoma 158 cDNA was amplified by PCR in 23 sepa
rate reactions. Immunoglobulin kappa chain variable region 10 EXAMPLE 10
(VK) cDNA was amplified using 11 VK primers (MKV1-11)
in combination with the kappa constant region primer MKC Construction of Chimeric Expression Vectors
(Table 6). Similarly, immunoglobulin heavy chain variable
region (VH) cDNA was amplified by PCR using 12 different Construction of chimeric expression vectors entails adding
VH primers (MHV1-12) in combination with a mix of the 15 a suitable leader sequence to VHand VK, preceded by a Hin
four IgG constant region primers (MHCG1/2a/2b/3: Table 7). dIII restriction site and a Kozak sequence. The Kozak
The result of the initial set of Igh PCR reactions was the sequence (Table 8) ensures efficient translation of the variable
single amplification product using MHV5 primer. None of the region sequence. It defines the correct AUG codon from
other 11 primer pairs gave a PCR product. The product of the which a ribosome can commence translation, and the most
PCR reaction primed by the oligonucleotide primers: 20 critical base is the adenine at position-3, upstream of the AUG
MHV5+(MHCG1/2a/2b/3 mixture) was ligated into the start. The leader sequence is selected as the most similar
pCR2.1(R)-TOPOR) vector using the TOPO-TA cloning R kit. mouse leader sequence in the Kabat database. These addi
The result of the initial set of IgK PCR reactions was two tions are encoded within the forward primers (Table 9). Fur
single amplification products using primers MKV1 and thermore, the construction of the chimeric expression vectors
MKV2 with MKC. The other 9 primer pairs generated no 25 entails introducing a 5' fragment of the human y1 constant
product. The products of the PCR reaction primed by the region, up to a natural Apa I restriction site, contiguous with
oligonucleotide primers: MKV 1 or MKV2+MKC were the 3' end of the J region of 158. The CH is encoded in the
ligated into the pCR2.1(R)-TOPOR) vector using the TOPO expression vector downstream of the inserted VH sequence
TA cloning Rkit. E.coli TOP10 bacteria transformed with the but lacks the V-Cintron. For the light chain, the natural splice
ligated vector were cloned on LB/ampicillin/X-gal agar 30 donor site (Table 8) and a Bam HI site is added downstream of
plates, by picking onto agar grid and into PCR screening the V region. The splice donor sequence facilitates splicing
mixture. The cloned plasmid inserts were screened by PCR out the kappa V:C intron which is necessary for in-frame
amplification. The PCR products were gel electrophoresed attachment of the VK to the constant region. The mouse VH
and clones producing the correct-sized PCR amplification and VK genes were analysed to identify any unwanted splice
product (500 bp approx) were identified. Overnight cultures 35 donor sites, splice acceptor sites, Kozak sequences and for the
(5 ml) of each clone were processed using the QIAprep Spin presence of any extra Sub-cloning restriction sites which
Miniprep Kit Protocol, to produce DNA plasmid minipreps. would later interfere with the subcloning and/or expression of
9.4—cDNA Sequence Determination functional whole antibody. In this case none were found.
The complete cycle of RT-PCR, cloning, and DNA 10.1—Expression Vectors
sequence analysis was repeated to obtain three completely 40 Plasmid DNA preparations of the expression vectors
independent sets of sequence information for each immuno pKN100, and pG1D200 were purified using Qiagen Maxikits
globulin chain. Plasmid clones from each independent set of following the manufacturers protocol. Plasmid DNA Purifi
RT-PCR reactions were sequenced in both directions using cation using QIAGEN Plasmid Midi and Maxi Kits, from 500
the 1212 and 1233 primers (Table 10). Plasmids were ml cultures of TOP10 bacteria transfected with either vector.
sequenced using the BigDye(R) Terminator v3.0 Cycle 45 The vector maps are shown in FIGS. 10 and 11.
Sequencing Ready Reaction Kit (ABI), cycled on a Gene 10.2 The Light Chain Chimerisation Primers
Amp9600 PCR machine and analysed on an ABI 310 capil The mouse leader sequence K5.1 it was incorporated into
lary sequencer. the design of the chimeric 158 VK. Primers were designed to
9.5–158 VK DNA Sequence generate a PCR product containing this complete leader, and
Sequences of VK clones generated using PCR primers 50 158 VK, with terminal restriction sites HindIII and Bam HI
MKV2 and MKC on 1 strand cDNAs rounds 1 and 2, were for cloning into the pKN100 expression vector (Table 9). The
identical to a sterile kappa transcript originating from the forward primer 158vl introduces a HindIII restriction site; a
myeloma fusion partner such as MOPC-21, SP2 and Ag8. Kozak site and the K5.1 it leader sequence. The back primer
This is a sterile transcript The consensus sequence (158 VK) 158vlrev introduces: a splice donor site and a Bam HI restric
of VK clones generated using PCR primers MKV1 and MKC 55 tion site.
on 1 strand cDNAs rounds 1-3 is shown in Table 11. This is 10.3—The Heavy Chain Chimerisation Primers
a functional rearrangement. Table 11 shows some differences The leader sequence NL-1 was incorporated into the
from the sequence shown in Tables 1, 4 and 5. These differ design of the chimeric 158 VH. Primers were designed to
ences are in the FW1 region where the PCR primer was generate a PCR product containing this leader, and the 158
located. The mouse VK leader sequence most identical to the 60 VH region, with terminal restriction sites HindIII and Apa I
fragment of leader in 158 VK, not encoded by our primers, for cloning into the pG1D200 expression vector. These are
was K5.1# (Table 12). The prediction for the signal peptide to shown in Table 9. The forward primer, 158vh, introduces a
cleave correctly the HK5.1 signal sequence was done by a HindIII restriction site; a Kozak translation initiation site and
prediction program. Most likely predicted cleavage site was the NL-1 leader sequence. The back primer, 158vhrev, intro
correctly between amino acid residue 19 and 20. (Table 13). 65 duces the 5' end of the Y1 C region and a natural Apa I
The chimeric 158VK protein and DNA sequence is shown in restriction site. The signal peptide cleavage site prediction for
Table 14. K5.1 leader sequence of VK is shown in Table 17.
 US 8,025,878 B2
19 20
10.4 Generation of the Chimeric 158 VH Construct: The chimeric antibody binds amyloid AB monomer and
pG1D200158VH protofibrils as shown by the direct binding ELISA and the
The 158 VHDNA fragment was amplified with primers: competition ELISA respectively. This evidence confirms that
158vh and 158vhrev (Table 9). The 450 bp (approx) PCR the combination of 158 VH and 158 VK chains encodes the
product was T-Aligated into the vector pCR2.1 and used to anti-LSAP antibody 158 and indicates that these sequences
transform chemically competent TOP10 bacteria. Clones are Suitable for the humanisation procedure to generate a
were selected by appropriate insert size and sequenced using humanised 158 antibody.
the 1212 primer (Table 10). The correct expression insert was
subcloned into pG1D200 expression vector and the correct EXAMPLE 12
Subclone was selected by DNA sequencing using primer 10
BDSH61R (Table 10). This clone was grown in 200 ml cul Humanised Antibody Design and Discussion
ture to produce plasmid DNA using the Qiagen Maxi Kit
using the manufacturers protocol. The chimeric 158VH pro
tein and DNA sequence is shown in Table 18. Abbreviations and definitions
10.5 Generation of the Chimeric 158 VK Construct: 15
pKN100158VK 158 mouse monoclonal anti-LSAPTM antibody 158
The 158 VK DNA fragment was amplified with primers 158 VH VH of mouse 158 antibody
158vl and 158vlrev (Table 9). The 450 bp (approx) PCR 158 VK VK of mouse 158 antibody
158RKASS Humanised version of 158 VK
product was T-A ligated into vector pCR2.1 and used to retaining cryptic splice sites
transform chemically competent TOP10 bacteria. Clones 158RKA Humanised version of 158 VK with
were selected by insert size and sequenced using the 1212 cryptic splice sites removed
primer (Table 10). The correct clone was subcloned into 158RHASS Humanised version of 158 VH retaining
cryptic splice sites
pKN100 expression vector. The correct subclone was 158RHA Humanised version of 158 VH with
selected by Screening for insert size and DNA sequencing cryptic splice sites removed
using primer Hu-K2 (Table 10). This clone was grown in 200 25 A.
bp
Adenine
base pairs
ml culture to produce plasmid DNA using the Qiagen Maxi C Cytosine
Kit using the manufacturers protocol. CDR Complementarity determining region
in the immunoglobulin variable regions,
EXAMPLE 11 defined using the Kabat numbering system
30 D-gene Diversity gene
DNA Deoxyribonucleic acid
Production and Binding Properties of Chimeric 158 FW Framework region: the immunoglobulin
Antibody variable regions excluding the CDR
regions
11.1 COS 7 Cell Transformation and Cell Culture G Guanine
IgG Immunoglobulin G
One vial of COS 7 cells was thawed and grown in DMEM 35
J-gene Joining gene
supplemented with 10% Fetal clone I serum and antibiotics. Kabat an immunoglobulin alignment and numbering
One week later, cells (0.8 ml at 10"/ml) were electroporated system pioneered by Elvin A
with pG1D200158VH plus pKN100158VK (10 ug DNA mAb
Kabat
monoclonal antibody
each). The cells were grown in 8ml of growth medium in petri MRCT Medical Research Council Technology
dishes for 3 days. 40 T Thymine
11.2 Chimeric Antibody Production VCI Framework residue classified as vernier
A sandwich ELISA was used to measure antibody concen or canonical or VH-VL interface
V-gene The gene segment that is rearranged together
trations in the COS 7 supernatants. Chimeric 158 VHx158 with a J (and D for VH) gene to
VKantibody was expressed at 0.3 ug/ml and Subsequently at generate a complete VH or VK
3.7 g/ml (Table 19) in transiently co-transfected COS cell 45 V region The segment of IgG chains which is
conditioned media. variable in sequence between different
antibodies. It extends to Kabat residue 109
11.3—Chimeric Antibody Activity in the light chain and 113 in the
Two ELISAs was used to analyse the antigen binding of heavy chain.
chimeric 158. Using the 3.7 ug/ml chimeric antibody condi Immunoglobulin heavy chain variable region
tioned medium, binding to AB monomer was measured by a 50 Immunoglobulin kappa light chain variable region
direct ELISA protocol (FIG. 12) and compared to the mouse
158 IgG. Secondly, a competition ELISA was done using
either monomer or protofibril mixed in the fluid phase with
antibody, which Subsequently bound to AB monomer in the
solid phase (FIG. 13). These showed that the chimeric 158 55
Equipment
antibody binds to amyloid AB monomer and protofibril simi
larly to the original 158 mouse antibody. Hardware & Software Origin
Comment
Later sequencing has shown that the mouse antibody SGWO2 computer Silicon Graphics
PC computer Hewlett Packard
sequence data, as shown in Tables 1 and 4 contain errors in 60 SR 7.6
Steve Searle, Wellcome Trust
both VH and VK chains at the 5' end. We suggest that this is Sanger Institute, Cambridge.
due to the use of primers located within the V region. In later Lasergene 6.0 DNAStar Inc
sequencing, primers located within the leader sequences, Modeler 9.0
SignalP
Accelrys Ltd.
Center for Biol. Sequence Analysis,
which cannot introduce mutations within the V regions, were Technical University of Denmark website
used. The later sequencing showed sequence differences (see 65 BlastP NCBI website
Tables 15 and 11). Said differences are however not located
within the CDR regions.
 US 8,025,878 B2
21
12.1—Human V Gene Databases
The protein sequences of human and mouse immunoglo
bulins from the International Immunogenetics Database 2006
and the Kabat Database Release 5 of Sequences of Proteins of
Immunological Interest (last update 17 Nov. 1999) were used
to compile a database of immunoglobulin protein sequences
in Kabat alignment. Our database contains 9322 human VH
and 2689 human VK sequences. The sequence analysis pro
gram, SR 7.6, was used to query the human VH and VK
databases with 158 VHand 158 VK protein sequences (Table
20).
12.2—Selection of a Human Framework for 158RHA
12.2.1—Comparison of 158 VH with Human VHSequences
Human VH sequences with highest identity to 158 VH at
Vernier (Foote, J. and G. Winter. 1992. Antibody framework
residues affecting the conformation of the hypervariable
loops. J Mol. Biol. 224:487-499.), Canonical (Morea, V., A.
M. Lesk, and A. Tramontano. 2000. Antibody modeling:
implications for engineering and design. Methods 20:267
279.) and VH-VL Interface (Chothia, C.J. Novotny, R. Bruc
colleri, and M. Karplus. 1985. Domain association in immu
noglobulin molecules. The packing of variable domains. J
Mol. Biol. 186:651-663.) (VCI) residues, located within the
V-region framework (FW), are shown in Table 21. The num
ber of VCI residues (VCI score) and FW residues (FW score)
identical to 158 are also shown. All these VH sequences share
identical VCI residues, and CDR lengths, as shown in Table
22. AJ556669 has an unusual Pro74 not seen in the other
human sequences in this dataset, leading us to discount it in
the initial analysis. Pro74 is, however, present in the 158VH
sequence, so AJ556669 could be considered as an alternative
FW for humanisation, if the VH construct based on
AF062243 does not bind antigen. The alignment of these
sequences (Table 23) highlights their differences. AF062243
uniquely within this dataset has the conservative change
T(82a)S and the conservation of F79. The other features of
AF062243 are the conservative changes D1E, K19R, A23S,
T77S, S118T. All other FW changes were common to all the
frameworks in Table 23. AF062243 was selected as the
framework on which to base 158RHA.
12.3 Generation of 158RHA
The design of 158RHA is simply the grafting of CDR 1, 2
and 3 from 158 VH into the acceptor FW of AF062243. The
human germline V-gene most identical to AF062243 is VH
M99649 (VH3-07), (Table 24) from which the leader peptide
was extracted (Table 25). The SignalPalgorithm (Nielsen, H.,
J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identi
fication of prokaryotic and eukaryotic signal peptides and
prediction of their cleavage sites. Protein Eng 10:1-6.) pre
dicted that it would cut appropriately with signal peptidase
(Table 26). Table 27 shows the scheme of grafting 158 VH
CDR 1, 2 and 3 into the AF062243 FW, to generate 158RHA
protein sequence. Table 28 shows the generation of the DNA
sequence 158RHASs from the natural DNA sequences of 158
VH and AF062243. Analysis of the 158RHASs DNA
sequence predicted the presence of splice donor sites, the
prediction scores of which are shown in Table 29. Non-coding
mutations were introduced to inactivate these predicted splice
sites, as shown in Table 30 to generate the final 158RHA DNA
sequence (Table 31).
12.4—Selection of a Human Framework for 158RKA
12.4.1—Comparison of 158 VK with Human VK Sequences
The human VK sequences with highest identity to 158VK
at VCI residues are shown in Table 32 together with the
number of VCI residues (VCI score) and FW residues (FW
score) identical to 158 VK. Eleven sequences have all VCI
residues identical to 158 VK. Table 33 shows that all these
22
sequences have CDR lengths identical to 158 VK. Table 34
highlights their differences, showing that K45 is retained in
AB064.054 only, which also retains 185. The G100P change is
unremarkable because P100 is common, having an incidence
5 of 15% in our human VK database. The two substitutions:
T7S and K74R, are conservative, and all other substitutions
are common to all the sequences in Table 34. For these rea
sons AB064054 was selected to generate 158RKA.
12.5 Generation of 158RKA
10 The design of 158RKA is the simple grafting of the CDRs
1, 2 and 3 from 158 VK into the acceptor FW of human
AB064.054. The nearest germline V-gene to AB064054 is A19
(Table 35), from which the leader peptide was extracted
(Table 36). The SignalP algorithm predicted appropriate cut
15 ting (Table 37) of this leader peptide. Table 38 shows the
generation of the protein sequence of 158RKA by intercala
tion of the 158VK CDRS into the FW of ABO64054. Table 39
shows the generation of the DNA sequence of 158RKAss
from the natural DNA sequence of 158 VK and AB064.054.
Analysis of the 158RKASs predicted the presence of splice
donor sites, the scores of which are shown in Table 40. Non
coding mutations (41) were introduced to inactivate these
sites and generate the final 158RKA DNA construct (Table
42).
25 12.6 Humanized Antibody (BAN2401) Binding Activity
The 158RKA and 158RHA genes were inserted into an
expression vector containing the IgG1 constant region. This
construct was expressed in COS cells to generate the human
ized 158 antibody. The humanized 158 antibody was tested
30 for binding activity and specificity in a competitive ELISA.
The humanised antibody exhibited identical binding proper
ties as to mAb158 and the 158 chimeric antibody (see FIG.
14.)
12.7 Additional Mutations in the 158RHA and 158RKA
35 Chains.
By comparing mouse germline V genes VHAAK71612 to
158 VH a single somatic mutation A60G in the CDR2 was
identified. Furthermore, the molecular model of antibody 158
which contains three VHFW residues within 5 A of CDR
40 residues which are unconserved in 158RHA. These substitu
tions are D1E, P74A and T82S (Table 43). Similarly, there are
two VK FW residues within 5 A of CDR residues which is
unconserved in 158RKA. This substitution is L3V and
G100P (Table 44). Introduction of back mutations at posi
45 tions VH-1, VH-74, VH-82, VK-3 and VK-100 into 158RHA
and 158RKA, in humanised versions 158RHB, 158RHC,
158RHD, 158RKB and 158RKC are shown in Table 43 and
44.
50 REFERENCES
Bacskai et al., Nat. Med. 7:369-372, 2001.
Bard et al., Nat. Med. 6:916-919, 2000.
Bayer et al., Neurology 64:94-101, 2005.
55 Brendza et al., J. Clin. Invest. 115:428-33, 2005.
Chen et al., Nature, 408:975-9, 2000.
Chothia, C. etal, J Mol. Biol., 186:651-663, 1985.
Ester W. P. Science 293, 1449-1459, 2001.
Gullberg et al., Proc. Natl AcadSci, 101:8420-4, 2004.
60 Foote, J. et al., J Mol. Biol., 224:487-499, 1992.
Hoshi et al. Proc. Natl Acad. Sci, 100:6370-6375, 2003.
Jarret J.T., Biochemistry, 32, 4693-4697, 1993.
Leatherbarrow R. J. et al., Mol. Immunol. 22, 407, 1985.
Lord et al., Neurobiol. Aging, 27:67-77, 2006.
65 McLean et al., Ann. Neurol. 46:860-866, 1999.
Morea, V. et al., Methods 20:267-279, 2000.
Mullan et al., Nat Genet. 1:345-347, 1992.
 US 8,025,878 B2
23 24
Nielsen, H. et al. Protein Eng 10: 1-6, 1997. TABL 2- Continued
Nilsberth et al., Nat Neurosci. 4:887-893, 2001.
Näslund et al., JAMA, 283:1571-1577, 2000. Amino acid sequences of CDR1-2 regions from VH
Pfeifer et al., Science 298:1379, 2002. chain from a protofibrill selective antibody and
Racke et al., J. Neurosci 25:629-36, 2005. 5 amino acid substitutions that are compatible with
Schenk D. et al. Nature, 400, 173-177, 1999. high affinity binding to human wildtype A proto
Stenh et al., Ann. Neurol. 58:147-50, 2005. fibrils. (SEQ ID NOS: 25-32)
Walsh D. M. et al., 272, 22364-22372, 1997
Walsh D. M. et al., Nature, 416, 535-9, 2002. - - - T- - - - G-YT--P-S- - - - - - Substitutions
Wilcock et al., J. Neurosci., 23:3745-51, 2003. 10 (SEQ ID NO : 29)
Wright A. et al., J. of Immunology, 3393-3402, 1998.
Xu Y. et al. J. Biol. Chem. 269,3469-3474, 1994.
TABLE 1.

Amino acid sequence of variable regions of the heavy

chain (VH) and light chain (VL/VK) from six different monoclonal
antibodies specific for human Wild-type AB protofibrils. (SEQ ID NOS : 13-24)
(SEO ID VH-BA1: EWKLVESGGGLVOPGGSRKLSCAASGFTFSSFGMRVWROAPEKGLEWVAYISSGSSTIYYADTVKGRFT
NO: 13) X731 ISRDNPKNPLPLOKTSLRSEDTAMYYCARYGNYAM- - - - - - -DYWGOGTSVTVSS

(SEO ID VH-BA2: EWKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVROTPEKRLEWVATISSGGSYTYYPDSVKGRFT

NO: 14) X736 ISRDNAKNTLYLOKSTSLRSEDAMYYCARNYGSRRYF- - - - -DVWGASTSYTVSS

(SEO ID VH-BA3: OVHLOOSGPELVKPGASVEMSCKASGYTFTSYVNHWWKOKPGOGLEWIGYINPYNDSTKYNEKFKGKAT

NO: 5) X745 LTSDKSSSTAYKELSSLTSEDSAVYYCARRVSPLTSYAM- - -DYWGOGTSVTVSS

(SEO ID VH-BA7: OVOLKESGPGLVAPPOSLSITCTVSGFSLTSYGVHWVROPPGKGLEWLGVIWAGGSTNYNSALMS-RLS

NO: 6) X746 ISKDNSKSOVPLKHNSLOTDDTAMYYCARGRYDGKTRFA----YWGDGTLVTVSS

(SEO ID VH-BA4: EWKLMESGGGLVOPGGSRKLSCAASGFTFSGFGMHEVROAPEKGLEWVAYISSGSSTTYYADYWKGRFT

NO: 7) X748 ISRDNPKNTLPLOMTSLRSEDTAMYYCARGDSF - - - - - - - - -DYWGDGTTL TVSS

(SEO ID VH-BA9 : EVORVESGGGLVOPGGSRKLSCAASGFTFSSFGMHWVROAPEKGLEWVAYISSGSSTIYYGDTVKGRFT

NO: 8) X758 ISRDNPKNTLPLOKTSLRSEDTAMYYCAREGGYYYGRSYYTMDYEGOSTSVTVSS

(SEQ ID VK-BA1: DVVMFQFFLSLPVSLGDQASISCRSSQSIYSSNGNYYLS-WYLQKPGQSPKLLIYKVSNRFSGVPDRFS

NO: 9) X731 GSGSGTDFTLKISRVEAEDLGVYYCFQGSNVPPFFGGGTKLEIK
(SEO ID Vk-BA2: DIWMTOAFKFLLWSAGDEVTTTCKASOSVSNDVA- - - - - - WYOOKPGOSPKLLIYYASNRYTGVPDRFT
NO: 2O) X736 GSGYGTDFTFTISTVOAEDLAVYFCOODYSSPFFFGSGTKLEIK
(SEO ID Vk-BA3 : DIWMTOAPSSLAVSAGEKVTMSCKSSOSLLNSRTRKNYLAWYOOKPGOSPKLLIYAASTRESGVPDRFT
NO: 21) X745 GSGSGTDPTLTISSVOAEDLAVYYCKOSYNL-WTFGGGTKLRIK
(SEO ID Vk-BA7: ENVLTOSPAINSASPGEKVTNECRASSSVSSSYLH- - - - -WYOOKSGASPKLWIYSTSNLASGVPARFS
NO: 22) X746 GSGSGTSYSLTISSVEAEDAATYYCOOYSGYPLTPGAGTKLELK
(SEO ID Vk-BA4: DIWMTOAPLSLPWSLEDOASISCRSSOSLVRSNGNFYLH-WYLOKPGOSLKLLKYKVSNRFSGVPDRPSV
NO. 23) X748 GSGSGTDFTLKISREAEDLSWYFCSOSTAVPLTFGAGTKLELK
(SEO ID Vk-BAs : DFVKTOAPLSLPWSLGDOASISCRSSOSIVHSNGNTYL-SHYLOKPGOSPKLLIYKVSNRFSGVPDRFSG
NO: 24) X758 SGSGTDPTLKISRVEAEDLGIYYCFOGSHVPPTFGGGYKLEIK
*Position of the various CDR regions (1-3) are underlined in VL and VH. The boundaries of the CDR regions
(1-3) are shown in Table 3 and Table 4. Antibody BA9, also named 158 in the patent application. , is an example
of a high affinity protofibrill specific antibody according to the invention.

50
TABL 2 TABLE 2 - continued
Amino acid sequences of CDR1-2 regions from VH Amino acid sequences of CDR1-2 regions from VH
chain from a protofibrill selective antibody and chain from a protofibrill selective antibody and
amino acid substitutions that are compatible with amino acid substitutions that are compatible with
high affinity binding to human wildtype A proto 55 high affinity binding to human wildtype A proto
fibrils. (SEQ ID NOS: 25-32) fibrils. (SEQ ID NOS: 25-32)

VH chain CDR-1 region VH chain CDR-3 region

AASGFTFSSFGMHWVR (SEO ID NO: 25) Antibody 158 CAREG-GYYYGRSYY-TMDYWGO Antibody 158
. . . . . . . . . YA-S- - - (SEQ ID NO: 26) Substitutions 60 (SEQ ID NO : 3 O)
CARYGXxxxxNYxxxxAMDYWGQ Substitutions
VH chain CDR-2 region (SEQ ID NO: 31) and deletions
CARNYXXXXGSRRXXXYFDWWGA Substitutions
WWAYISSGSSTIYYGDTWKGRFT Antibody 158 (SEQ ID NO: 32) and deletions
(SEO ID NO: 27)
. . . . . . . . . . . . . . A- - - - - - - - Substitutions 65 The amino acid Substitutions (other amino acid than in
antibody 158) are shown with one amino acid letter code
(SEQ ID NO: 28) Deletions are shown with (x).
 US 8,025,878 B2
25 26
TABLE 3 TABLE 3 - continued
Amino acid sequences of CDR 1-3 regions from V/L Amino acid sequences of CDR 1-3 regions from V/L
chain from a protofibrill selective antibody and chain from a protofibrill selective antibody and
amino acid substitutions that are compatible with amino acid substitutions that are compatible with
high affinity binding to human wildtype A proto 5
fibrils (SEQ ID NOS: 33-38) high affinity binding to human wildtype A proto
fibrils (SEQ ID NOS: 33-38)
VL chain CDR-1 region
VL chain CDR-3 region
ISCRSSOSIVHSNGNTYLEWYL Antibody 158
(SEQ ID NO: 33) 10 YYCFOGSHVPPTFGG (SEO ID NO : 37) Antibody 158
ITCKASOSVxxSNDXXXVAWYO Substitutions and
(SEQ ID NO: 34) deletions -F-Q-DYSS-F- - - S (SEQ ID NO: 38) Substitutions
VL chain CDR-2 region
The amino acid substitutions (other amino acid than in
LIYKWSNRFSGWP (SEQ ID NO: 35) Antibody 158 antibody 158) are shown with one amino acid letter code.
Deletions are shown with (x).
- - -YA- - - YT- - - (SEQ ID NO: 36) Substitutions

TABL E 4

Amino acid sequence of mouse framework regions of the mouse and human variable light
chain (VL) region from protofibril specific antibodies (SEQ ID NOS: 39-47)
Mouse framework* VL regions
Divmtgaplislpwslgdoasiscwylckpgp spiklliygvpdrfsg.sgsgtoftliki Srveaedlgiyyc antibody 158 (SEO ID NO : 39
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - BA9 VL fr123 (SEQ ID NO: 4 O)

w .. t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . w BA1 VL fr123 (SEQ ID NO: 41)

- - - - - - - - kf.l.. a . . rvt. t . . . g . . . . . . . . . . . . . . . . . . t. . . Y. . . . . ft. . t. C av. f. BA2 VL fr123 (SEQ ID NO: 42)

Human framework VL regions
- - - - - - t . . . . . .tp ep . . . . . . . . . . . . . . . q. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v. v . . . WKII-3-1 - (1) - O11 (SEQ ID NO : 43)
- - - - - - S. . . . . .tp.ep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .V.V WKII-4-1- (1) - A19 (SEQ ID NO: 44)

- - - - - - t. s.t. Cp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .V.V WKII-4-1- (1) - A18 (SEQ ID NO: 45)

- - - - - - t. s.t. Cp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .V.V WKII-4-1- (1) - A2 (SEQ ID NO: 46

w . . is . . . . . . t. Cp . . . . . . for . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v. v . . . WKII- 4 - 1 - (1) - A17 (SEQ ID NO: 47)

Framework region is the region outside the CDR regions. The CDR regions has been deleted for Clarity.

TABLE 5

Amino acid sequence of mouse and human framework regions of the mouse and human
variable light heavy (VH) region from protofibril specific antibodies (SEQ ID NOS: 48-56)
Mouse framework* VH regions
Evklimesggglvopggsrkls caas Wvrqapekglew varfti Srdnpkintilflgmtslrseditamyycar antibody 158 (SEQ ID
NO: 48)
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - BA9 VH fr123 (SEQ ID

NO: 49)
W. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BA1 VH fr123 (SEQ ID
NO: 5O)
W . . . . . . . k. •l . . . . . . . . . . . t . . . . . . . . . . . . . a. Y . . . is . . . . . . . . . . . . . . BA2 VH fr123 (SEQ ID
NO: 51)
Human framework VH regions
C. W. . . . . . . . . . . . lir. . . . . . . . . . . . 9 . . . . . . . . . . . . . . . a . . S.y a. V. . . . . VH3-7 fr123 (SEQ ID
NO: 52)
C. W. . . . . . l . . . . . lir. . . . . . . . . . . . 9 . . . . . . is . . . . . . . . S y a. V. . . . . VH3-53 fr123 (SEQ ID
NO: 53)
d:l . . . . . . . . . . . . lir. . . . . . . . . . . . 9 . . . . . . is . . . . . . . . S y l. . . a . w k VH3-23 fr123 (SEQ ID
NO: 54)

C. W. . . . . . . . . . . . lir. . . . . . . . . . . . 9 . . . . . . is . . . . . . . . a . . S.y c V. . . . . VH3-48 fr123 (SEQ ID

NO: 55)
 US 8,025,878 B2
27 28
TABLE 5- continued
Amino acid sequence of mouse and human framework regions of the mouse and human
variable light heavy (VH) region from protofibril specific antibodies (SEQ ID NOS: 48-56)
. C. W. . . . . . . . . . . . . lir. . . . . . . . . . . . 9 . . . W. . . is . . . . . . . . a . . . . Y. . . . . . . . a . . . . W. . . . . . VH3-74 fr123 (SEQ ID
NO: 56)
* Framework region is the region outside the CDR regions. The CDR regions has been deleted for Clarity.

TABLE 6 10 TABLE 7-continued

PCR primers for cloning mouse VK

PCR primers for cloning mouse heavy VH
(SEO ID NOS: 57-68)
(SEQ ID NOS: 69-84)
Name Sequence (5' -> 3') 15
Name Sequence (5' -> 3')
KW1 ATGAAGTTGVVTGTTAGGCTGTTGGTGCTG (SEO ID NO : 57)

KV2 ATGGAGWCAGACACACTCCTGYTATGGGTG (SEO ID NO : 58) HV 11 ATGGATTTTGGGCTGATTTTTTTTATTG (SEQ ID NO: 79)

r 2O
KV3 ATGAGTGTGCTCACTCAGGTCCTGGSGTTG (SEO ID NO. 59) HV12 ATGATGGTGTTAAGTCTTCTGTACCTG (SEO ID NO: 80)

KW4 ATGAGGRCCCCTGCTCAGWTTYTTGGMWTC (SEO ID NO : 6O)

TTG HCG1 CAGTGGATAGACAGATGGGGG (SEQ ID NO: 81)

r 25
KVs ATGGATTTWAGGTGCAGATTWTCAGCTTC (SE D NO : 61
(SEQ ) HCG2a CAGTGGATAGACCGATGGGGC (SEQ ID NO: 82)
KW6 ATGAGGTKCKKTGKTSAGSTSCTGRGG (SEQ ID NO: 62)
HCG2b CAGTGGATAGACTGATGGGGG (SEQ ID NO: 83)
KV7 ATGGGCWTCAAGATGGAGTCACAKWYYCW (SEO ID NO : 63)
GG 30
HCG3 CAAGGGATAGACAGATGGGGC (SEQ ID NO: 84)
KV8 ATGTGGGGAYCTKTTTYCMMTTTTTCAAT (SEQ ID No. 64)
TG Legend: Wobble bases are defined in Abbreviations (Section 2).

KV9 ATGGTRTCCWCASCTCAGTTCCTTG (SEO ID NO : 65) 35

TABLE 8
KV10 ATGTATATATGTTTGTTGTCTATTTCT (SEQ ID NO: 66)
Sequences important for efficient expression of
KV11 ATGGAAGCCCCAGCTCAGCTTCTCTTCC (SEO ID NO : 67)
40 immunoglobulin in mammalian cells
KC ACTGGATGGTGGGAAGATGG (SEQ ID NO: 68) (SEQ ID NOS: 85-88)

Name Consensus DNA. Sequence (5' -> 3')

TABLE F
45 Kozak transla - G C C G C C R C C A' U G G
PCR primers for cloning mouse heavy VH tion initiation (SEQ ID NO: 85)
(SEQ ID NOS: 69-84)
site
Name Sequence (5' -> 3')
HV1 ATGAAATGCAGCTGGGGCATSTTCTTC (SEQ ID NO: 69) 50 Kappa light A C : : G T R A. G. T.
HW2 ATGGGATGGAGCTRTATCATSYTCTT (SEO ID NO : 7O) chain splice (SEQ ID NO: 86)
donor site
HW3 ATGAAGWTGTGGTTAAACTGGGTTTTT (SEO ID NO : 71.)
HW4 ATGRACTTTGGGYTCAGCTTGRTTT (SEO ID NO : 72) ss Heavy chain M. A. G. : : G T R A. G. T.
li c SEO ID NO : 87
HV5 ATGGACTCCAGGCTCAATTTAGTTTTC (SEQ ID NO: 73) spilce donor (SEQ )
CTT site

HW6 ATGGCTGTCYTRGSGCTRCTCTTCTGC (SEQ ID NO: 74)

60 Immunoglobulin Y Y Y Y Y Y Y Y Y Y Y N C A G : : G
HV7 ATGGRATGGAGCKGGRTCTTTMTCTT SE D NO : 75
(SEQ ) splice acceptor (SEQ ID NO: 88)
HV8 ATGAGAGTGCTGATTCTTTTGTG (SEO ID NO : 76) site

HW9 ATGGMTTGGGTGTGGAMCTTGCTATTC (SEQ ID NO: 77)

CTG Legend: Bases shown in bold are considered to be invariant
65 within each Consensus sequence. Splice Sites are defined by the
symbol : :". Wobble bases are defined in Abbreviations (see
HV1 O ATGGGCAGACTTACATTCTCATTCCTG (SEQ ID NO: 78) Examples 9-11).
 US 8,025,878 B2
29 30
TABLE 9
Oligonucleotide primers used to generate
chimeric 158 (SEQ ID NOS. 89-92)
Oligonucletotide name Sequence (5' -> 3')
158 wh(SEO ID NO: 89) AAGCTTGCCGCCACCATGGACTCCAGGCTC
158 whrew GGGCCCTTGGTGGAGGCTGAGGAGACGGTGACTGAGG
(SEO ID NO: 90)
158 vl (SEO ID NO: 91) AAGCTTGCCGCCACCATGAAGTTGCCTGTTAGG

158 vilrev (SEO ID NO: 92) GGATCCACTCACGTTTGATTTCCAGCTTGG

Legend: Restriction sites are underlined. Kozak sequences are in bold type.

TABLE 1.O TABLE 1.O- continued

Oligonucleotide primers used for sequencing Oligonucleotide primers used for sequencing
(SEQ ID NOS: 93-96) 2O (SEQ ID NOS: 93-96)
Oligonucletotide name Sequence (5'-->3') Oligonucletotide name Sequence (5'-->3')
1212 (17mer) GTTTTCCCAGTCACGAC Hu-K2 (1.7 CTCATCAGATGGCGGGA
(SEO ID NO: 93) l (17mer)
(SEO ID NO: 95)
1233 (24 mer) AGCGGATAACAATTTCACACAGGA 25
(SEQ ID NO: 94) BDSH61R (SEO ID NO: 96) CGCTGCTGAGGGAGTAGAGTC

TABLE 11

DNA sequence of 158 VK, primer MKV1 and the VK sequence derived using primers located within the V
region (SEQ ID NOS: 97-99)

(SEO ID NO: 97)

ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTG 158 WK
(SEO ID NO: 98)
MKV1
(SEO ID NO: 99)

CCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTAC 158 WK

18 CTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGA. 158
WK

27 TCAGGGACAGATTTCACACTGAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCTCCG 158 WK

158 WK
* + k WK

Legend: Residues identical to 158 WK are indicated by a dot.

*** Sequencing using primers located within the W region.

TABL E 12

Chimeric WK leader sequence selection - K5. 1: leader

selection for the chimeric WK (SEQ ID NOS: 100-102)
158 WK MKLPVRLLVLMFWIPASSS (SEO ID NO : 1.OO)
K5.1EProtein MKLPVRLLVLMFWIPASSS (SEO ID NO : 101)
K5.1DNA ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCT
GCTTCCAGCAGT (SEO ID NO : 102)
 US 8,025,878 B2
31 32
TABLE 13 TABLE 13-continued
SignalP result 6 for K5.1# leader (SEQID NO: 103) SignalP result 6 for K5.1# leader (SEOID NO: 103
>Sequence length = 40 5 >
Sequence len :
40
# Measure Position Value Cut off signal peptide?
# Measure Position Value Cut off signal peptide?
mean S 1-19 O.954 O.48 YES
8X. C 2O O.970 O.32 YES D 1-19 O.922 O.43 YES
max. Y 2O O.890 O.33 YES
8X. S 13 O.989 O.87 YES 10 # Highest probability for cleavage is between amino acid residue 19 and 20 (SSS-DV)

TABLE 1.4

Protein and DNA sequence of chimeric 158 VK construct (SEQ ID NOS: 104-105)
HindIII

(SEQ ID NO: AAGCTTGCCGCCACCATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTG

104) H 81
HkozakHK5.1: leader e- 158 VK
(SEQ ID NO: K. L. A. A T M K L P W R L. L. W. L. M. F W I P A S S S D W L
105) ATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTA
162
158 WK
M. T. O. T P L S L P W S L G D Q A S I S C R S S Q S I V
CATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCC
243
158 WK

H S N G N T Y L E W Y L Q K P G O S P K L L I Y K V S
AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAG
- lllllllllllllllllllllllllllllllllllllllllllllllllllllllll- 324
158 WK
N. R. F. S. G W P D R F S. G. S G. S G T D F T L K I S R W E
GCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATC
405
158 WK
A E D L G I Y Y C F O G. S H V P P T F G G G T K L E I
BamHI

AAACGTGAGTGGATCC

TABL E 15

DNA sequence of 158 VH, primer MHV5 and the sequence derived using primers
located within the V region (SEQ ID NOS: 106-108)
(SEQ ID NO: 106)
1 ATGGACTCCAGGCTCAATTTAGTTTTCCTTGTCCTTATTTTAAAAGGTGTCCAGTGTG 158 WH
ATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTA

(SEO ID NO : 107)
1. 'k k k WH

(SEQ ID NO: 108)

1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MHV5

91 GTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTA 158 WH
GCTTTGGAATGCACTGGGTTCGTCAGGCTCCA

34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 'k k k WH

181 GAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACCATCTACTATG 158 WH

GAGACACAGTGAAGGGCCGATTCACCATCTCC
 US 8,025,878 B2
33 34
TABLE 15- continued

DNA sequence of 158 VH, primer MHV5 and the sequence derived using primers
located within the W region (SEQ ID NOS: 106-108)
'k k k WH

271 AGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACA 158 WH

CGGCCATGTATTACTGTGCAAGAGAGGGGGGA

361 TATTACTACGGTAGGAGTTACTATACTATGGACTACTGGGGTCAAGGAACCTCAGTCAC 158 WH

CGTCTCCTCAGCCAAAACAACAGCCCCA

Legend: Residues identical to 158 WH are indicated by a dot.

***Sequencing using primers located within the V region.

2O
TABL E 16 TABLE 17
Chimeric WH leader selection SignalP result 6 for NL-1 VH leader sequence (SEQ ID NO. 112
NL-1 WH leader sequence (SEQ ID NO: 109-111) # Measure Position Value Cut off signal peptide?
159 WH leader MDSRLNLVFLVLILKGVOC (SEQ ID NO: 109) 25 8X. C O.775 O.32 YES
8X. Y 0.795 O.33 YES
NL-1 protein MDSRLNLVFLVLILKGVOC (SEQ ID NO: 110) 8X. S 13 O.953 O.87 YES
(8. S 1-19 O.866 O48 YES
NL-1 DNA ATGGACTCCAGGCTCAATTTAGTTTTCCTTGTCCTTA D 1-19 O.830 O43 YES
TTTTAAAAGGTGTCCAGTGT (SEQ ID NO: 111)
30 # Highest probability for cleavage is between amino acid residue 19 and 20 (VQC-DV) 19
and 20:VQC-DV

TABL E 18

Protein and DNA sequence of chimeric 158 WH (SEQ ID NOS: 113-114)

HindIII

(SEQ ID NO: 113) AAGCTTGCCGCCACCATGGACTCCAGGCTCAATTTAGTTTTCC TTGTCCTTATTTTAAAAGGTGTCCAGTGTGATGTGCAG

- ill ill- 81
kozakh NL-1 leader 158 WK
(SEQ ID NO: 114) K. L. A. A. T M D S R L. N. L. W. F W L I L K G W Q C D W Q
CTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCC GGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGT
162
158 W K
L W E S G G G L W Q. P G G S R. K. L. S. C. A. A. S. G. F. T. F. S
AGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACC
11 - 243
158 W

S F. G M H W W R O A P E K. G E W W A Y I S S G. S S T
ATCTACTATGGAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACC
| | | | | | | | | | | | | | | | | | | | | | | | | || - 324
158 W
Y Y G D T V K G R F S R D N P K N T L F L Q M T
AGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGAGGGGGGATATTACTACGGTAGGAGTTACTATACTATG
4 OS
158 W
S L R S E D T A M Y Y C A R E G G Y Y Y G. R. S Y Y T M.

ppal
GACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCC
it - 461
158 WH XHHum HC region -
D Y W G O G T S W T V S S A. S T K G P
 US 8,025,878 B2
35 36
TABLE 19 TABLE 2 O

Expression of chimeric 158 antibody in COS cells Amino acid sequence of 158 WH and 158 VK
(SEQ ID NOS : 115-116)
Expression Antibody 5 (SEQ ID NO: 115)
Number of Vector Constructs Concentration VH DVOLVESGGGLVOPGGSRKLSCAASGFTFSSFGMHWVROAPEKGL
Co-transfections Co-Transfected (ng ml) EWVAYISSGSSTIYYGDTVKGRFTISRDNPKNTLFLOMTSLRSED
TAMYYCAREGGYYYGRSYYTMDYWGOGTSVTVSS
2 pooled pG1 D200158 and pKN100158 3OO
2 pooled pG1 D200158 and pKN100158 3700 (SEQ ID NO: 116)
10 VK DVLMTOTPLSLPVSLGDOASISCRSSQSIVHSNGNTYLEWYLOKP
GOSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGI
Legend: Antibody concentration was measured by ELISA in 3-day cultures of transfected YYCFOGSHVPPTFGGGTKLEIK
COS 7 cells. COS cells were co-transformed with 10 ug each of the heavy and light chain
chimeric expression vectors pG1D200158 and pKN100158.

TABL E 21

Best human VH framework. VCI scores compared with 158 VH

Kabat Number 2 24 26 27 28 29 30 37 39 45 47 48 49 6f 69 f1 73 78 91 93 94 103
Canonical Residue - 1 1 1 - 1 - - 2 - - 1 -
Wernier Residue" k k k k k k k k k k k k k k k
Interface Residue” I I I I - - I I - I
Sequence FW WCI
ale S. Coe SCC e WCI Residues

158 WH 87 22 W. A. G. F T S W. Q. L. W. W. A. F I R. N. L. Y. A R W (SEQ ID NO :
7)
386.87 79 22 . (SEQ ID NO :
8)
ABO 2152O 77 22 . (SEQ ID NO :
8)
AJS 56 669 77 22 . (SEQ ID NO :
8)
386.72 77 22 . (SEQ ID NO :
8)
386.73 77 22 . (SEQ ID NO :
8)
DQ322738 77 22 . (SEQ ID NO :
8)
ABO 67108 76 22 . (SEQ ID NO :
8)
ABO 21531 76 22 . (SEQ ID NO :
8)
ABO 21532 76 22 . (SEQ ID NO :
8)
ABO 63892 76 22 . (SEQ ID NO :
8)
ABO 67237 76 22 . (SEQ ID NO :
8)
ABO 215. Of 76 22 . (SEQ ID NO :
8)
AF471.177 76 22 . (SEQ ID NO :
8)

AF471184 76 22 . (SEQ ID NO :
8)

AFO 62243 76 22 . (SEQ ID NO :

8)

AF174 O3 O 76 22 . (SEQ ID NO :
8)

AF4 66141 76 22 . (SEQ ID NO :

8)
 US 8,025,878 B2
39 40
TABLE 22 - continued
Sequences of best VCI-scoring human VH, compared with 158 WH (SEQ ID NOS: 119-135)
AJS 56 669 EDTAWYYCARERGHDFWSIYYTH- - - - - - - - - - - - - - FDYWGOGALWTVSS (SEO ID NO: 121)

DQ322738 EDTAWYYCARDGGSI - - - - - - - - - - - - - - - - - - - - - - FDYWGOGTLWTVSS (SEO ID NO: 122)

ABO 67108 EDTAWYYCARARDYYYYP - - - - - - - - - - - - - - - - - - - MDWWGOGTTWTVSS (SEO ID NO: 123)

ABO 21531 EDTAVYYCARDOSWSRIAAAGTPPSL - - - - - - - - - - - FDPWGOGTLWTVSs (SEO ID NO: 124)

ABO 21532 DEDTAWYYCARARNYYDSSGYS- - - - - - - - - - - - - - - FDYWGOGTLWTVSS (SEO ID NO: 125)

ABO 63892 EDTAWYYCARWRRGS - - - - - - - - - - - - - - - - - - - - - - GDSWGOGTLVTVSS (SEO ID NO: 126)

ABO 67237 EDTAVYYCAREQOLGPHNW- - - - - - - - - - - - - - - - - - FDPWGOGTLWTVSS (SEO ID NO: 127)

ABO 215. Of EDTAVYYCARDOETGTTFDYYYYG- - - - - - - - - - - - - MDWWGOGTTWTVSS (SEO ID NO: 128)

AF471.177 EDTAWYYCARDPMITTWWKPSLAT- - - - - - - - - - - - - - NDYWGOGTLVTVSS (SEO ID NO: 129)

AF471184 EDTAWYYCARDCWGAL.GA- - - - - - - - - - - - - - - - - - - FDIWGOGTMVTVSS (SEO ID NO : 13 O)

AFO 62243 EDTAWYYCARANS - - - - - - - - - - - - - - - - - - - - - - - - LDVWGOGTTWTVSS (SEO ID NO : 131)

AF174 O3 O EDTAWYYCARDGDIGDWW- - - - - - - - - - - - - - - - - - - FDPWGOGTLWTVSS (SEO ID NO : 132)

AF4 66141 EDTAWYYCARDKGYYDYWWGSYRSNPKNDA- - - - - - - FDIWGOGTMVTVSS (SEO ID NO : 133)

AF4 66142 EDTAWYYCARDKGYYDYWWGSYRSNPKNDA- - - - - - - FDIWGOGTMVTVSS (SEO ID NO : 134)

AJ245.279 EDTAWYYCARDRFF- - - - - - - - - - - - - - - - - - - - - - - FDNWGOGTLWTVSS (SEO ID NO : 135)

TABLE 23

Alignment of 158 WH with the best VCI-scoring human VH (SEQ ID NO: 136-147)
2 3 4. 5 6 7 S.
12345 6 F89 O123456789 O12345 6 F89 O123456789 O123456789 O2A3456789 O123456789 O123456789 O2 ABC345 6 F8
(SEO ID NO: 136) DVOLVESGGGLVOPGGSRKLSCAASGFTFSS OAPEKGLEWVAYISS RDNPKNTLFLOMTSLRSEDTA
(SEQ ID NO: 137) E. . . . . . . . . . . . . . . . II . . . . . . . . . . . . . Y& G . . . . . . . N. KO . . A. . S.Y. . .N. . . A. .
(SEQ ID NO: 138) L. . . . . . . . . . . . . . . . IR... . . . . . . . . . . . G . . . . . . . W.W.Y . S. . . . Y. . . N. . . A. .
(SEO ID NO: 139) E. . . . . . . . . W. . . . . . TR . . . . . . . . . . . N G . . . . . . . W.Y . . S. . . . Y. . . N. . . A . .
(SEO ID NO: 14 O. O. . . . . . . . . W. . . . R.T. . . . . . . . . . . . . . G . . . . . . . W.Y. . . S. . . . Y. . . N. . . A . .
(SEQ ID NO: 141) Q. . . . . . . . . W. . . . R.T. . . . . . . . . . . . . . G . . . . . . . W.Y. . . S. . . . Y. . . N. . . A . .
(SEQ ID NO: 142) E. . . . . . . . . . . . . . . . IR... . . . . . . . . . . . G . . . . . . . N. KO . . A. . S.Y. . .N. . . A. .
(SEQ ID NO: 143) E. . . . . . . . . . . . . . . . IR... . . . . . . . . . . . G . . . . . . . N. KO . . A. . S.Y. . .N. . . A. .
(SEQ ID NO: 144) Q. . . . . . . . . W. . . . R.T. . . . . . . . . . . . . . G . . . . . . . W.Y. . . S. . . . Y. . . N. . . A . .
(SEQ ID NO: 145) E. . . . . . . . . . . . . . . . IR... . . . . . . . . . . . G . . . . . . . N. KO . . A. . S.Y. . .N. . . A. .
(SEQ ID NO: 146) E. . . . . . . . . . . . . . . . IR... . . . . . . . . . . . G . . . . . . . N. KO .A. . S.Y. . .N. . . A. .
(SEQ ID NO: 147) E. . . . . . . . . . . . . . . . LR S. . . . . . . TY S . . . . . S. . . A. .

158 WH
ABO 2152O
DQ322738
ABO 67108
ABO 21531
ABO 21532
ABO 63 892
ABO 67237
ABO 21507
AF471.177
AF471184
AFO 62243

Legend: Residues identical to 158 WH are represented by a dot. CDRS are grey-shaded
 US 8,025,878 B2
41 42
TABLEE 24

VH signal peptide selection-5. 5.1 V-gene alignment of 158 WH,

human AFO62243 and human germline M99649 (VH3- 07) (SEQ ID NOS: 148-150)

DVOLVESGGGLVOPGGSRLSCAASGFTFSSFGMHWVROAPEKGLEWVAYIS 158 VH (SEQ ID NO: 148)

E. . . . . . . . . . . . . . . . LR. . . S. . . . . . TYW.T. . . . . . G. . . . . . . N.K AFO 62243 (SEQ ID NO: 149)

E. . . . . . . . . . . . . . . . LR. . . . . . . . . . . YW. S. . . . . . G. . . . . . . N. K. M99649 (SEQ ID NO: 150)

SGSSTIYYGDTVKGRFTISPDNPKNTLFLOMTSLRSEDTAMYYCAR 158 WH (SEQ ID NO: 148)

PHCG.E.A. W. S. . . . . . . . . . . A. . S. . . . . S. . . A. . . . W. . . . . AFO 62243 (SEQ ID NO: 149)

QDG.EK..W. S. . . . . . . . . . . A. . S.Y. . .N. . .A. . . . W. . . . . M99649 (SEQ ID NO: 150)

Legend: W-gene residues identical to 158 WH are represented by a dot.

TABLE 25 TABLE 26

Signal peptide of M99649 human germline VH gene 25

M99649 signal peptide cutting prediction (SEQ ID NO: 153
(SEQ ID NOS : 151-152) # Measure Position Value Cutoff signal peptide?
8X. C 2O O.909 O.32 YES
DNA ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTA 8X Y 2O O.836 O.33 YES
GAAGGTGTCCAGTGT (SEO ID NO : 151) 8X. S 13 O.953 O.87 YES
30 (8. S 1-19 O.859 O.48 YES
D 1-19 O848 O.43 YES
protein MELGLSWVFLVAILEGVQC (SEQ ID NO: 152)
#Highest probability for cleavage is between amino acid residue 19-20: VOC-DV,

TABLE 27
Generation of 158RHA protein sequence (SEQID NOS: 154-163)

1. 2 3 4. 5 6 7
O 12345678901234567890123456789012345AB678 90123456789 O12ABC3456789 O123456789 O

(SEO ID NO: 155 (SEQ ID NO: 156):

158RHA -EVOLVESGGGLVOPGGSLRLSCSASGFTFS -WVROAPGKGLEWVA
AFO 62243 FW (SEQ ID NO: 159) -EVOLVESGGGLVOPGGSLRLSCSASGFTFS WWRQAPGKGLEWVA (SEQ ID NO: 16O) RFTIS
AFO 62243 CEVOLVESGGGLVOPGGSLRLSCSASGFTFSTYWMT--WVROAPGKGLEWVANIKIP--HGSEAYYVDSVKGRFTIS

7 8 9 1O 1.
O123456789 O12ABC34567890123456789 OABCDEFGHIJK123456789 O123

158RHA

AFO 62243 FW RDNAKNSLFLOMSSLRAEDTAVYYCAR (SEO ID NO: 161). WGOGT

AFO 62243 RDNAKNSLFLQMSSLRAEDTAVYYCARANS - - - - - - - - - - - - - LDWWGQGT
 US 8,025,878 B2
45 46
TABL E 3 O

Mutations in 158RHA removing splice sites in 158RHAss

(SEO ID NOS : 175-176)

(SEO ID NO : 175)
ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAGGGAGT 58RHA
(SEO ID NO : 176)
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A. .T. . 58RHAss

CCAGTGCGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTG 58RHA
- - - - - - T. G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58RHAss

10 GGGGGTCCCTGAGACTCTCCTGTTCAGCCTCTGGATTCACCTTTAGTAGC 58RHA
10 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 58RHAss

15 TTTGGAATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGT 58RHA
15 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G. . . . . 58RHAss

GGCCTACATTAGTAGTGGCAGTAGTACCATCTACTATGGAGACACCGTGA 58RHA
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A. . . . 58RHAss

25 AGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTTTCTG 58RHA

25 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 58RHAss

3O CAAATGAGCAGCCTGAGAGCCGAGGACACGGCCGTGTATTATTGTGCGAG 58RHA
3O - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 58RHAss

35 AGAGGGGGGATATTACTACGGAAGGAGTTACTATACTATGGACTACTGGG 58RHA
35 - - - - - - - - - - - - - - - - - - - - - T. . . . . . . . . . . . . . . . . . . . . . . . . . . . 58RHAss

4O GCCAAGGGACCACGGTCACCGTCTCC 58RHA
4O - - - - - - - - - - - - - - - - - - - - - - - - - - 58RHAss

Legend 158RHA DNA sequence Compared to 158RHASS (Table 5. 7. 2) which contains predicted
splice sites. Positions identical to 158RHA are identified as a dot.

TABL E 31

DNA and protein sequence of 158RHA (SEQ ID NOS: 177-178)

(SEO ID NO: 177) ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAGGGAGTCCAGTGCGAAGTGCAGCTGGTGGAGTCTGGG 81
O7 leader e- 158RHA FW1
(SEO ID NO: 178) M E L G L S W V F L. W. A. I L E G W Q C E W Q L W E S G
GAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTTCAGCCTCTGGATTCACCTTTAGTAGCTTTGGAATGCAC 162

G G L W Q. P G G S R L S C S A. S. G. F. T. F. S. S. F. G. M. H.
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTGGCCTACATTAGTAGTGGCAGTAGTACCATCTACTATGGAGAC 243
158RHA FW2 CDR2
W W R O A P G K. G E W W A Y I S S G. S S T I Y Y G D
ACCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTTTCTGCAAATGAGCAGCCTGAGAGCCGAG 324
CDR2 158RHA FW3
T V K G R F T I S R D N A. K. N S L F L Q M S S L R A E
GACACGGCCGTGTATTATTGTGCGAGAGAGGGGGGATATTACTACGGAAGGAGTTACTATACTATGGACTACTGGGGCCAA 4 OS
158RHA FW3 HCDR3 H158RHA E
D. T. A. W. Y Y C A R E G G Y Y Y G R S Y Y T M D Y W G O
GGGACCACGGTCACCGTCTCC 426

G T T W T W S
 US 8,025,878 B2
47 48
TABLEE 32

Best VCI scores of human VK compared with 158 WK

(SEQ ID NOS: 179-182)
Kabat Number 2 4 35 36 .38 44 46 47 48 49 64 66 68 69 71. 87 98
Canonical Residue 1 2 2 1.
Wernier Residue" k k k k k k k k k k k k k
Interface Residue I I I I I I

Fw WCI
Sequence scorescore WCI residues

158 WK 8O 7 VM W Y Q P L. L. I Y G G G T F Y F (SEO ID NO : 179)

ABO 64 O54 71 7 W . . . (SEQ ID NO: 18O

ABO 63934 70 7 W . . . (SEQ ID NO: 18 O)

ABO641. Os 70 7 W . . . (SEQ ID NO: 18O

AY94, 1999 70 7 W . . . (SEQ ID NO: 18 O)

AX805665 69 7 W . . . (SEQ ID NO: 18O

ABO641.04 69 7 W . . . (SEQ ID NO: 18O

AY942O57 69 7 W . . . (SEQ ID NO: 18O

ABO 64 O55 68 7 W . . . (SEQ ID NO: 18O

AX742B74 68 7 W . . . (SEQ ID NO: 18O

AY685343 67 7 W . . . (SEQ ID NO: 18O

AY6853 53 67 7 W . . . (SEQ ID NO: 18O

DO1875 O6 7.O 6 . (SEQ ID NO: 181)

DO1876.79 7O 6 . (SEQ ID NO: 181)

AYO431. Of 69 6 . (SEQ ID NO: 181)

AJ3886.39 69 6 . . . W . (SEQ ID NO: 181)

AJ38864 6 69 6 . (SEQ ID NO: 181)

AJ388642 69 6 . (SEQ ID NO: 181)

M7447O 69 6 . (SEQ ID NO: 181)

X724 66 69 6 . (SEQ ID NO: 181)

U95244 69 6 . (SEQ ID NO: 181)

AAA51016 69 6 . (SEQ ID NO: 181)

X890.54 69 6 . (SEQ ID NO: 181)

DO1875 O5 69 6 . (SEQ ID NO: 181)

DQ187683 69 6 . (SEQ ID NO: 181)

DQ187691 69 6 . (SEQ ID NO: 181)

AX805669 68 6 . (SEQ ID NO: 181)

AF4555 62 68 6 . (SEQ ID NO: 181)

Legend: Canonical residues are numbered in this table according to which CDR they are associated.
FW score and VCI score are the number of residues in the FW or VCI definition respectively, which are
identical to their Counterpart in 158.
Residues identical to 158 WK are indicated by a dot.
 US 8,025,878 B2
55 56
TABLEE 35

WK signal peptide selection - Alignment of 158 WK

with human ABO64054 and human germline A19 (SEQ ID NOS: 234-236)
DVLMTOTPLSLPWSLGDOASISCRSSOSIVHSNGNTYLEWYLOKPGOSPKLLIYKVSN 158 WK (SEQ ID NO: 234)

.W. . . S. . . . . . TPAP. . . . . . . . . LL.T. . . WNF.D. . . . . . . . . . . . . . . . L.A.H ABO 64 O54 (SEQ ID NO: 235)

. IV. . . S. . . . . . TP. EP. . . . . . . . . LL. . . . YN . . D. . . . . . . . . . . Q. . . . IG. . A19 (SEQ ID NO: 236)

RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFOGSHVPPTFGGGTKLEIK 158 WK (SEQ ID NO: 234)

. A . . . . . . . . . . . . . . . . . . R. . . . . . . . W. . . . . M. . LOT. F. . .P. . . . . . N ABO 64 O54 (SEQ ID NO: 235)

. A . . . . . . . . . . . . . . . . . . . . . . . . . . . W.W. . . M. ALOT. A19 (SEQ ID NO: 236)

2O
TABLE 36 TABLE 37

Signal peptide of human A19 A19 signal peptide cutting prediction (SEQ ID NO. 239
(WK2-28; X63397) germlie VK (SEQ ID NOS: 237-238) >Sequence length = 50
25 re?
VK A19 leader sequence # Measure Position Value Cutoff signal peptide:
8X. C 21 O.853 O.32 YES
DNA ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGG max. Y 21 O.831 O.33 YES
GTCTCTGGATCCAGTGGG (SEO ID NO. 237) 8X. S 13 O.990 O.87 YES
(8. S 1-20 O.932 O.48 YES
30 D 1-2O O.881 O.43 YES
protein MRLPAQLLGLLMLWVSGSSG (SEQ ID NO: 238)
# Most likely cleavage site between pos, 20 and 21; SSG-DV

TABLE 38

Generation of 158RKA Protein Sequence (SEQID NOS: 240-249)

1. 2 3 4. 5 6 7
1234567 O90123456789 O1234567 ABCDEF89 O123456789 O123456789 O123456789 O123456789 O

SEQ ID NO: 241 (SEQ ID NO: 242)

158RHA DVVMTQSPLSLPVTPGAPASISC YLQKPGQSPKLLIYE SGVPDRFSGSGSGTD
FW (SEO ID NO: 245) DVVMTOSPLSLPWTPGAPASISC (SEO ID NO: 246) WYLOKPGOSPKLLIY GWPDRFSGSGSGTDF
ABO 64 O54 DVVMTOSPLSLPWTPGAPASISCRSSOSLLHT-NGVNFLDWYLOKPGOSPKLLIYLASHRASGVPDRFSGSGSGTD

8 9 1O
123456789 O123456789012345ABCDEF678 901234567
L3
(SEQ ID NO:
(SEQ ID NO:
FTLRISRWEAEDWGIYY FGPGTKLEIK (SEQ ID NO :
TLRISRVEAEDVGIYYC (SEO ID NO: 247) FGPGTKLEIK (SEO ID NO :
ABO 64 O54 FTLRISRVEAEDVGIYYCMOGLOTP- - - - - - FTFGPGTKLEIK (SEO ID NO: 249)

Legend: 158 WK CDRs intercalated in human ABO 64054 FW to generate 158RKA

 US 8,025,878 B2
59 60
TABLE 41 - continued

Mutations in 158RKA removing splice sites in 158RKA (SEQ ID NOS: 261-262)

121 ATCTCCTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAGTGG 58RKA

121 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. . . 58RKAss

181 TATCTTCAAAAGCCAGGGCAGTCTCCAAAGCTCCTGATCTATAAAGTTTCCAACCGATTT 58RKA

181 . . . . . G. G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58RKAss

241 TCTGGAGTCCCTGACAGGTTCAGTGGAAGTGGATCAGGCACAGATTTTACACTGAGAATC 58RKA

241 . . . . . . . . . . . . . . . . . . . . . . . . . . C. . . . . G. . . . . . . . . . . . . . . . . . . . . . . . . . . 58RKAss

301 AGCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCT 58RKA

301 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58RKAss

361 CCGACGTTCGGCCCTGGGACCAAATTGGAAATCAAA 58RKA

361 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58RKAss

Legend: 158RKA DNA sequence Compared to 158RKASS (Table 5.13. 2) which contains predicted splice sites. Residues identical to
158RKA are identified by a dot.

TABL E 42

DNA and protein sequence of 158RKA (SEQ ID NOS: 263-264)

(SEO ID NO: 263) ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTGGAAGCAGTGGGGATGTTGTGATGACTCAGTCT 81
A19 leader sequence He- 158RHA FW1
(SEO ID NO: 264) M. R. L. P. A. Q. L. L. G. L. L. M. L. W. V S G S S G D V V M T Q S
CCACTCTCCCTGCCCGTCACCCCTGGAGCGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGA. 162
158RHA FW1 CDR1

P L S L P W T P G A P A S I S C R S S Q S I V H S N G
AACACCTATTTAGAGTGGTATCTTCAAAAGCCAGGGCAGTCTCCAAAGCTCCTGATCTATAAAGTTTCCAACCGATTTTCT 243
CDR1-- 158RHA FW2 > CDR2 ce
N T Y L E W Y L Q K P G O S P K L L I Y K V S N R F S
GGAGTCCCTGACAGGTTCAGTGGAAGTGGATCAGGCACAGATTTTACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTT 324
158RHA FW3
G W P D R F S. G. S. G. S G T D F T L R I, S R W E A E D W
GGAATTTATTACTGCTTTCAAGGTTCACATGTTCCTCCGACGTTCGGCCCTGGGACCAAATTGGAAATCAAA 396
158RHA FW3 CDR3 e- 158RHA FW4
G I Y Y C F O G S H V P P T F G P G T K L E I K

TABL E 43

Further version of humanized 158WHA (158RHB, 158RHC, 158RHD) (SEQ ID NO: 265-268)
Kabat 1. 2 3 4. 5 6 7 8
Number 123456789 O123456789 O123456789 O123456789 O123456789 O12A3456789012345678901234567890 12ABC3456789
158RHA EVOLVESGGGLVOPGGSLRLSCSASGFTFSSFGMHWVROAPGKGLEWVAYISSGSSTIYYSDTVKGRFTISRDNAKNSL FLOMSSLRAEDTAV
158RHB EVOLVESGGGLVOPGGSLRLSCSASGFTFSSFGMHWVROAPGKGLEWVAYISSGSSTIYYGDTVKGRFTISRDNAKNSL FLOMSSLRAEDTAV
158RHC EVOLVESGGGLVOPGGSLRLSCSASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTIYYGDTVKGRFTISRDNRKNSL FLOMSSLRAEDTAV
158RHD EVOLVESGGGLVQPGGSLRLSCSASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTIYYGDTVKGRFTISRDNAKNSLFLQMESLRAEDTAV

Kabat 9 10 11
Number O123456789 OABCDEFW123456789 O123
158RHA YYCAREGGYYYGRSYYTMDYWGOGTTWTVS (SEO ID NO: 265)
158RHB YYCAREGGYYYGRSYYTMDYWGOGTTWTVS (SEO ID NO: 266)
158RHC YYCAREGGYYYGRSYYTMDYWGOGTTWTVS (SEO ID NO: 267)
158RHD YYCAREGGYYYGRSYYTMDYWGOGTTWTVS (SEO ID NO: 268)
 US 8,025,878 B2
61 62
TABLE 44

Further version of humanized 158VKA (158RKB,158RKC) (SEQID NO: 269-271)

Kabat 1. 2 3 4. 5 6 7 8
Number 123456789 O123456789 O1234567 ABCDE89012345678901234567890123456789012345678901234567890123456789
158RKA DVVMTOSPLSLPVTPGAPASISCRSSOSIVHSNGNTYLEWYLOKPGOSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCF
158RKB DVEMTQSPLSLPVTPGAPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCF
158RKC DVVMTOSPLSLPVTPGAPASISCRSSOSIVHSNGNTYLEWYLOKPGOSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCF
Kabat 9 10 11
Number O123456789 OABCDEFW123456789 O123
158RHA YYCAREGGYYYGRSYYTMDYWGOGTTWTVS (SEO ID NO: 265)
158RHB YYCAREGGYYYGRSYYTMDYWGOGTTWTVS (SEO ID NO: 266)
158RHC YYCAREGGYYYGRSYYTMDYWGOGTTWTVS (SEO ID NO: 267)
158RHD YYCAREGGYYYGRSYYTMDYWGOGTTWTVS (SEO ID NO: 268)

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 271

<210s, SEQ ID NO 1
&211s LENGTH: 5
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
<4 OOs, SEQUENCE: 1

Ser Phe Gly Met His

1. 5

<210s, SEQ ID NO 2
&211s LENGTH: 17
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
<4 OOs, SEQUENCE: 2

Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val Lys
1. 5 1O 15

Gly

<210s, SEQ ID NO 3
&211s LENGTH: 15
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
<4 OOs, SEQUENCE: 3
Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp Tyr
1. 5 1O 15

<210s, SEQ ID NO 4
&211s LENGTH: 16
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
<4 OOs, SEQUENCE: 4

Arg Ser Ser Glin Ser Ile Val His Ser Asn Gly Asn Thr Tyr Lieu. Glu
1. 5 1O 15
 US 8,025,878 B2
63 64
- Continued

<210s, SEQ ID NO 5
&211s LENGTH: 7
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
<4 OOs, SEQUENCE: 5

Llys Val Ser Asn Arg Phe Ser

1. 5

<210s, SEQ ID NO 6
&211s LENGTH: 9
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus region
<4 OOs, SEQUENCE: 6

Phe Glin Gly Ser His Val Pro Pro Thr

1. 5

<210s, SEQ ID NO 7
&211s LENGTH: 16
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
<4 OO > SEQUENCE: 7

Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg
1. 5 1O 15

<210s, SEQ ID NO 8
&211s LENGTH: 23
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
<4 OOs, SEQUENCE: 8
Trp Val Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp
1. 5 1O 15

Thr Val Lys Gly Arg Phe Thr

2O

<210s, SEQ ID NO 9
&211s LENGTH: 21
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
<4 OOs, SEQUENCE: 9
Cys Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met
1. 5 1O 15

Asp Tyr Trp Gly Glin

2O

<210s, SEQ ID NO 10
&211s LENGTH: 22
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
 US 8,025,878 B2
65
- Continued
<4 OOs, SEQUENCE: 10

Ile Ser Cys Arg Ser Ser Glin Ser Ile Val His Ser Asn Gly Asn Thr
1. 5 1O 15

Tyr Lieu. Glu Trp Tyr Lieu.

2O

<210s, SEQ ID NO 11
&211s LENGTH: 13
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
<4 OOs, SEQUENCE: 11

Lieu. Ile Tyr Llys Val Ser Asn Arg Phe Ser Gly Val Pro
1. 5 1O

<210s, SEQ ID NO 12
&211s LENGTH: 15
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody consensus sequence
<4 OOs, SEQUENCE: 12

Tyr Tyr Cys Phe Glin Gly Ser His Val Pro Pro Thr Phe Gly Gly
1. 5 1O 15

<210s, SEQ ID NO 13
&211s LENGTH: 117
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 13

Glu Val Lys Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 1O 15

Ser Arg Llys Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
2O 25 3O

Gly Met His Trp Val Arg Glin Ala Pro Glu Lys Gly Lieu. Glu Trp Val
35 4O 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Pro Lys Asn. Thir Lieu. Phe
65 70 7s 8O

Lieu Gln Met Thr Ser Lieu. Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95

Ala Arg Tyr Gly Asn Tyr Ala Met Asp Tyr Trp Gly Glin Gly Thr Ser
1OO 105 11 O

Wall. Thir Wal Ser Ser

115

<210s, SEQ ID NO 14
&211s LENGTH: 119
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 14

Glu Val Lys Lieu Val Glu Ser Gly Gly Gly Lieu Val Llys Pro Gly Gly
1. 5 1O 15
 US 8,025,878 B2
67
- Continued

Ser Leu Lys Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
2O 25 3O

Ala Met Ser Trp Val Arg Glin Thr Pro Glu Lys Arg Lieu. Glu Trp Val
35 4O 45

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn. Thir Lieu. Tyr
65 70 7s 8O

Lieu Gln Met Ser Ser Lieu. Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95

Ala Arg Asn Tyr Gly Ser Arg Arg Tyr Phe Asp Val Trp Gly Ala Gly
1OO 105 11 O

Thir Ser Wall. Thir Wal Ser Ser

115

<210s, SEQ ID NO 15
&211s LENGTH: 121
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 15

Glin Val His Lieu. Glin Glin Ser Gly Pro Glu Lieu Val Llys Pro Gly Ala
1. 5 1O 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr
2O 25 3O

Val Met His Trp Val Lys Gln Llys Pro Gly Glin Gly Lieu. Glu Trp Ile
35 4O 45

Gly Tyr Ile Asin Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
SO 55 6O

Lys Gly Lys Ala Thr Lieu. Thir Ser Asp Llys Ser Ser Ser Thir Ala Tyr
65 70 7s 8O

Trp. Glu Lieu Ser Ser Lieu. Thir Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95

Ala Arg Arg Val Ser Pro Leu. Thir Ser Tyr Ala Met Asp Tyr Trp Gly
1OO 105 11 O

Gln Gly. Thir Ser Val Thr Val Ser Ser

115 12 O

<210s, SEQ ID NO 16
&211s LENGTH: 119
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 16

Glin Val Glin Leu Lys Glu Ser Gly Pro Gly Lieu Val Ala Pro Pro Glin
1. 5 1O 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Lieu. Thir Ser Tyr
2O 25 3O

Gly Val His Trp Val Arg Glin Pro Pro Gly Lys Gly Lieu. Glu Trp Lieu.
35 4O 45

Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met
SO 55 6O

Ser Arg Lieu. Ser Ile Ser Lys Asp Asn. Ser Lys Ser Glin Val Phe Lieu.
65 70 7s 8O
 US 8,025,878 B2
69 70
- Continued

Lys Met Asn Ser Lieu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95

Arg Gly Arg Tyr Asp Gly Llys Thr Arg Phe Ala Tyr Trp Gly Glin Gly
105 11 O

Thir Lieu Val Thir Wal Ser Ser

115

SEO ID NO 17
LENGTH: 115
TYPE : PRT
ORGANISM: Artificial
FEATURE:
OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 17
Glu Val Llys Lieu. Met Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 15

Ser Arg Lys Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Phe
25

Gly Met His Trp Wall Arg Glin Ala Pro Glu Gly Lell Glu Trp Wall
35 4O 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thir Ile Tyr Ala Asp Thir Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Pro Asn Thir Luell Phe
65 70 7s

Lell Glin Met Thir Ser Lell Arg Ser Glu Asp Thir Ala Met Tyr Cys
85 90 95

Ala Arg Gly Asp Ser Phe Asp Trp Gly Glin Gly Thir Thir Luell Thir
1OO 105 11 O

Wall Ser Ser

115

SEQ ID NO 18
LENGTH: 124
TYPE : PRT
ORGANISM: Artificial
FEATURE:
OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 18
Glu Val Glin Arg Wall Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 15

Ser Arg Lys Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Phe
2O 25

Gly Met His Trp Wall Arg Glin Ala Pro Glu Gly Lell Glu Trp Wall
35 4O 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thir Ile Tyr Gly Asp Thir Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Pro Asn Thir Luell Phe
65 70

Lell Glin Met Thir Ser Lell Arg Ser Glu Asp Thir Ala Met Tyr Cys
85 90 95

Ala Arg Glu Gly Gly Gly Arg Ser Thir Met Asp
105 11 O

Trp Gly Glin Gly Thir Ser Wall Thir Wall Ser Ser
115 12 O

<210s, SEQ ID NO 19
 US 8,025,878 B2
71
- Continued
&211s LENGTH: 112
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 19

Asp Val Val Met Thr Glin Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1. 5 1O 15

Asp Glin Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Ile Val His Ser
2O 25 3O

Asn Gly Asn. Thir Tyr Lieu. Glu Trp Tyr Lieu. Glin Llys Pro Gly Lys Ser
35 4O 45

Pro Llys Lieu. Lieu. Ile Tyr Llys Val Ser Asn Arg Phe Ser Gly Val Pro
SO 55 6O

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile
65 70 7s 8O

Ser Arg Val Glu Ala Glu Asp Lieu. Gly Val Tyr Tyr Cys Phe Glin Gly
85 90 95

Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile Llys
1OO 105 11 O

<210s, SEQ ID NO 2 O
&211s LENGTH: 107
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 2O

Asp Ile Val Met Thr Glin Ala Pro Llys Phe Lieu. Lieu Val Ser Ala Gly
1. 5 1O 15

Asp Arg Val Thir Ile Thr Cys Lys Ala Ser Glin Ser Val Ser Asn Asp
2O 25 3O

Val Ala Trp Tyr Glin Gln Llys Pro Gly Glin Ser Pro Llys Lieu. Lieu. Ile
35 4O 45

Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
SO 55 6O

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Glin Ala
65 70 7s 8O

Glu Asp Leu Ala Val Tyr Phe Cys Glin Glin Asp Tyr Ser Ser Pro Phe
85 90 95

Thr Phe Gly Ser Gly Thr Lys Lieu. Glu Ile Llys
1OO 105

<210s, SEQ ID NO 21
&211s LENGTH: 112
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 21

Asp Ile Val Met Thr Glin Ala Pro Ser Ser Leu Ala Val Ser Ala Gly
1. 5 1O 15

Glu Lys Val Thr Met Ser Cys Llys Ser Ser Glin Ser Lieu. Lieu. Asn Ser
2O 25 3O

Arg Thr Arg Lys Asn Tyr Lieu Ala Trp Tyr Glin Gln Llys Pro Gly Glin
35 4O 45

Ser Pro Llys Lieu. Lieu. Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
 US 8,025,878 B2
73 74
- Continued
SO 55 6O

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thir Luell Thir
65 70 8O

Ile Ser Ser Val Glin Ala Glu Asp Lieu Ala Val Tyr Tyr Cys Lys Glin
85 90 95

Ser Tyr Asn Lieu. Trp Thr Phe Gly Gly Gly Thr Lys Lieu Glu Ile Lys
105 11 O

<210s, SEQ ID NO 22
&211s LENGTH: 108
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223s OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 22
Glu Asn. Wall Lieu. Thir Glin Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1. 5 1O 15

Glu Lys Val Thr Asn Thr Cys Arg Ala Ser Ser Ser Wall Ser Ser Ser
2O 25

Tyr Lieu. His Trp Tyr Glin Gln Lys Ser Gly Ala Ser Pro Luell Trp
35 4O 45

Ile Tyr Ser Thr Ser Asn Lieu. Ala Ser Gly Wall Pro Ala Arg Phe Ser
SO 55 6O

Gly Ser Gly Ser Gly Thr Ser Ser Luell Thir Ile Ser Ser Wall Glu
65 70

Ala Glu. Asp Ala Ala Thr Tyr Glin Glin Ser Gly Tyr Pro
85 90 95

Lieu. Thir Phe Gly Ala Gly Thr Luell Glu Luell
1OO 105

<210s, SEQ ID NO 23
&211s LENGTH: 112
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223s OTHER INFORMATION: Antibody variable region
<4 OOs, SEQUENCE: 23

Asp Ile Val Met Thr Glin Ala Pro Luell Ser Luell Pro Wall Ser Luell Gly
1. 5 15

Asp Glin Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Lell Wall His Ser
2O 25

Asn Gly Asn Thr Tyr Lieu. His Trp Luell Glin Lys Pro Gly Glin Ser
35 4O 45

Lieu Lys Lieu. Lieu. Ile Tyr Lys Wall Ser Asn Arg Phe Ser Gly Wall Pro
SO 55 6O

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thir Asp Phe Thir Luell Ile
65 70

Ser Arg Val Glu Ala Glu Asp Luell Gly Wall Tyr Phe Ser Glin Ser
85 90 95

Thir His Wall Pro Lieu. Thir Phe Gly Ala Gly Thir Lell Glu Luell Lys
1OO 105 11 O

<210s, SEQ ID NO 24
&211s LENGTH: 112
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223s OTHER INFORMATION: Antibody variable region
 US 8,025,878 B2
75 76
- Continued

<4 OOs, SEQUENCE: 24

Asp Ile Val Met Thr Glin Ala Pro Leu Ser Leu Pro Val Ser Leu Gly
1. 5 1O 15

Asp Glin Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Ile Val His Ser
2O 25 3O

Asn Gly Asn Thr Tyr Lieu. Glu Trp Tyr Lieu Gln Lys Pro Gly Glin Ser
35 4O 45

Pro Llys Lieu. Lieu. Ile Tyr Llys Val Ser Asn Arg Phe Ser Gly Val Pro
SO 55 6O

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile
65 70 7s 8O

Ser Arg Val Glu Ala Glu Asp Lieu. Gly Ile Tyr Tyr Cys Phe Glin Gly
85 90 95

Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile Llys
1OO 105 11 O

<210s, SEQ ID NO 25
&211s LENGTH: 16
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VH CDR1
<4 OOs, SEQUENCE: 25

Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg
1. 5 1O 15

<210s, SEQ ID NO 26
&211s LENGTH: 16
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VH CDR1 (Substitutions)
<4 OOs, SEQUENCE: 26

Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg
1. 5 1O 15

<210s, SEQ ID NO 27
&211s LENGTH: 23
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VH CDR2
<4 OOs, SEQUENCE: 27
Trp Val Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp
1. 5 1O 15

Thr Val Lys Gly Arg Phe Thr

2O

<210s, SEQ ID NO 28
&211s LENGTH: 23
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VH CDR2 (Substitutions)
<4 OOs, SEQUENCE: 28

Trp Val Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp
1. 5 1O 15

Thr Val Lys Gly Arg Phe Thr

 US 8,025,878 B2
77 78
- Continued

<210s, SEQ ID NO 29
&211s LENGTH: 23
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VH CDR2 (Substitutions)
<4 OOs, SEQUENCE: 29

Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp
1. 5 1O 15

Ser Val Lys Gly Arg Phe Thr

2O

<210s, SEQ ID NO 3 O
&211s LENGTH: 21
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VH CDR3
<4 OOs, SEQUENCE: 30
Cys Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met
1. 5 1O 15

Asp Tyr Trp Gly Glin

2O

<210s, SEQ ID NO 31
&211s LENGTH: 14
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VH CDR3 (Substitutions)
<4 OOs, SEQUENCE: 31
Cys Ala Arg Tyr Gly Asn Tyr Ala Met Asp Tyr Trp Gly Glin
1. 5 1O

<210s, SEQ ID NO 32
&211s LENGTH: 16
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VH CDR3 (Substitutions)
<4 OOs, SEQUENCE: 32
Cys Ala Arg Asn Tyr Gly Ser Arg Arg Tyr Phe Asp Val Trp Gly Ala
1. 5 1O 15

<210s, SEQ ID NO 33
&211s LENGTH: 22
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VL CDR1
<4 OOs, SEQUENCE: 33

Ile Ser Cys Arg Ser Ser Glin Ser Ile Val His Ser Asn Gly Asn Thr
1. 5 1O 15

Tyr Lieu. Glu Trp Tyr Lieu.

2O

<210s, SEQ ID NO 34
&211s LENGTH: 17
212. TYPE: PRT
 US 8,025,878 B2
79 80
- Continued
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VL CDR1 (Substitutions)
<4 OOs, SEQUENCE: 34

Ile Thr Cys Lys Ala Ser Glin Ser Val Ser Asn Asp Val Ala Trp Tyr
1. 5 1O 15

Glin

<210s, SEQ ID NO 35
&211s LENGTH: 13
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VL CDR2
<4 OOs, SEQUENCE: 35

Lieu. Ile Tyr Llys Val Ser Asn Arg Phe Ser Gly Val Pro
1. 5 1O

<210s, SEQ ID NO 36
&211s LENGTH: 13
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VL CDR2 (Substitutions)
<4 OOs, SEQUENCE: 36
Lieu. Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro
1. 5 1O

<210s, SEQ ID NO 37
&211s LENGTH: 15
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VL CDR3
<4 OO > SEQUENCE: 37

Tyr Tyr Cys Phe Glin Gly Ser His Val Pro Pro Thr Phe Gly Gly
1. 5 1O 15

<210s, SEQ ID NO 38
&211s LENGTH: 15
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Antibody 158 VL CDR3 (Substitutions)
<4 OOs, SEQUENCE: 38

Tyr Phe Cys Glin Glin Asp Tyr Ser Ser Pro Phe Thr Phe Gly Ser
1. 5 1O 15

<210s, SEQ ID NO 39
&211s LENGTH: 70
212. TYPE: PRT
<213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 39

Asp Ile Val Met Thr Glin Ala Pro Leu Ser Leu Pro Val Ser Leu Gly
1. 5 1O 15

Asp Glin Ala Ser Ile Ser Cys Trp Tyr Lieu. Glin Llys Pro Gly Glin Ser
2O 25 3O

Pro Llys Lieu. Lieu. Ile Tyr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
35 4O 45
 US 8,025,878 B2
81 82
- Continued
Ser Gly Thr Asp Phe Thir Lieu Lys Ile Ser Arg Val Glu Ala Glu Asp
SO 55 6O

Lieu. Gly Ile Tyr Tyr Cys

65 70

<210s, SEQ ID NO 4 O
&211s LENGTH: 70
212. TYPE: PRT
&213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 4 O

Asp Ile Val Met Thr Glin Ala Pro Luell Ser Luell Pro Val Ser Leu Gly
1. 5 1O 15

Asp Glin Ala Ser Ile Ser Trp Tyr Luell Glin Llys Pro Gly Glin Ser
2O 25 3O

Pro Llys Lieu. Lieu. Ile Gly Wall Pro Asp Arg Phe Ser Gly Ser Gly
35 4O 45

Ser Gly Thr Asp Phe Thir Lell Ile Ser Arg Val Glu Ala Glu Asp
SO 55 6O

Lieu. Gly Ile Tyr Tyr Cys

65 70

<210s, SEQ ID NO 41
&211s LENGTH: 70
212. TYPE: PRT
&213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 41

Asp Val Val Met Thr Glin Thir Pro Luell Ser Luell Pro Val Ser Leu Gly
1. 5 1O 15

Asp Glin Ala Ser Ile Ser Trp Tyr Luell Glin Llys Pro Gly Glin Ser
2O 25 3O

Pro Llys Lieu. Lieu. Ile Gly Wall Pro Asp Arg Phe Ser Gly Ser Gly
35 4O 45

Ser Gly Thr Asp Phe Thir Lell Ile Ser Arg Val Glu Ala Glu Asp
SO 55 6O

Lieu. Gly Val Tyr Tyr Cys

65 70

<210s, SEQ ID NO 42
&211s LENGTH: 70
212. TYPE: PRT
&213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 42

Asp Ile Val Met Thr Glin Ala Pro Lys Phe Luell Lieu Val Ser Ala Gly
1. 5 1O 15

Asp Arg Val Thir Ile Thir Trp Tyr Glin Glin Llys Pro Gly Glin Ser
2O 25 3O

Pro Llys Lieu. Lieu. Ile Gly Wall Pro Asp Arg Phe Thr Gly Ser Gly
35 4O 45

Tyr Gly Thr Asp Phe Thir Phe Thir Ile Ser Thir Val Glin Ala Glu Asp
SO 55 6O

Lieu Ala Val Tyr Phe Cys

65 70

<210s, SEQ ID NO 43
&211s LENGTH: 70
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
 US 8,025,878 B2
83
- Continued

<4 OOs, SEQUENCE: 43

Asp Ile Val Met Thr Glin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1. 5 1O 15

Glu Pro Ala Ser Ile Ser Cys Trp Tyr Lieu Gln Lys Pro Gly Glin Ser
2O 25 3O

Pro Gln Leu Lieu. Ile Tyr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
35 4O 45

Ser Gly Thr Asp Phe Thr Lieu Lys Ile Ser Arg Val Glu Ala Glu Asp
SO 55 6O

Val Gly Val Tyr Tyr Cys

65 70

<210s, SEQ ID NO 44
&211s LENGTH: 70
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 44

Asp Ile Val Met Thr Glin Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1. 5 1O 15

Glu Pro Ala Ser Ile Ser Cys Trp Tyr Lieu Gln Lys Pro Gly Glin Ser
2O 25 3O

Pro Gln Leu Lieu. Ile Tyr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
35 4O 45

Ser Gly Thr Asp Phe Thr Lieu Lys Ile Ser Arg Val Glu Ala Glu Asp
SO 55 6O

Val Gly Val Tyr Tyr Cys

65 70

<210s, SEQ ID NO 45
&211s LENGTH: 70
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 45

Asp Ile Val Met Thr Glin Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1. 5 1O 15

Gln Pro Ala Ser Ile Ser Cys Trp Tyr Lieu Gln Lys Pro Gly Glin Ser
2O 25 3O

Pro Gln Leu Lieu. Ile Tyr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
35 4O 45

Ser Gly Thr Asp Phe Thr Lieu Lys Ile Ser Arg Val Glu Ala Glu Asp
SO 55 6O

Val Gly Val Tyr Tyr Cys

65 70

<210s, SEQ ID NO 46
&211s LENGTH: 70
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 46

Asp Ile Val Met Thr Glin Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1. 5 1O 15

Gln Pro Ala Ser Ile Ser Cys Trp Tyr Lieu Gln Lys Pro Gly Glin Pro
2O 25 3O

Pro Gln Leu Lieu. Ile Tyr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
35 4O 45
 US 8,025,878 B2
85 86
- Continued

Ser Gly Thr Asp Phe Thr Lieu Lys Ile Ser Arg Val Glu Ala Glu Asp
SO 55 6O

Val Gly Val Tyr Tyr Cys

65 70

<210s, SEQ ID NO 47
&211s LENGTH: 70
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 47

Asp Val Val Met Thr Glin Ser Pro Luell Ser Luell Ser Val Thr Lieu. Gly
1. 5 1O 15

Glin Pro Ala Ser Ile Ser Trp Phe Glin Glin Arg Pro Gly Glin Ser
2O 25 3O

Pro Arg Arg Lieu. Ile Tyr Gly Wall Pro Asp Arg Phe Ser Gly Ser Gly
35 4O 45

Ser Gly Thr Asp Phe Thir Lell Ile Ser Arg Val Glu Ala Glu Asp
SO 55 6O

Val Gly Val Tyr Tyr Cys

65 70

<210s, SEQ ID NO 48
&211s LENGTH: 71
212. TYPE: PRT
&213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 48

Glu Val Lys Lieu Met Glu Ser Gly Gly Gly Lieu. Val Glin Pro Gly Gly
1. 5 1O 15

Ser Arg Llys Lieu. Ser Ala Ala Ser Trp Val Arg Glin Ala Pro Glu
2O 25 3O

Lys Gly Lieu. Glu Trip Wall Ala Arg Phe Thir Ile Ser Arg Asp Asin Pro
35 4O 45

Lys Asn Thr Lieu Phe Lell Glin Met Thir Ser Luell Arg Ser Glu Asp Thr
SO 55 6O

Ala Met Tyr Tyr Cys Ala Arg

65 70

<210s, SEQ ID NO 49
&211s LENGTH: 71
212. TYPE: PRT
&213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 49

Glu Val Lys Lieu Met Glu Ser Gly Gly Gly Lieu. Val Glin Pro Gly Gly
1. 5 1O 15

Ser Arg Llys Lieu. Ser Ala Ala Ser Trp Val Arg Glin Ala Pro Glu
2O 25 3O

Lys Gly Lieu. Glu Trip Wall Ala Arg Phe Thir Ile Ser Arg Asp Asin Pro
35 4O 45

Lys Asn Thr Lieu Phe Lell Glin Met Thir Ser Luell Arg Ser Glu Asp Thr
SO 55 6O

Ala Met Tyr Tyr Cys Ala Arg

65 70

<210s, SEQ ID NO 50
&211s LENGTH: 71
212. TYPE: PRT
 US 8,025,878 B2
87
- Continued
<213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 50

Glu Val Lys Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 1O 15

Ser Arg Llys Lieu. Ser Cys Ala Ala Ser Trp Val Arg Glin Ala Pro Glu
2O 25 3O

Lys Gly Lieu. Glu Trp Val Ala Arg Phe Thir Ile Ser Arg Asp Asn Pro
35 4O 45

Lys Asn Thr Lieu Phe Leu Gln Met Thr Ser Lieu. Arg Ser Glu Asp Thr
SO 55 6O

Ala Met Tyr Tyr Cys Ala Arg

65 70

<210s, SEQ ID NO 51
&211s LENGTH: 71
212. TYPE: PRT
<213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 51

Glu Val Lys Lieu Val Glu Ser Gly Gly Gly Lieu Val Llys Pro Gly Gly
1. 5 1O 15

Ser Lieu Lys Lieu. Ser Cys Ala Ala Ser Trp Val Arg Glin Thr Pro Glu
2O 25 3O

Lys Arg Lieu. Glu Trp Val Ala Arg Phe Thir Ile Ser Arg Asp Asn Ala
35 4O 45

Lys Asn Thr Lieu. Tyr Lieu Gln Met Ser Ser Lieu. Arg Ser Glu. Asp Thr
SO 55 6O

Ala Met Tyr Tyr Cys Ala Arg

65 70

<210s, SEQ ID NO 52
&211s LENGTH: 71
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 52

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Trp Val Arg Glin Ala Pro Gly
2O 25 3O

Lys Gly Lieu. Glu Trp Val Ala Arg Phe Thir Ile Ser Arg Asp Asn Ala
35 4O 45

Lys Asn. Ser Lieu. Tyr Lieu Gln Met Asn. Ser Lieu. Arg Ala Glu Asp Thr
SO 55 6O

Ala Val Tyr Tyr Cys Ala Arg

65 70

<210s, SEQ ID NO 53
&211s LENGTH: 71
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 53

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu. Ile Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Trp Val Arg Glin Ala Pro Glu
2O 25 3O

Lys Gly Lieu. Glu Trp Val Ser Arg Phe Thir Ile Ser Arg Asp Asn. Ser
 US 8,025,878 B2
89 90
- Continued
35 4O 45

Lys Asn. Thir Lieu. Tyr Lieu Gln Met Asn. Ser Lieu. Arg Ala Glu Asp Thr
SO 55 6O

Ala Val Tyr Tyr Cys Ala Arg

65 70

<210s, SEQ ID NO 54
&211s LENGTH: 71
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 54

Glu Val Glin Lieu. Lieu. Glu Ser Gly Gly Gly Lieu. Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Trp Val Arg Glin Ala Pro Gly
2O 25 3O

Lys Gly Lieu. Glu Trp Val Ser Arg Phe Thir Ile Ser Arg Asp Asn Ser
35 4O 45

Lys Asn Thr Lieu. Tyr Lieu Gln Met Asn Ser Luell Arg Ala Glu Asp Thir
SO 55 6O

Ala Val Tyr Tyr Cys Ala Lys

65 70

<210s, SEQ ID NO 55
&211s LENGTH: 71
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 55

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu. Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Trp Val Arg Glin Ala Pro Gly
2O 25 3O

Lys Gly Lieu. Glu Trp Val Ser Arg Phe Thir Ile Ser Arg Asp Asn Ala
35 4O 45

Lys Asn Thr Lieu. Tyr Lieu Gln Met Asn Ser Luell Arg Asp Glu Asp Thir
SO 55 6O

Ala Val Tyr Tyr Cys Ala Arg

65 70

<210s, SEQ ID NO 56
&211s LENGTH: 71
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 56

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu. Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Trp Val Arg Glin Ala Pro Gly
2O 25 3O

Lys Gly Lieu Val Trp Val Ser Arg Phe Thir Ile Ser Arg Asp Asn Ala
35 4O 45

Lys Asn Thr Lieu. Tyr Lieu Gln Met Asn Ser Luell Arg Ala Glu Asp Thir
SO 55 6O

Ala Val Tyr Tyr Cys Ala Arg

65 70

<210s, SEQ ID NO 57
&211s LENGTH: 30
 US 8,025,878 B2
91 92
- Continued
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OO > SEQUENCE: 57

atgaagttgv wtgttaggct gttggtgctg 3O

<210s, SEQ ID NO 58
&211s LENGTH: 30
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 58

atggagWCag acacactic ct gy tatgggtg 3O

<210s, SEQ ID NO 59
&211s LENGTH: 30
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OO > SEQUENCE: 59

atgagtgtgc tcacticaggit cctggsgttg 3O

<210s, SEQ ID NO 60
&211s LENGTH: 33
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 60

atgaggrocc ctdct cagwt tyttggmwtc ttg 33

<210s, SEQ ID NO 61
&211s LENGTH: 29
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 61

atggattitwa gotgcagatt Witcagcttic 29

<210s, SEQ ID NO 62
&211s LENGTH: 27
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 62

atgaggtkck ktgktsagst Sct grgg 27

<210s, SEQ ID NO 63
&211s LENGTH: 31
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 63

atgggcWitca agatggagtic acakWyycWg g 31

 US 8,025,878 B2
93 94
- Continued

<210s, SEQ ID NO 64
&211s LENGTH: 31
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 64

atgtggggay ctktttycmm tttittcaatt g 31

<210s, SEQ ID NO 65
&211s LENGTH: 25
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 65

atgg trtc cw cascticagtt cottg 25

<210s, SEQ ID NO 66
&211s LENGTH: 27
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 66

atgtatatat gtttgttgtc. tatttct 27

<210s, SEQ ID NO 67
&211s LENGTH: 28
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OO > SEQUENCE: 67

atggaag.ccc cagcticagot totct tcc 28

<210s, SEQ ID NO 68
&211s LENGTH: 2O
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 68

actggatggit gggaagatgg

<210s, SEQ ID NO 69
&211s LENGTH: 27
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 69

atgaaatgca gctggggcat Stt Cttic 27

<210s, SEQ ID NO 70
&211s LENGTH: 26
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer
 US 8,025,878 B2
95 96
- Continued

<4 OO > SEQUENCE: 7 O

atgggatgga gctrtatic at Sytctt 26

<210s, SEQ ID NO 71
&211s LENGTH: 27
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 71

atgaagWtgt ggittaaactg ggtttitt 27

<210s, SEQ ID NO 72
&211s LENGTH: 25
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 72

atgractittg ggytcagott grtitt 25

<210s, SEQ ID NO 73
&211s LENGTH: 30
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OO > SEQUENCE: 73

atgg acticca ggct caattt agtttitcc tt 3O

<210s, SEQ ID NO 74
&211s LENGTH: 27
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 74

atggctgtcy trgsgctrct cittctgc 27

<210s, SEQ ID NO 75
&211s LENGTH: 26
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OO > SEQUENCE: 75

atggratgga gckggrtcitt timt citt 26

<210s, SEQ ID NO 76
&211s LENGTH: 23
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OO > SEQUENCE: 76

atgaga.gtgc tigattcttitt gtg 23

<210s, SEQ ID NO 77
&211s LENGTH: 30
 US 8,025,878 B2
97 98
- Continued
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OO > SEQUENCE: 77

atggmttggg ttggamctt gct attic ct 3O

<210s, SEQ ID NO 78
&211s LENGTH: 27
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OO > SEQUENCE: 78

atgggcagaci ttacattct c attcc tig 27

<210s, SEQ ID NO 79
&211s LENGTH: 28
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OO > SEQUENCE: 79

atggattittg ggctgattitt ttittattg 28

<210s, SEQ ID NO 8O
&211s LENGTH: 27
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 80

atgatggtgt taagttcttct gtacctg 27

<210s, SEQ ID NO 81
&211s LENGTH: 21
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 81

Cagtggatag acagatggggg 21

<210s, SEQ ID NO 82
&211s LENGTH: 21
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 82

Cagtggatag acagatggggg 21

<210s, SEQ ID NO 83
&211s LENGTH: 21
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 83

Cagtggatag actgatggggg 21
 US 8,025,878 B2
99 100
- Continued

<210s, SEQ ID NO 84
&211s LENGTH: 21
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 84

Caagggatag acagatgggg C 21

<210s, SEQ ID NO 85
&211s LENGTH: 13
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: DNA/RNA Consensus Sequence
22 Os. FEATURE:
<221 > NAMEAKEY: misc feature
<222s. LOCATION: (11) . . (11)
223s OTHER INFORMATION: n is ul

<4 OOs, SEQUENCE: 85

gcc.gc.crcca ngg 13

<210s, SEQ ID NO 86
&211s LENGTH: 8
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Consenus Sequence
<4 OOs, SEQUENCE: 86
acgtragt

<210s, SEQ ID NO 87
&211s LENGTH: 9
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Consensus Sequence
<4 OO > SEQUENCE: 87
magg tragt

<210s, SEQ ID NO 88
&211s LENGTH: 16
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Consensus Sequence
22 Os. FEATURE:
<221 > NAMEAKEY: misc feature
<222s. LOCATION: (12) ... (12)
<223> OTHER INFORMATION: n is a, c, g, t or u.
<4 OOs, SEQUENCE: 88
yyyyyyyyyy yncagg 16

<210s, SEQ ID NO 89
&211s LENGTH: 30
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 89

aagcttgc.cg ccaccatgga citcCaggctic 3O

 US 8,025,878 B2
101 102
- Continued

<210s, SEQ ID NO 90
&211s LENGTH: 37
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 90

gggCCCttgg tdgaggctga gaga.cggtg actgagg 37

<210s, SEQ ID NO 91
&211s LENGTH: 33
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 91

aagcttgc.cg ccaccatgaa gttgcctgtt agg 33

<210s, SEQ ID NO 92
&211s LENGTH: 30
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 92

ggat.ccactic acgtttgatt to cagcttgg 3O

<210s, SEQ ID NO 93
&211s LENGTH: 17
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 93

gttitt.cccag toacgac 17

<210s, SEQ ID NO 94
&211s LENGTH: 24
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 94

agcggataac aattt Cacac agga 24

<210s, SEQ ID NO 95
&211s LENGTH: 17
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OO > SEQUENCE: 95

Ctcatcagat ggcggga 17

<210s, SEQ ID NO 96
&211s LENGTH: 21
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer
 US 8,025,878 B2
103 104
- Continued

<4 OOs, SEQUENCE: 96

cgctgctgag ggagtagagt c 21

<210s, SEQ ID NO 97
&211s LENGTH: 4 OO
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: PCR Template
<4 OO > SEQUENCE: 97
atgaagttgc Ctgttaggct gttggtgctg atgttctgga titcctgcttic Cagcagtgat 6O

gttittgatga cccaaactico act ct coctd cctgtcagtic ttggagat.ca agcct coat c 12 O

tcttgcagat Ctagt cagag cattgtacat agtaatggaa acaccitattt agaatgg tac 18O

Ctgcagaaac Caggc.cagtic tccaaagctic citgat ctaca aagtttic caa ccgattittct 24 O

gggg.tcc.cag acaggttcag tggcagtgga t cagggacag attitcacact Caagat cagc 3OO

agagtggagg Ctgaggat ct gggaattitat tactgct titc alaggttcaca tgttcct cog 360

acgttcggtg gaggcaccala gctggaaatc aaacgggctg 4 OO

<210s, SEQ ID NO 98
&211s LENGTH: 30
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 98

atgaagttgv wtgttaggct 3O

<210s, SEQ ID NO 99
&211s LENGTH: 336
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: PCR Product

<4 OOs, SEQUENCE: 99

gatattgttga tigacccaggc to cact ct co ctgcctgtca gtc.ttggaga t caagcc to c 6O

atct cittgca gatctagt ca gag cattgta catagtaatg gaaac accta tittagaatgg 12 O

tacctgcaga aaccaggc.ca gtc. tccaaag ctic ct gatct acaaagttt c caa.ccgattit 18O

tctggggtco Cagacaggtt Cagtggcagt ggat Caggga cagattt cac act caagat c 24 O

agcagagtgg aggctgagga tctgggaatt tattactgct ttcaaggttc acatgttcct 3OO

ccgacgttcg gtggaggcac Caagctggaa atcaaa. 336

<210s, SEQ ID NO 100

&211s LENGTH: 19
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Chimeric Protein leader sequence
<4 OOs, SEQUENCE: 1.OO

Met Lys Lieu Pro Val Arg Lieu. Lieu Val Lieu Met Phe Trp Ile Pro Ala
1. 5 15

Ser Ser Ser

<210s, SEQ ID NO 101

 US 8,025,878 B2
105 106
- Continued
&211s LENGTH: 19
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Chimeric protein leader sequence
<4 OOs, SEQUENCE: 101

Met Lys Lieu Pro Val Arg Lieu. Lieu Val Lieu Met Phe Trp Ile Pro Ala
1. 5 1O 15

Ser Ser Ser

<210s, SEQ ID NO 102

&211s LENGTH: 57
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Chimeric protein DNA sequence
<4 OOs, SEQUENCE: 102
atgaagttgc Ctgttaggct gttggtgctg atgttctgga titcCtgctt C Cagcagt

<210s, SEQ ID NO 103

&211s LENGTH: 4 O
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Chimeric Protein leader sequence
<4 OOs, SEQUENCE: 103

Met Lys Lieu Pro Val Arg Lieu. Lieu Val Lieu Met Phe Trp Ile Pro Ala
1. 5 1O 15

Ser Ser Ser Asp Val Lieu Met Thr Glin Thr Pro Leu Ser Leu Pro Val
2O 25 3O

Ser Lieu. Gly Asp Glin Ala Ser Ile

35 4O

<210s, SEQ ID NO 104

&211s LENGTH: 421
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Chimeric 158 VK construct DNA sequence
<4 OOs, SEQUENCE: 104
aagcttgc.cg ccaccatgaa gttgcctgtt aggctgttgg totgatgtt Ctggatt Cot 6O

gct tccagca gtgatgttitt gatgacccaa act coactict coctogcc tigt cagtc.ttgga 12 O

gatgaagcct coat ct cittg cagat ctagt cagagcattg tacatagitaa toggaaac acc 18O

tatttagaat gigtacctgca gaalaccaggc cagt citccaa agctic ct gat ctacaaagtt 24 O

tcca accgat tttctggggt CCC agaCagg ttcagtggca gtggat Cagg gacagattt C 3OO

acacticaaga t cagoaga.gt ggaggctgag gatctgggala titt attactg. Ctttcaaggt 360

t cacatgttc Ctc.cgacgtt C9gtggaggc accaa.gctgg aaatcaaacg tagtggat C

c 421

<210s, SEQ ID NO 105

&211s LENGTH: 14 O
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Chimeric 158 VK protein sequence
<4 OOs, SEQUENCE: 105
 US 8,025,878 B2
107 108
- Continued
Lieu Ala Ala Thr Met Lys Lieu Pro Val Arg Lell Lell Wall Luell Met
15

Trp Ile Pro Ala Ser Ser Ser Asp Val Lieu. Met Thir Glin Thir Pro
2O 25

Lell Ser Lieu Pro Val Ser Lieu. Gly Asp Glin Ala Ser Ile Ser
35 4O 45

Ser Ser Glin Ser Ile Val His Ser Asn Gly Asn Thir Luell Glu Trp
SO 55 6O

Tyr Lieu. Glin Llys Pro Gly Glin Ser Pro Llys Lieu. Lell Ile
65 70 7s

Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
85 90 95

Gly Thr Asp Phe Thr Lieu Lys Ile Ser Arg Val Glu Ala Glu Asp Lieu.
1OO 105 11 O

Gly Ile Tyr Tyr Cys Phe Glin Gly Ser His Val Pro Pro Thir Phe Gly
115 12 O 125

Gly Gly. Thir Lys Lieu. Glu Ile Lys Arg Glu Trp Ile
13 O 135 14 O

<210s, SEQ ID NO 106

&211s LENGTH: 447
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: PCR Template
<4 OOs, SEQUENCE: 106

atgg acticca ggct caattt agtttitcc tt gtcct tattt taalaaggtgt Ccagtgtgat 6O

gtgcagctgg tdgagtctgg gaggaggctta gttgcagcCtg gagggtc.ccg gaaact citcc 12 O

tgtgcago: ct ctdgatt cac titt cagtagc tittggaatgc actgggttcg t caggct coa 18O

gaga aggggc tiggagtgggit cqcatacatt agtag togca gtag taccat c tactatgga 24 O

gacacagtga agggc.cgatt cac catct Co agaga caatc c caagaacac cctgttcctg 3OO

caaatgacca gtctaagg to taggacacg gcc atgt att actgtgcaa.g agagggggga 360

tatt actacg gtaggagitta ctatactatg gact actggg gtcaaggaac cticagt cacc

gtct cotcag ccaaaacaac agc.ccca 447

<210s, SEQ ID NO 107

&211s LENGTH: 390
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: PCR Product

<4 OOs, SEQUENCE: 107

gaggtgaagc tigatggaatc tiggggaggc titagtgcago CtggagggtC ccggaaactic 6O

t cct gtgcag cct ctdgatt cactitt cagt agctittggaa tgcactgggt tcgtcaggct 12 O

ccagagaagg ggctggagtg ggtcgcatac attagtagtg gcagtag tac Cat Ctactat 18O

ggagacacag taagggc.cg attcaccatc. tccagaga.ca atcc.caagaa caccctgttc 24 O

Ctgcaaatga C cagtictaag gtctgaggac acggc catgt attactgtgc aagagagggg 3OO

ggat attact acgg taggag titactatact atggact act ggggt Caagg aac ct cagtic 360

accotctic ct cagccaaaac aac agc.ccca 390

<210s, SEQ ID NO 108

&211s LENGTH: 30
 US 8,025,878 B2
109 110
- Continued
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223s OTHER INFORMATION: PCR Primer

<4 OOs, SEQUENCE: 108

atgg acticca ggct caattt agtttitcc tt 3O

<210s, SEQ ID NO 109

&211s LENGTH: 19
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: 158 WH Leader sequence
<4 OOs, SEQUENCE: 109

Met Asp Ser Arg Lieu. Asn Lieu Val Phe Lieu Val Lieu. Ile Lieu Lys Gly
1. 5 1O 15

Val Glin Cys

<210s, SEQ ID NO 110

&211s LENGTH: 19
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: NL-1 protein
<4 OOs, SEQUENCE: 110

Met Asp Ser Arg Lieu. Asn Lieu Val Phe Lieu Val Lieu. Ile Lieu Lys Gly
1. 5 1O 15

Val Glin Cys

<210s, SEQ ID NO 111

&211s LENGTH: 57
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: NL-1 DNA

<4 OOs, SEQUENCE: 111

atgg acticca ggct caattt agtttitcc tt gtcct tattt taaaaggtgt ccagtgt f

<210s, SEQ ID NO 112

&211s LENGTH: 30
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: NL-1 Leader Sequence
<4 OOs, SEQUENCE: 112

Met Asp Ser Arg Lieu. Asn Lieu Val Phe Lieu Val Lieu. Ile Lieu Lys Gly
1. 5 1O 15

Val Glin Cys Asp Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu.
2O 25 3O

<210s, SEQ ID NO 113

&211s LENGTH: 461
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: Chimeric 158 WH DNA. Sequence
<4 OOs, SEQUENCE: 113
aagcttgc.cg ccaccatgga citccaggctic aatttagttt to cittgtcct tattittaaaa 6O

ggtgtcCagt gttgatgttgca gctggtggag tictaggggag gCttagtgca gCCtggaggg 12 O

 US 8,025,878 B2
111 112
- Continued

tcc.cggaaac t ct cotgtgc agcct Ctgga tt cact t t ca. gtagctttgg aatgcactgg 18O

gttctgtcagg Ctccagagaa ggggctggag tgggit cqcat acattagtag tggcagtagt 24 O

acCatctact atggagacac agtgaagggc cgatt cacca t ct coagaga caatcc.caag 3OO

aacaccctgt tcc tigcaaat gaccagticta aggtotgagg acacggc cat gtatt actgt 360

gcaa.gagagg ggggat atta Ctacgg tagg agittactata ctatogacta Ctggggt caa

ggaacct cag to accotctic cticagoct co accalagggcc c 461

SEQ ID NO 114
LENGTH: 154
TYPE : PRT
ORGANISM: Artificial
FEATURE:
OTHER INFORMATION: Chimeric 158 VH Protein sequence
SEQUENCE: 114

Llys Lieu Ala Ala Thr Met Asp Ser Arg Lieu. Asn Lell Wall Phe Lieu Wall
1. 5 1O 15

Lell Ile Luell Lys Gly Val Glin Cys Asp Val Glin Lell Wall Glu Ser Gly
25

Gly Gly Luell Val Glin Pro Gly Gly Ser Arg Llys Lell Ser Ala Ala
35 4O 45

Ser Gly Phe Thir Phe Ser Ser Phe Gly Met His Trp Wall Arg Glin Ala
SO 55 6O

Pro Glu Gly Lieu. Glu Trp Val Ala Tyr Ile Ser Ser Gly Ser Ser
65 70 7s 8O

Thir Ile Tyr Gly Asp Thr Val Phe Thir Ile Ser Arg
85 90 95

Asp Asn Pro Lys Asn Thr Lieu Phe Leul Glin Met Thir Ser Luell Arg Ser
105 11 O

Glu Asp Thir Ala Met Tyr Tyr Cys Ala Arg Glu Gly Gly
115 12 O 125

Gly Arg Ser Tyr Tyr Thr Met Asp Glin Gly Thir Ser Wall
13 O 135 14 O

Thir Wall Ser Ser Ala Ser Thr Lys Gly Pro
145 150

SEQ ID NO 115
LENGTH: 124
TYPE : PRT
ORGANISM: Artificial
FEATURE:
OTHER INFORMATION: 158 WH amino acid sequence
<4 OOs, SEQUENCE: 115

Asp Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu. Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Arg Lys Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Phe
25

Gly Met His Trp Val Arg Glin Ala Pro Glu Lys Gly Lell Glu Trp Val
35 4O 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thir Ile Tyr Tyr Gly Asp Thir Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp ASn Pro Asn Thir Lieu. Phe
65 70 7s 8O

Lell Glin Met Thir Ser Lieu. Arg Ser Glu Asp Thr Ala Met Tyr Cys
85 90 95
 US 8,025,878 B2
113 114
- Continued

Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp
1OO 105 11 O

Tyr Trp Gly Glin Gly. Thir Ser Val Thr Val Ser Ser
115 12 O

<210s, SEQ ID NO 116

&211s LENGTH: 112
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: 158 VK amino acid sequence
<4 OOs, SEQUENCE: 116

Asp Val Lieu Met Thr Glin Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1. 5 1O 15

Asp Glin Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Ile Val His Ser
2O 25 3O

Asn Gly Asn Thr Tyr Lieu. Glu Trp Tyr Lieu Gln Lys Pro Gly Glin Ser
35 4O 45

Pro Llys Lieu. Lieu. Ile Tyr Llys Val Ser Asn Arg Phe Ser Gly Val Pro
SO 55 6O

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile
65 70 7s 8O

Ser Arg Val Glu Ala Glu Asp Lieu. Gly Ile Tyr Tyr Cys Phe Glin Gly
85 90 95

Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile Llys
1OO 105 11 O

<210s, SEQ ID NO 117

&211s LENGTH: 22
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158 WH framework

<4 OOs, SEQUENCE: 117

Val Ala Gly Phe Thr Phe Ser Val Glin Leu Trp Val Ala Phe Ile Arg
1. 5 1O 15

Asn Lieu. Tyr Ala Arg Trp

2O

<210s, SEQ ID NO 118

&211s LENGTH: 22
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 118

Val Ala Gly Phe Thr Phe Ser Val Glin Leu Trp Val Ala Phe Ile Arg
1. 5 1O 15

Asn Lieu. Tyr Ala Arg Trp

2O

<210s, SEQ ID NO 119

&211s LENGTH: 124
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158 WH

<4 OOs, SEQUENCE: 119

Asp Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 1O 15
 US 8,025,878 B2
115 116
- Continued

Ser Arg Llys Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Phe
2O 25 3O

Gly Met His Trp Val Arg Glin Ala Pro Glu Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thir Ile Tyr Gly Asp Thir Wall
SO 55 6O

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Asn Thir Luell Phe
65 70 7s

Lell Gln Met Thr Ser Lieu. Arg Ser Glu Asp Thir Ala Met Tyr Cys
85 90 95

Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Thir Met Asp
1OO 105 11 O

Trp Gly Glin Gly Thr Ser Val Thir Wall Ser Ser
115 12 O

<210s, SEQ ID NO 120

&211s LENGTH: 124
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 120
Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu Lys Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25

Trp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Asn. Ile Lys Glin Asp Gly Ser Glu Tyr Wall Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Ser Luell Tyr
65 70 7s

Lell Gln Met Asn. Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Pro Asp Asp Ser Ser Gly Tyr Ser Ala Glu Tyr Phe Glin
1OO 105 11 O

His Trp Gly Glin Gly Thr Lieu Val Thir Wall Ser Ser
115 12 O

<210s, SEQ ID NO 121

&211s LENGTH: 125
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 121
Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25

Trp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Asn. Ile Lys Glu Asp Gly Gly Glu Phe Tyr Wall Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Asn Ser Luell Phe
65 70

Lell Gln Met Asn. Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95
 US 8,025,878 B2
117 118
- Continued
Ala Arg Glu Arg Gly His Asp Phe Trp Ser Ile Tyr Tyr Thr His Phe
1OO 105 11 O

Asp Tyr Trp Gly Glin Gly Ala Leu Val Thr Val Ser Ser
115 12 O 125

<210s, SEQ ID NO 122

&211s LENGTH: 118
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 122

Pro Leu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly
1. 5 1O 15

Gly Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
2O 25 3O

Tyr Trp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp
35 4O 45

Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Llys Tyr Tyr Ala Asp Ser
SO 55 6O

Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn. Ser Lys Asn. Thir Lieu
65 70 7s 8O

Tyr Lieu Gln Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr
85 90 95

Cys Ala Arg Asp Gly Gly Ser Ile Phe Asp Tyr Trp Gly Glin Gly Thr
1OO 105 11 O

Leul Wall. Thir Wal Ser Ser

115

<210s, SEQ ID NO 123

&211s LENGTH: 120
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 123

Glu Val Glin Leu Val Glu Ser Gly Gly Gly Val Val Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
2O 25 3O

Ala Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val
35 4O 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Llys Tyr Tyr Ala Asp Ser Val
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn. Ser Lys Asn. Thir Lieu. Tyr
65 70 7s 8O

Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95

Ala Arg Ala Arg Asp Tyr Tyr Tyr Tyr Pro Met Asp Val Trp Gly Glin
1OO 105 11 O

Gly. Thir Thr Val Thr Val Ser Ser

115 12 O

<210s, SEQ ID NO 124

&211s LENGTH: 128
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 124

Glin Val Glin Lieu Val Glu Ser Gly Gly Gly Val Val Glin Pro Gly Arg
1. 5 1O 15
 US 8,025,878 B2
119 120
- Continued

Ser Luell Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25 3O

Ala Met His Trp Wall Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Wall Ile Ser Tyr Asp Gly Ser Asn Tyr Ala Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ser Asn Thir Luell Tyr
65 70 7s

Lell Glin Met Asn Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Asp Glin Ser Trp Ser Arg Ile Ala Ala Ala Gly Thir Pro Pro
105 11 O

Ser Luell Phe Asp Pro Trp Gly Glin Gly Thir Luell Wall Thir Wall Ser Ser
115 12 O 125

<210s, SEQ ID NO 125

&211s LENGTH: 123
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 125
Glin Wall Glin Lieu Wall Glu Ser Gly Gly Gly Wall Wall Glin Pro Gly Arg
1. 5 1O 15

Ser Luell Lys Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
25

Ala Met His Trp Wall Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Wall Ile Ser Tyr Asp Gly Ser Asn Tyr Ala Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ser Asn Thir Luell Tyr
65 70 7s

Lell Glin Met Asn Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Ala Arg Asn Tyr Asp Ser Ser Gly Tyr Ser Phe Asp Tyr
105 11 O

Trp Gly Glin Gly Thir Lell Wall Thir Wall Ser Ser
115 12 O

SEQ ID NO 126
LENGTH: 117
TYPE : PRT
ORGANISM: Homo sapiens
< 4 OOs SEQUENCE: 126
Glu Wall Glin Lieu Wall Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Luell Arg Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25

Trp Met Ser Trp Wall Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Asn Ile Glin Asp Gly Ser Glu Tyr Wall Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Asn Ser Luell Tyr
65 70 7s

Lell Glin Met Asn Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95
 US 8,025,878 B2
121 122
- Continued
Ala Arg Val Arg Arg Gly Ser Gly Asp Ser Trp Gly Glin Gly. Thir Lieu
105 11 O

Wall. Thir Wal Ser Ser

115

<210s, SEQ ID NO 127

&211s LENGTH: 121
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 127
Glu Wall Glin Lieu. Val Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Luell Arg Luell Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25

Trp Met Ser Trp Val Arg Glin Ala Pro Gly Gly Lell Glu Trp Wall
35 4O 45

Ala Asn Ile Glin Asp Gly Ser Glu Tyr Wall Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Asn Ser Luell Tyr
65 70 7s

Lell Glin Met Asn Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Glu Glin Gln Leu Gly Pro His Asn Trp Phe Asp Pro Trp Gly
105 11 O

Glin Gly Thir Luell Wall. Thir Wal Ser Ser

115 12 O

<210s, SEQ ID NO 128

&211s LENGTH: 126
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 128
Glin Wall Glin Lieu. Val Glu Ser Gly Gly Gly Wall Wall Glin Pro Gly Arg
1. 5 1O 15

Ser Luell Lys Luell Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25 3O

Ala Met His Trp Val Arg Glin Ala Pro Gly Gly Lell Glu Trp Wall
35 4O 45

Ala Wall Ile Ser Tyr Asp Gly Ser Asn Tyr Ala Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ser Asn Thir Luell Tyr
65 70 7s

Lell Glin Met Asn Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Asp Glin Glu Thr Gly Thr Thir Phe Asp Tyr Gly
105 11 O

Met Asp Wall Trp Gly Glin Gly Thr Thir Wall Thir Wall Ser Ser
115 12 O 125

SEQ ID NO 129
LENGTH: 125
TYPE : PRT
ORGANISM: Homo sapiens
< 4 OOs SEQUENCE: 129

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 15
 US 8,025,878 B2
123 124
- Continued

Ser Luell Arg Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25

Trp Met Ser Trp Wall Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Asn Ile Glin Asp Gly Ser Glu Tyr Wall Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Asn Ser Luell Tyr
65 70 7s

Lell Glin Met Asn Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Asp Pro Met Thir Thir Wall Wall Pro Ser Lell Ala Thir Asn
105 11 O

Asp Trp Gly Glin Gly Thir Luell Wall Thir Wall Ser Ser
115 12 O 125

<210s, SEQ ID NO 130

&211s LENGTH:
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 13 O
Glu Wall Glin Lieu. Wall Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Luell Arg Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25

Trp Met Ser Trp Wall Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Asn Ile Glin Asp Gly Ser Glu Tyr Wall Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Asn Ser Luell Tyr
65 70 7s

Lell Glin Met Asn Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Asp Cys Wall Gly Ala Luell Gly Ala Phe Asp Ile Trp Gly Glin
105 11 O

Gly Thir Met Wall Thir Wall Ser Ser

115 12 O

SEQ ID NO 131
LENGTH: 116
TYPE : PRT
ORGANISM: Homo sapiens
< 4 OOs SEQUENCE: 131

Cys Glu Val Glin Lell Wall Glu Ser Gly Gly Gly Lell Wall Glin Pro Gly
1. 5 1O 15

Gly Ser Luell Arg Lell Ser Ser Ala Ser Gly Phe Thir Phe Ser Thir
25

Tyr Trp Met Thir Trp Wall Arg Glin Ala Pro Gly Gly Luell Glu Trp
35 4O 45

Wall Ala Asn Ile Lys Pro His Gly Ser Glu Ala Tyr Wall Asp Ser
SO 55 6O

Wall Gly Arg Phe Thir Ile Ser Arg Asp ASn Ala Asn Ser Luell
65 70

Phe Luell Glin Met Ser Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Tyr
85 90 95
 US 8,025,878 B2
125 126
- Continued
Cys Ala Arg Ala Asn. Ser Lieu. Asp Val Trp Gly Glin Gly. Thir Thr Val
105 11 O

Thir Wal Ser Ser

115

<210s, SEQ ID NO 132

&211s LENGTH: 121
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 132
Cys Glu Wall Glin Lieu Val Glu Ser Gly Gly Gly Lell Wall Glin Pro Gly
1. 5 15

Gly Ser Luell Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser
2O 25

Tyr Trp Met Ser Trp Val Arg Glin Ala Pro Gly Gly Luell Glu Trp
35 4O 45

Wall Ala Asn Ile Lys Glin Asp Gly Ser Glu Lys Tyr Wall Asp Ser
SO 55 6O

Wall Gly Arg Phe Thir Ile Ser Arg Asp ASn Ala Asn Ser Luell
65 70

Luell Glin Met Asn Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr Tyr
85 90 95

Ala Arg Asp Gly Asp Ile Gly Asp Trp Trp Phe Asp Pro Trp Gly
1OO 105 11 O

Glin Gly Thir Luell Wall. Thir Wal Ser Ser

115 12 O

<210s, SEQ ID NO 133

&211s LENGTH: 132
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 133
Glin Wall Glin Lieu Val Glu Ser Gly Gly Gly Wall Wall Glin Pro Gly Arg
1. 5 1O 15

Ser Luell Arg Luell Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
25 3O

Gly Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Wall Ile Trp Tyr Asp Gly Ser Asn Tyr Ala Asp Ser Ala
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ser Asn Thir Luell Phe
65 70 7s

Lell Glin Met Asn Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Asp Lys Gly Tyr Tyr Asp Tyr Wall Trp Gly Ser Tyr Arg Ser
105 11 O

Asn Pro Lys Asn Asp Ala Phe Asp Ile Trp Gly Glin Gly Thir Met Wall
115 12 O 125

Thir Wall Ser Ser

13 O

SEQ ID NO 134
LENGTH: 132
TYPE : PRT
ORGANISM: Homo sapiens
< 4 OOs SEQUENCE: 134
 US 8,025,878 B2
127 128
- Continued

Glin Wall Glin Luell Wall Glu Ser Gly Gly Gly Wall Wall Glin Pro Gly Arg
1O 15

Ser Luell Arg Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
25

Gly Met His Trp Wall Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Wall Ile Trp Tyr Asp Gly Ser Asn Tyr Tyr Ala Asp Ser Ala
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ser Asn Thir Luell Phe
65 70

Lell Glin Met Asn Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Asp Lys Gly Tyr Asp Tyr Wall Trp Gly Ser Tyr Arg Ser
105 11 O

Asn Pro Lys Asn Asp Ala Phe Asp Ile Trp Gly Glin Gly Thir Met Wall
115 12 O 125

Thir Wall Ser Ser

13 O

<210s, SEQ ID NO 135

&211s LENGTH: 116
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 135
Glin Wall Glin Lieu Wall Glu Ser Gly Gly Gly Wall Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Luell Arg Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
25

Gly Met His Trp Wall Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Wall Ile Trp Tyr Asp Gly Ser Asn Tyr Tyr Ala Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ser Asn Thir Luell Tyr
65 70

Lell Glin Met Asn Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Asp Arg Phe Phe Phe Asp Asn Trp Gly Glin Gly Thir Luell Wall
105 11 O

Thir Wall Ser Ser

115

SEQ ID NO 136
LENGTH: 124
TYPE : PRT
ORGANISM: Artificial
FEATURE:
OTHER INFORMATION: 158 WH

SEQUENCE: 136

Asp Val Glin Lieu Val Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Arg Llys Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Phe
25 3O

Gly Met His Trp Val Arg Glin Ala Pro Glu Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thir Ile Tyr Gly Asp Thir Wall
 US 8,025,878 B2
129 130
- Continued
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Pro Asn Thir Luell Phe
65 70 7s 8O

Lell Glin Met Thir Ser Lell Arg Ser Glu Asp Thir Ala Met Tyr Tyr Cys
85 90 95

Ala Arg Glu Gly Gly Tyr Gly Arg Ser Thir Met Asp
1OO 105 11 O

Trp Gly Gln Gly Thir Ser Wall Thir Wall Ser Ser
115 12 O

<210s, SEQ ID NO 137

&211s LENGTH: 124
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 137
Glu Wall Glin Lieu Wall Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Luell Lys Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25

Trp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Asn Ile Lys Glin Asp Gly Ser Glu Tyr Wall Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Asn Ser Luell Tyr
65 70 7s

Lell Glin Met Asn. Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Tyr Cys
85 90 95

Ala Arg Pro Asp Asp Ser Ser Gly Tyr Ser Ala Glu Tyr Phe Glin
1OO 105 11 O

His Trp Gly Gln Gly Thir Lell Wall Thir Wall Ser Ser
115 12 O

<210s, SEQ ID NO 138

&211s LENGTH: 117
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 138
Lieu Wall Glin Lieu. Wall Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Luell Arg Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25

Trp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Wall Ile Trp Tyr Asp Gly Ser Asn Tyr Ala Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ser Asn Thir Luell Tyr
65 70 7s

Lell Glin Met Asn. Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Tyr Cys
85 90 95

Ala Arg Asp Gly Gly Ser Ile Phe Asp Trp Gly Glin Gly Thir Luell
1OO 105 11 O

Wall Thir Wall Ser Ser

115

<210s, SEQ ID NO 139

 US 8,025,878 B2
131 132
- Continued
&211s LENGTH: 120
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 139
Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Wall Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Asn
2O 25

Ala Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Tyr Ala Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn Thir Luell Tyr
65 70 7s

Lell Gln Met Asn. Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr
85 90 95

Ala Arg Ala Arg Asp Tyr Tyr Tyr Tyr Pro Met Asp Wall Trp Gly Glin
1OO 105 11 O

Gly Thir Thir Wall. Thir Wal Ser Ser

115 12 O

<210s, SEQ ID NO 140

&211s LENGTH: 128
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 140
Glin Val Glin Lieu Val Glu Ser Gly Gly Gly Wall Wall Glin Pro Gly Arg
1. 5 1O 15

Ser Lieu Lys Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser
2O 25

Ala Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Tyr Ala Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn Thir Luell Tyr
65 70 7s

Lell Gln Met Asn. Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr
85 90 95

Ala Arg Asp Glin Ser Trp Ser Arg Ile Ala Ala Ala Gly Thir Pro Pro
1OO 105 11 O

Ser Leu Phe Asp Pro Trp Gly Glin Gly Thir Luell Wall Thir Wall Ser Ser
115 12 O 125

<210s, SEQ ID NO 141

&211s LENGTH: 123
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 141
Glin Val Glin Lieu Val Glu Ser Gly Gly Gly Wall Wall Glin Pro Gly Arg
1. 5 1O 15

Ser Lieu Lys Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25 3O

Ala Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Ala Asp Ser Wall
 US 8,025,878 B2
133 134
- Continued
SO 55 6O

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thir Luell Tyr
65 70 7s 8O

Lell Gln Met Asn. Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr Tyr Cys
85 90 95

Ala Arg Ala Arg Asn Tyr Tyr Asp Ser Ser Gly Tyr Ser Phe Asp Tyr
105 11 O

Trp Gly Glin Gly Thr Lieu Val Thr Wall Ser Ser
115 12 O

<210s, SEQ ID NO 142

&211s LENGTH: 117
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 142
Glu Wall Glin Lieu Val Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Luell Arg Luell Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25

Trp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Asn Ile Glin Asp Gly Ser Glu Tyr Wall Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Asn Ser Luell Tyr
65 70 7s

Lell Glin Met Asn Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr Tyr Cys
85 90 95

Ala Arg Wall Arg Arg Gly Ser Gly Asp Ser Trp Gly Glin Gly Thir Luell
105 11 O

Wall Thir Wall Ser Ser

115

<210s, SEQ ID NO 143

&211s LENGTH: 121
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 143
Glu Wall Glin Lieu. Val Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 1O 15

Ser Luell Arg Luell Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Tyr
2O 25

Trp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Asn Ile Glin Asp Gly Ser Glu Tyr Wall Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Asn Ser Luell Tyr
65 70 7s

Lell Glin Met Asn Ser Lieu. Arg Ala Glu Asp Thir Ala Wall Tyr Tyr Cys
85 90 95

Ala Arg Glu Glin Gln Leu Gly Pro His Asn Trp Phe Asp Pro Trp Gly
105 11 O

Glin Gly Thir Luell Wall. Thir Wal Ser Ser

115 12 O

<210s, SEQ ID NO 144

 US 8,025,878 B2
135 136
- Continued
&211s LENGTH: 126
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 144

Glin Val Glin Lieu Val Glu Ser Gly Gly Gly Val Val Glin Pro Gly Arg
1. 5 1O 15

Ser Leu Lys Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
2O 25 3O

Ala Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val
35 4O 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Llys Tyr Tyr Ala Asp Ser Val
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn. Ser Lys Asn. Thir Lieu. Tyr
65 70 7s 8O

Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95

Ala Arg Asp Glin Glu Thr Gly. Thir Thr Phe Asp Tyr Tyr Tyr Tyr Gly
1OO 105 11 O

Met Asp Val Trp Gly Glin Gly Thr Thr Val Thr Val Ser Ser
115 12 O 125

<210s, SEQ ID NO 145

&211s LENGTH: 125
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 145

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
2O 25 3O

Trp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val
35 4O 45

Ala Asn. Ile Lys Glin Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn. Ser Lieu. Tyr
65 70 7s 8O

Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95

Ala Arg Asp Pro Met Thr Thr Val Val Llys Pro Ser Leu Ala Thr Asn
1OO 105 11 O

Asp Tyr Trp Gly Glin Gly Thr Lieu Val Thr Val Ser Ser
115 12 O 125

<210s, SEQ ID NO 146

&211s LENGTH: 125
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 146

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
2O 25 3O

Trp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val
35 4O 45

Ala Asn. Ile Lys Glin Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val
 US 8,025,878 B2
137 138
- Continued
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Asn Ser Luell Tyr
65 70 7s 8O

Lell Glin Met Asn. Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Asp Pro Met Thir Thir Wall Wall Pro Ser Lell Ala Thir Asn
1OO 105 11 O

Asp Tyr Trp Gly Glin Gly Thir Luell Wall Thir Wall Ser Ser
115 12 O 125

<210s, SEQ ID NO 147

&211s LENGTH: 115
212. TYPE : PRT
&213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 147
Glu Wall Glin Lieu Wall Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 15

Ser Luell Arg Luell Ser Ser Ala Ser Gly Phe Thir Phe Ser Thir Tyr
2O 25

Trp Met Thir Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Asn Ile Llys Pro His Gly Ser Glu Ala Tyr Wall Asp Ser Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Asn Ser Luell Phe
65 70 7s

Lell Glin Met Ser Ser Lell Arg Ala Glu Asp Thir Ala Wall Tyr Cys
85 90 95

Ala Arg Ala Asn. Ser Lell Asp Wall Trp Gly Glin Gly Thir Thir Wall Thir
1OO 105 11 O

Wall Ser Ser

115

SEQ ID NO 148
LENGTH: 98
TYPE : PRT
ORGANISM: Artificial
FEATURE:
OTHER INFORMATION: 158 WH

SEQUENCE: 148

Asp Val Glin Lieu Val Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 15

Ser Arg Lys Luell Ser Ala Ala Ser Gly Phe Thir Phe Ser Ser Phe
2O 25

Gly Met His Trp Val Arg Glin Ala Pro Glu Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thir Ile Tyr Gly Asp Thir Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Pro Asn Thir Luell Phe
65 70 7s

Lell Glin Met Thir Ser Lell Arg Ser Glu Asp Thir Ala Met Tyr Cys
85 90 95

Ala Arg

<210s, SEQ ID NO 149

&211s LENGTH: 98
212. TYPE : PRT
 US 8,025,878 B2
139 140
- Continued
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 149

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Thr Tyr
2O 25 3O

Trp Met Thr Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val
35 4O 45

Ala Asn. Ile Llys Pro His Gly Ser Glu Ala Tyr Tyr Val Asp Ser Val
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn. Ser Lieu. Phe
65 70 7s 8O

Lieu. Glin Met Ser Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95

Ala Arg

<210s, SEQ ID NO 150

&211s LENGTH: 98
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 150

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
2O 25 3O

Trp Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val
35 4O 45

Ala Asn. Ile Lys Glin Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn. Ser Lieu. Tyr
65 70 7s 8O

Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95

Ala Arg

<210s, SEQ ID NO 151

&211s LENGTH: 57
&212s. TYPE: DNA
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 151
atggaattgg ggctgagctg ggttitt CCtt gttgctattt tagaaggtgt C cagtgt f

<210s, SEQ ID NO 152

&211s LENGTH: 19
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 152

Met Glu Lieu. Gly Lieu. Ser Trp Val Phe Lieu Val Ala Ile Lieu. Glu Gly
1. 5 1O 15

Val Glin Cys

<210s, SEQ ID NO 153

&211s LENGTH: 44
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
 US 8,025,878 B2
141 142
- Continued

<4 OOs, SEQUENCE: 153

Met Glu Lieu. Gly Lieu Ser Trp Val Phe Lieu Val Ala Ile Lieu. Glu Gly
1. 5 15

Val Glin Cys Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin
25

Pro Gly Gly Ser Lieu. Arg Lieu. Ser Cys Ser Ala Ser
35 4O

<210s, SEQ ID NO 154

&211s LENGTH: 124
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158 WH

<4 OOs, SEQUENCE: 154

Asp Val Glin Lieu Val Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly
1. 5 15

Ser Arg Llys Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Ser Phe
2O 25

Gly Met His Trp Val Arg Glin Ala Pro Glu Lys Gly Lell Glu Trp Wall
35 4O 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thir Ile Tyr Gly Asp Thir Wall
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Pro Asn Thir Luell Phe
65 70 7s

Lieu Gln Met Thr Ser Lieu. Arg Ser Glu Asp Thir Ala Met Tyr Cys
85 90 95

Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Thir Met Asp
1OO 105 11 O

Tyr Trp Gly Glin Gly. Thir Ser Val Thir Wall Ser Ser
115 12 O

<210s, SEQ ID NO 155

&211s LENGTH: 5
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158 WH CDR1

<4 OO > SEQUENCE: 155

Ser Phe Gly Met His

1. 5

SEQ ID NO 156
LENGTH: 17
TYPE : PRT
ORGANISM: Artificial
FEATURE:
OTHER INFORMATION: 158 WH CDR2

<4 OOs, SEQUENCE: 156

Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val Lys
1. 5 15

Gly

SEO ID NO 157
LENGTH: 15
TYPE : PRT
ORGANISM: Artificial
FEATURE:
 US 8,025,878 B2
143 144
- Continued
223 OTHER INFORMATION: 158 WH CDR3

<4 OO > SEQUENCE: 157

Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp Tyr
1. 5 1O 15

<210s, SEQ ID NO 158

&211s LENGTH: 123
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158RHA

<4 OOs, SEQUENCE: 158

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe
2O 25 3O

Gly Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val
35 4O 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val
SO 55 6O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn. Ser Lieu. Phe
65 70 7s 8O

Lieu. Glin Met Ser Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95

Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp
1OO 105 11 O

Tyr Trp Gly Glin Gly. Thir Thr Val Thr Val Ser
115 12 O

<210s, SEQ ID NO 159

&211s LENGTH: 30
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: AFO62243 FW1

<4 OOs, SEQUENCE: 159

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly
1. 5 1O 15

Ser Lieu. Arg Lieu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser
2O 25 3O

<210s, SEQ ID NO 160

&211s LENGTH: 14
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: AFO62243 FW2

<4 OOs, SEQUENCE: 160

Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val Ala
1. 5 1O

<210s, SEQ ID NO 161

&211s LENGTH: 32
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: AFO62243 FW3

<4 OOs, SEQUENCE: 161

 US 8,025,878 B2
145 146
- Continued
Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn. Ser Lieu. Phe Lieu. Glin
1. 5 1O 15

Met Ser Ser Lieu. Arg Ala Glu Asp Thir Ala Val Tyr Tyr Cys Ala Arg
2O 25 3O

<210s, SEQ ID NO 162

&211s LENGTH: 10
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: AFO62243 FW4

<4 OOs, SEQUENCE: 162

Trp Gly Glin Gly Thr Thr Val Thr Val Ser
1. 5 1O

<210s, SEQ ID NO 163

&211s LENGTH: 116
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: AFO62243

<4 OOs, SEQUENCE: 163

Cys Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly
1. 5 1O 15

Gly Ser Lieu. Arg Lieu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Thr
2O 25 3O

Tyr Trp Met Thr Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp
35 4O 45

Val Ala Asn. Ile Llys Pro His Gly Ser Glu Ala Tyr Tyr Val Asp Ser
SO 55 6O

Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn. Ser Lieu
65 70 7s 8O

Phe Leu Gln Met Ser Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr
85 90 95

Cys Ala Arg Ala Asn. Ser Lieu. Asp Val Trp Gly Glin Gly. Thir Thr Val
1OO 105 11 O

Thir Wal Ser Ser

115

<210s, SEQ ID NO 164

&211s LENGTH: 57
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: WH3- Of leader

<4 OOs, SEQUENCE: 164

atggaattgg ggctgagctg ggttitt CCtt gttgctattt tagaaggtgt C cagtgt f

<210s, SEQ ID NO 165

&211s LENGTH: 19
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: WH3- Of leader

<4 OOs, SEQUENCE: 165

Met Glu Lieu. Gly Lieu. Ser Trp Val Phe Lieu Val Ala Ile Lieu. Glu Gly
1. 5 1O 15

Val Glin Cys

 US 8,025,878 B2
147 148
- Continued

<210s, SEQ ID NO 166

&211s LENGTH: 90
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: AFO62243 FW1

<4 OOs, SEQUENCE: 166

gaggtgcagc tiggtggagtic tiggggaggc titggtCcagc CtggggggtC CCtgagactic 6O

t cct gttcag cct ctdgatt cacctittagt 9O

<210s, SEQ ID NO 167

&211s LENGTH: 15
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158 CDR1

<4 OOs, SEQUENCE: 167

agctttggaa toac 15

<210s, SEQ ID NO 168

&211s LENGTH: 42
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: AFO62243 FW2

<4 OOs, SEQUENCE: 168

tgggtcc.gc.c aggctcCagg gaaggggctg gagtgggtgg cc. 42

<210s, SEQ ID NO 169

&211s LENGTH: 51
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158 CDR2

<4 OOs, SEQUENCE: 169

tacatt agta gtggcagtag taccatctac tatggagaca Cagtgaaggg C 51

<210s, SEQ ID NO 170

&211s LENGTH: 96
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: AFO62243 FW3

<4 OOs, SEQUENCE: 170

cgatt cacca tot coagaga caacgc.caag aact cactgt ttctgcaaat gag cagoctd 6O

agagcc.gagg acacggcc.gt gtatt attgt gcgaga 96

<210s, SEQ ID NO 171

&211s LENGTH: 45
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158 CDR3

<4 OOs, SEQUENCE: 171

gaggggggat attact acgg taggagttac tatactatgg act ac 45

<210s, SEQ ID NO 172

&211s LENGTH: 30
&212s. TYPE: DNA
 US 8,025,878 B2
149 150
- Continued
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: AFO62243 FW4

<4 OOs, SEQUENCE: 172

tggggccaag ggaccacggt caccgt.ct Co 3O

<210s, SEQ ID NO 173

&211s LENGTH: 426
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: 158RHAss DNA sequence
<4 OOs, SEQUENCE: 173
atggaattgg ggctgagctg ggttitt CCtt gttgctattt tagaaggtgt C cagtgtgag 6O

gtgcagctgg tagt ctgg gggaggcttg gtc.ca.gc.ctg ggggg.tc.cct gagactic to C 12 O

tgttcagcct Ctggattcac ctittagtagc tittggaatgc actgggtc.cg C caggct coa 18O

gggaaggggc tiggagtgggt ggcct acatt agtag togca gtag taccat Ctact atgga 24 O
gacacagtga agggc.cgatt cac catct Co agaga caacg C caagaactic actgtttctg 3OO
caaatgagca gcctgaga.gc cgaggacacg gcc.gtgtatt attgttgc gag agagggggga 360
tatt actacg gtaggagitta ctatactatg gact actggg gccaagggaC cacggtcacc 42O

gtct co 426

<210s, SEQ ID NO 174

&211s LENGTH: 142
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
<223> OTHER INFORMATION: 158RHAss protein sequence
<4 OOs, SEQUENCE: 174

Met Glu Lieu. Gly Lieu. Ser Trp Val Phe Lieu Val Ala Ile Lieu. Glu Gly
1. 5 1O 15

Val Glin Cys Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin
2O 25 3O

Pro Gly Gly Ser Lieu. Arg Lieu Ser Cys Ser Ala Ser Gly Phe Thr Phe
35 4O 45

Ser Ser Phe Gly Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu.
SO 55 6O

Glu Trp Val Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly
65 70 7s 8O

Asp Thr Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95

Ser Lieu. Phe Lieu. Glin Met Ser Ser Lieu. Arg Ala Glu Asp Thir Ala Val
1OO 105 11 O

Tyr Tyr Cys Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr
115 12 O 125

Thr Met Asp Tyr Trp Gly Glin Gly Thr Thr Val Thr Val Ser
13 O 135 14 O

<210s, SEQ ID NO 175

&211s LENGTH: 426
&212s. TYPE: DNA
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158RHA

<4 OO > SEQUENCE: 175

 US 8,025,878 B2
153 154
- Continued
1. 5 1O 15

Val Glin Cys Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin
2O 25 3O

Pro Gly Gly Ser Lieu. Arg Lieu Ser Cys Ser Ala Ser Gly Phe Thr Phe
35 4O 45

Ser Ser Phe Gly Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu.
SO 55 6O

Glu Trp Val Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly
65 70 7s 8O

Asp Thr Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95

Ser Lieu. Phe Lieu. Glin Met Ser Ser Lieu. Arg Ala Glu Asp Thir Ala Val
1OO 105 11 O

Tyr Tyr Cys Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr
115 12 O 125

Thr Met Asp Tyr Trp Gly Glin Gly Thr Thr Val Thr Val Ser
13 O 135 14 O

<210s, SEQ ID NO 179

&211s LENGTH: 17
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158 WK

<4 OO > SEQUENCE: 179

Val Met Trp Tyr Gln Pro Leu Lieu. Ile Tyr Gly Gly Gly Thr Phe Tyr
1. 5 1O 15

Phe

<210s, SEQ ID NO 18O

&211s LENGTH: 17
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 18O
Val Met Trp Tyr Gln Pro Leu Lieu. Ile Tyr Gly Gly Gly Thr Phe Tyr
1. 5 1O 15

Phe

<210s, SEQ ID NO 181

&211s LENGTH: 17
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 181

Ile Met Trp Tyr Glin Pro Leu Lieu. Ile Tyr Gly Gly Gly Thr Phe Tyr
1. 5 1O 15

Phe

<210s, SEQ ID NO 182

&211s LENGTH: 17
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 182

Val Met Trp Tyr Gln Pro Leu Lieu Val Tyr Gly Gly Gly Thr Phe Tyr
1. 5 1O 15

Phe
 US 8,025,878 B2
155 156
- Continued

<210s, SEQ ID NO 183

&211s LENGTH: 112
212. TYPE: PRT
<213> ORGANISM: Artificial
22 Os. FEATURE:
223 OTHER INFORMATION: 158 WK

<4 OOs, SEQUENCE: 183

Asp Val Lieu Met Thr Glin Thr Pro Luell Ser Luell Pro Wall Ser Luell Gly
1. 5 15

Asp Glin Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Ile Wall His Ser
2O 25

Asn Gly Asn Thr Tyr Lieu. Glu Trp Luell Glin Pro Gly Glin Ser
35 4O 45

Pro Llys Lieu. Lieu. Ile Tyr Llys Val Ser Asn Arg Phe Ser Gly Wall Pro
SO 55 6O

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thir Asp Phe Thir Luell Ile
65 70

Ser Arg Val Glu Ala Glu Asp Lieu Gly Ile Tyr Phe Glin Gly
85 90 95

Ser His Val Pro Pro Thr Phe Gly Gly Gly Thir Lell Glu Ile Lys
1OO 105 11 O

<210s, SEQ ID NO 184

&211s LENGTH: 112
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 184

Asp Val Val Met Thr Glin Ser Pro Luell Ser Luell Pro Wall Thir Pro Gly
1. 5 15

Ala Pro Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Lell Luell His Thir
2O 25

Asn Gly Val Asn. Phe Lieu. Asp Trp Luell Glin Pro Gly Glin Ser
35 4O 45

Pro Llys Lieu. Lieu. Ile Tyr Lieu Ala Ser His Arg Ala Ser Gly Wall Pro
SO 55 6O

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thir Asp Phe Thir Luell Arg Ile
65 70

Ser Arg Val Glu Ala Glu Asp Wall Gly Ile Tyr Met Glin Gly
85 90 95

Lieu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thir Lell Glu Ile Lys
1OO 105 11 O

<210s, SEQ ID NO 185

&211s LENGTH: 112
212. TYPE: PRT
<213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 185

Asp Val Val Met Thr Glin Ser Pro Luell Ser Luell Pro Wall Thir Pro Gly
1. 5 1O 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Lell Luell His Ser
2O 25 3O

Asn Gly Tyr Asn Tyr Lieu. Asp Trp Luell Glin Pro Gly Glin Ser
35 4O 45

Pro Glin Lieu. Lieu. Ile Tyr Lieu. Gly Ser Asn Arg Ala Ser Gly Wall Pro
SO 55 6O