Note: Descriptions are shown in the official language in which they were submitted.
CA 02607771 2007-11-01
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CA 02607771 2007-11-01
1 HUMANIZED ANTI-VENEZUELAN EQUINE ENCEPHALITIS VIRUS
2 RECOMBINANT ANTIBODY
3 FIELD OF THE INVENTION
4 [0001] The present invention relates to a humanized antibody (Ab) and, more
specifically, to a
humanized recombinant Ab (rAb) directed to the Venezuelan equine encephalitis
virus (VEEV).
6 BACKGROUND OF THE INVENTION
7 [0002] Venezuelan equine encephalitis virus (VEEV), a member of the
alphavirus genus of the
8 family Togaviridae, is an important mosquito-borne pathogen in humans and
equides ['t]. VEEV
9 infections mainly target the central nervous system and lymphoid tissues
causing severe
encephalitis in equines and a spectrum of human diseases ranging from
unapparent or sub-clinical
11 infection to acute encephalitis. Neurological disease appears in 4-14% of
cases. The incidence of
12 human infection during equine epizootics could be up to 30%. Mortality
associated with the
13 encephalitis in children is as high as 35%. Recent outbreaks in Venezuela
and Colombia in 1995
14 resulted in around 100,000 human cases with more than 300 fatal
encephalitis cases [2].
Furthermore, VEEV is highly infectious by aerosol inhalation in humans and
other animals.
16 However, there are no antiviral drugs available that are effective against
VEEV although currently
17 there are two forms of IND (investigational new drug) VEEV vaccines
available for human and
18 veterinary use: TC-83, a live-attenuated Trinidad donkey strain and C-84, a
formalin-inactivated TC-
19 83 [3,4]. However, for various reasons, these vaccines are far from
satisfactory. For example,
approximately 20% of recipients that receive the TC-83 vaccine fail to develop
neutralizing Abs,
21 while another 20% exhibit reactogenicity. In addition, the TC-83 vaccine
could revert to wild-type
22 form. The vaccine C-84 is well tolerated, but requires multiple
immunizations, periodic boosts, and
23 fails to provide protection against aerosol challenge in some rodent
models.
24 10003] Like the other alphaviruses, VEEV is an enveloped virus, consisting
of three structural
proteins: a capsid encapsidating the viral RNA genome, and two envelope
glycoproteins, El and
26 E2. El and E2 form heterodimers, which project from the virus envelope as
trimer spikes. Epitopes
27 on the spikes are the targets of neutralizing Abs. Studies have shown that
the viral neutralizing
28 epitopes are mainly located on the E2 protein, and that the E2c epitope
appears to be the hub of the
29 neutralization epitopes [5,6]. The murine monoclonal Ab (mAb) 1A1A4 [14] is
specific far E2c. This
mAb has been shown to be efficient in protecting animals from a lethal
peripheral challenge with
31 virulent VEEV [7].
32 [0004] Murine mAbs, however, have serious disadvantages as therapeutic
agents in humans
33 [8]. For example, one of the problems associated with using murine mAbs in
humans is that they
34 may induce an anti-mouse Ab response. Further, repeat administration of
murine mAbs may result
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CA 02607771 2007-11-01
I in rapid clearance of the murine mAbs and anaphylaxis, which can sometimes
be fatal. To
2 overcome this hurdle, the humanization of murine mAbs has been proposed, by
which process
3 murine Ab frameworks are replaced by human Ab ones in order to reduce
immunogenicity of Abs in
4 humans [9,10].
[0005] Thus, a need exists for a humanized anti-VEEV Ab.
6 SUMMARY OF THE INVENTION
7 100061 In one aspect, the present invention provides prophylaxis and post-
exposure therapy
8 against VEEV infection.
9 [0007] In one aspect, the invention provides a humanized rAb comprising a
human
immunoglobulin (!g) framework and having grafted thereon complementarity
determining regions
11 (CDRs) from the murine mAb 1A4A1.
12 [0008] In another aspect, the invention provides a humanized rAb having
specificity to the E2
13 envelope protein of VEEV. More specifically, the rAb has spectficity to the
E2c epitope of the E2
14 protein.
[0009] In another aspect, the inventlon provides a humanized rAb wherein the
complementarity
16 determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region
(VH) have the
17 foliowing amino acid sequences:
18 CDR1: SEQ ID NO: 1
19 CDR2: SEQ ID NO: 2
CDR3: SEQ ID NO: 3.
21 [0010] In another aspect, the invention provides a humanized rAb wherein
the compiementarity
22 determining regions CDRI, CDR2 and CDR3 of the light chain variable region
(VL) have the
23 foiiowing amino acid sequences:
24 CDR1: SEQ iD NO: 4
CDR2: SEQ ID NO: 5
26 CDR3: SEQ ID NO: 6.
27 [0011] In a further aspect, the invention provides a humanized rAb having a
VH comprising the
28 amino acid sequence of SEQ ID NO: 7.
29 [0012] In a further aspect, the Invention provides a humanized rAb having a
VL comprising the
amino acid sequence of SEQ ID NO: 8.
31 [0013] In another aspect, the invention provides a DNA sequence which
encodes a polypeptide
32 corresponding to a CDR grafted VH having the amino acid sequence according
to SEQ ID NO: 7_
33 10014] In another aspect, the invention provides a DNA sequence which
encodes a polypepfide
34 corresponding to a CDR grafted VL having the amino acid sequence according
to SEQ ID NO: 8.
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CA 02607771 2007-11-01
1 [0015] In a further aspect, the invention provides a DNA construct having a
nucleic acid
2 sequence according to SEQ ID NO:1 1 or SEQ ID NO:13.
3 [0016] In another aspect, the invention provides an expressed protein
comprising a humanized
4 rAb having an amino acid sequence according to SEQ ID NO: 12 or SEQ ID NO:
14.
[0017J The invention provides vectors containing such DNA sequences and host
cells
6 transformed thereby.
7 [0018] in other aspects, the invention provides methods and uses for
treatment or prophylaxis of
8 VEEV infection utiiizing the rAbs described herein. The invention also
provides pharmaceuticai
9 preparations for such treatment or prophylaxis.
BRIEF DESCRIPTION OF THE DRAWINGS
11 (0019] These and other features of the invention will become more apparent
in the following
12 detailed description in which reference is made to the appended drawings
wherein:
13 [0020] Figure 1 is a representation of the extemai structure of the VEEV.
14 [0021] Figures 2a to 2d schematically iliustrate murine, human, chimeric
and humanized Abs,
respectively.
16 [0022] Figures 3a to 3c schematically illustrate the humanization of the
murine Ab variabte
17 region.
18 [0023] Figure 4 schematically illustrates the cloning of the murine Ab VH
and VL.
19 [0024] Figure 5 schematically illustrates the humanization of the Ab VH and
shows its amino
acid sequence.
21 10025] Figure 6 schematically illustrates the humanization of the Ab VL and
shows its amino
22 acid sequence.
23 [0026] Figure 7 schematicaiiy iiiustrates the design of a full Hu1A4A1IgG1
rAb gene in a single
24 open reading frame with two versions, Hu1A4A1 igG1-furin and Hu1A4A1 IgG1-
2A.
[0027] Figure 8 schematicaiiy illustrates the cloning of the Hu1A4A1IgG1-furin
and
26 Hu1A4A1igG1-2A genes into an adenoviral vector respectively.
27 [0028] Figure 9 schematically illustrates expression and purification of
the Hu1A4A1IgG1 furin
28 and Hu1A4A1IgG1-2A rAbs.
29 [0029] Figures 10 and 11 illustrate the resutts from the SDS-PAGE
separation of the produced
Hu1A4A1IgG1 furin rAb.
31 [0030] Figure 12 illustrates the results from the sodium dodecyl sulfate-
polyacrylamide gel
32 electrophoresis (SDS-PAGE) separafion of the produced Hu1A4A11gG1 2A rAb.
33 [0031] Figure 13 illustrates the results of the enzyme-linked immunosorbent
assays (ELISA) for
34 the reactivity of the Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A rAbs.
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CA 02607771 2007-11-01
1 [0032] Figure 14 schematically illustrates Hu1A4A1 igG1-2A was cleaved
between the heavy
2 and light chains as expected, whereas Hu1A4A11gG1-furin was not cleaved.
3 [0033] Figure 15 schematically illustrates the neutralization assay used in
assessing the
4 neutralizing activity of the Hu1A4A11gG1-furin and Hu1A4A11gG1-2A rAbs
against VEEV.
DETAILED DESCRIPTION OF THE INVENTION
6 [0034] Figure 1 illustrates the external structure of the VEEV. As shown,
the virus 10 includes a
7 nucleocapsid 12 enveloping the viral RNA genome. The envelope comprises
glycoproteins El and
8 E2, arranged in the form of heterodimers 14. Protein E2, which is
responsible for viral atfachment to
9 the host cell, contains neutralizing epitopes.
[0035] As has been described in the prior art, the murine mAb 1A4A1 has been
found to be
11 specific to the VEEV E2 envelope protein and, further, has been found to
have a strong neutralizing
12 function against VEEV. The murine mAb, however, causes a sometimes fatal
allergenic reaction in
13 humans, resulting in the formation of human anti-mouse Abs (HAMA). It is
for this reason that the
14 present inventors have sought to humanize the 1A4A1 mAb so as to provide an
effective agent to
counter VEEV infection in humans.
16 [0036] In vivo efficacy studies in mice have demonstrated that treatment
with murine mAb
17 1A4A1 leads to protection of animals from a lethal peripherai challenge
with virulent VEEV. Thus,
18 the present invention builds upon these findings by providing a humanized
mAb 1A4A1 to reduce the
19 foreignness of murine mAb in humans. For doing this, the majority of the
non-human protein
sequence (in one embodiment, more than 90%) of mAb 1A4A1 is replaced with a
human Ab
21 sequence and the resultant whole humanized mAb gene is then synthesized and
cloned to an
22 adenoviral vector. The recombinant adenoviral vector can be delivered as a
therapeutic agent for
23 prophylaxis or treatment of VEEV infection in humans. One advantage of this
method is that the
24 vector can express the humanized Ab in the human body for a long period of
time. The humanized
Ab can also be produced In cell culture and delivered directly as a
therapeutic.
26 [0037] The humanization of the present anti-VEEV mAb 1A4A1 has not been
done previously
27 and particularly not for the prophylaxis or treatment of VEEV infection.
The present invention
28 provides in one embodiment a humanized Ab, referred to herein as
Hu1A4A1IgG1, that retains the
29 VEEV-binding specificity and neutralizing activity of murine 1A4A1 while
not eliciting a HAMA
response. As described further below, the humanized Ab comprises an Ig
framework of human
31 IgG1 and CDRs obtained from murine mAb 1A4A9. The rAb of the present
invention is specific to
32 an epitope of the E2 envelope glycoprotein of VEEV and, more specifically,
to the E2` epitope
33 thereon.
34 [0038] The construction of the humanized Ab of the invention is
schematically illustrated in
Figures 2a to 2d. Figure 2a illustrates schemat'scally the structure of a
murine Ab 16 containing
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CA 02607771 2007-11-01
1 murine CDRs 18 on the respective variable regions. Figure 2b shows a human
Ab 20 containing
2 human CDRs 22. As shown in Figure 2c, a chimeric Ab 26 would comprise the
murine variable
3 regions 24, containing the murine CDRs 18, joined to the constant regions of
the human Ab. On the
4 other hand, Figure 2d illustrates a humanized Ab 28 according to an
embodiment of the invention,
wherein only the murine CDRs 18 are grafted to the variabte regions of the
human Ab 20.
6 [0039] The substitution of the murine CDRs into the human ig framework is
illustrated also in
7 Figures 3a to 3c. As shown, the humanized Ab variabie region comprises the
grafted CDRs, 18,
8 from the murine Ab.
9 [0040] The protein sequences of the rAbs of the invention include linker
sequences. The
expressed rAbs of the invention have amino acid sequences as shown in SEQ ID
NO:12 and SEQ
11 ID NO:14. The nucleic acid constructs used in transfecting cells to express
the above rAbs are
12 shown in SEQ ID N0:11 and SEQ ID NO:13.
13 [0041] Examples
14 [0042] The following examples are provided to illustrate embodiments of the
present invention.
The examples are not intended to limit the scope of the invention in any way.
16 [0043] Example 1: Construction of Hu1A4A1iaG1 and In vitro studies
17 [0044] In the study described below, murine mAb 1A4A1 CDRs of VH, VL were
grafted onto the
18 frameworks of germline variabie and joining (V, J) gene segments of human
Ig heavy and light
19 chains, respectively, which were chosen based on the CDR simiiarities
between human Igs and
murine mAb 1A4A1. Furthermore, the humanized VH and VL were, respectivety,
grafted onto
21 human gamma 1 heavy chain constant regions (CHs) and kappa 1 light chain
constant region (CL)
22 to assemble the whole humanized Ab gene. The resultant whole humanized mAb
gene was
23 synthesized and cloned to an adenoviral vector. After the humanized Ab was
expressed in HEK 293
24 cells and purified with protein L column, the Ab was demonstrated to retain
antigen-binding
specificity and neutralizing activity.
26 [0045] Materials and Methods
27 [0046] Humanization of murine mAb 1A4A1
28 [0047] Murine mAb 1A4A1 was provided by Dr. J.T. Roehrig (Division of
Vector-borne Infectious
29 Diseases, Centers for Disease Control and Prevention, Fort Colins, CO,
USA). The VH and VL of
mAb 1A4A1 were cloned in a single chain variable fragment (ScFv) format, mA116
previously [7],
31 which showed to retain the same binding specificity as mAb 1A4A1 [11]. The
humanization of VH
32 and VL of murine mAb 1A4A1 was done by Absaius Inc. (Mountain View, CA,
USA). Sriefly, in order
33 to select human VH and VL frameworks 1-3, the VH and VL amino acid
sequences of murine 1A4A1
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1 were separately subjected to IgBiast and IMGT searches against the entire
human lg germline V
2 gene segments and then human heavy and tight chain germiine V gene segments
were selected
3 based on their highest CDR 1 and 2 similarities with those of murine 1A4A1
VH and VL without
4 consideration of framework sirnilarity. Both human VH and VL framework 4
were selected,
respectively, from human heavy and light chain J gene segments based on the
highest similarities
6 between human J gene segments and murine 1A4A1 VH and VL CDR3. Finally, CDRs
of murine
7 1A4A1 VH and VL were, respectively, grafted onto the frameworks of selected
germline V and J
8 gene segments of human Ab heavy and light chains, resulting in humanized
1A4A1 (Hu1A4A1).
9 Furthermore, the Hu1A4A1 VH and VL were, respectively, grafted onto human
gamma 1 heavy
chain CHs and kappa 1 light chain CL to assemble the whole humanized Ab gene,
resulting in
11 humanized 1A4A1IgG1 (Hu1A4A1 IgGI). This process is illustrated in Figures
3 to 6.
12 [0048] Construction, exoression and ourification of Hu1A4A1IaG1
(Hu1A4A1IaG1-furin and
13 Hu1A4A11aG1-2A)
14 [0049] The Hu1A4A11gG1 DNA sequence (-2 kb) is schematically illustrated in
Figure 7. The
nucleic acid sequence of the Hu1A4A1IgG1-furin rAb Is provided In SEQ ID NO:11
and the nucleic
16 acid sequence of the Hu1A4A1igG1-2A rAb is provided in SEQ ID NO:13.
17 [0050] The Hu1A4A1IgG1 DNA sequences were synthesized as follows. As shown
in Figure 7,
18 a light chain leader sequence was provided upstream from the light chain,
followed by a furin or 2A
19 linker (discussed further below) before the heavy chain. The whole DNA
sequence flanked by Kpn i
and Hind Iil was synthesized by GenScript Corporation (Scotch Plaines, NJ,
USA) and cloned into
21 pUC57 vector, resulting in pUC57-Hu1A4A1IgG1 furin or pUC57-Hu1A4A1IgG1-2A.
22 [0051] Recombinant adenovirus vectors expressing either Hu1A4A1IgG1 furin
or
23 Hu1A4A1IgG1-2A were constructed using AdEasyTM system (Qbiogene, Carlsbad,
CA, USA)
24 according to the manufacturer's protocol. Briefly, the Kpn I-Hind III
fragment of Hu1A4A11gG1 furin
or Hu1A4A1IgG1-2A was ligated to a Kpn 1-Hind III-digested pShuttle-CMV
vector. The resulting
26 pShuttle construct was co-transformed with the pAdEasy-1 vector into
Escherichia coil BJ5183 cells
27 to produce recombinant adenoviral genomic constructs for Hu1A4A1IgG1-furin
or Hu1A4A11gG1-2A
28 proteins. The recombinant adenoviral constructs, pAd-Hu1A4A1IgG1 furin and
pAd-Hu1A4A1IgG1-
29 2A were linearized with Pac I and transfected into HEK 293 cells (American
Type Culture Collection,
Manassas, VA, USA) cultured in Duibecco's Modified Eagle's Medium supplemented
with 5% fetal
31 bovine senim (FBS) for amplificafion and then the amplified adenovirus was
purified by a
32 chromatographic method. This procedure is illustrated in Figure 8.
33 [0052] As illustrated in Figure 9, the expression of Hu1A4A1IgG1-furin or
Hu1A4A1IgG1-2A was
34 achieved by first infecting HEK 293 cells with the recombinant adenovirus
pAd-Hu1A4A1 IgG1-furin
or pAd-Hu1A4A11gG1-2A at a multiplicity of infection (MOI) of 1. The infected
cells were cultured for
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CA 02607771 2007-11-01
1 one week and the culture supematant was harvested. The expressed Hu1A4A1
igG1 furin or
2 Hu1A4A1 igG1-2A was purified using protein L agarose gel from Pierce
(Brockville, Ont., Canada).
3 Briefly, culture supematant was dialyzed against phosphate buffer saline
(PBS) (Sigma-Aldrich,
4 Oakville, Ont., Canada) for 12h and then concentrated using PEG (Sigma-
Aidrich) to less than 50
ml. The concentrated sample was incubated with 2ml protein L agarose gel at 4
C for 1 h. The gel
6 and supernatant mixture was then loaded to an empty column, which was
subsequently washed with
7 binding buffer. Bound Hu1A4A11gG1-furin or Hu1A4A1IgG1-2A was eluted with
elution buffer. The
8 eiuted Ab was further desalted using an exceilulose column (Pierce) and then
concentrated by a
9 Centracon"" YM-30 (Millipore Corp., Bedford, MA, USA).
[0053] The amino acid sequence of the expressed Hu1A4A11gG1-furin is shown in
SEQ ID
11 NO:12 and the amino acid sequence of the expressed Hu1A4A1IgG1-7A is shown
in SEQ ID NO:14.
12 [0054] SDS-PAGE
13 [0055] Abs were separated by 10% SDS-PAGE gels using a Mini-PROTEANT"' II
apparatus
14 (Bio-Rad Laboratories, Mississauga, Ont., Canada). The bands were
visualized by SimplyBlueTM
safestain staining (invitrogen, Burlington, Ont., Canada). The moiecular
weights of the samples
16 were estimated by comparison to the relative mobility values of standards
of known molecular
17 weights. The SDS-PAGE analyses of the purified Hu1A4A1IgG1-furin are
illustrated in Figures 10
18 and 11. Figure 12 illustrates the SDS-PAGE analysis of the purified
Hu1A4A1IgG1-2A. As shown,
19 lanes I and 3 correspond to purified Hu1A4A11gG1 and control human IgG1 in
a non-reducing
condition and lanes 2 and 4 correspond to purifled Hu1A4A11gG1 and control
human IgG1 in a
21 reducing condition.
22 [0056] ELISA
23 [0057] The reacctivity of purfied Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A to
VEEV E2 antigen
24 was determined by ELISA. Nunc MaxisorpTM flat bottomed 96-well plates
(Canadian Life
Technologies, Burlington, Ont., Canada) were coated overnight at 4 C with
recombinant VEEV E2
26 antigen at a concentration of 10ug/mI in carbonate bicarbonate buffer, pH
9.6. The plates were
27 washed five times with PBS containing 0.1% Tween'" -20 (PBST) and then
blocked in 2% bovine
28 serum albumin fvr 2h at room temperature. After five washes with PBST, the
plates were incubated
29 for 2h at room temperature with various concentrations of Hu1A4A1IgG1-
furin, Hu1A4A1igG1-2A or
1A4A1 Abs diluted in PBST. Following five washes with PBST, the plates were
incubated for 2h at
31 room temperature with horseradish peroxidase (HRP)-conjugated rabbit anti-
human IgG fragment
32 crystallizable portion or HRP-conjugated rabbit anti-mouse IgG (Jackson
lmmunoResearch
33 Laboratories inc., West Grove, PA, USA) diluted 1:5000 in PBST. Finally,
the plates were washed
34 five times with PBST and developed for 10 min at room temperature with a
3,3',5,5'-
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CA 02607771 2007-11-01
1 tetramethylbenzidine substrate (Kirkegaard and Perry Laboratories). The
reactions were read at an
2 absorbance of 650 nm by a microplate autoreader (Molecular Devices,
Sunnyvale, CA, USA). The
3 results of the ELISA Hu1A4A1IgG1-antigen binding assay are illustrated in
Figure 13.
4 [0058] Neutralization assay in vitro
[0059] Neutralizing activity of each of Hu1A4A11gG1 furin and Hu1A4A11gG1-2A
against VEEV
6 (strain TC-83) was analyzed by a plague reduction assay. Briefly, each Ab
was serially two-fold
7 diluted and mixed with an equal volume containing 50 plaque-forming units of
virus per 100 pl. After
8 mixtures were incubated for I h at room temperature, 200 pl of the mixture
was inoculated in
9 duplicate into wells of six-well plates containing confluent Vero cell
monolayers and incubated at
37 C for I h. At the end of the incubation, the virus/Ab mixtures were removed
from the wells before
11 the wells were overlaid by tragacanth gum and then incubated for 2 days.
The wells were stained
12 with 0.3% crystal violet and plaques were counted. Neutralization titre was
expressed as the highest
13 Ab dilution that inhibited 50% of virus plaques. This procedure is
illustrated in Figure 15.
14 [0060] Results and Discussion
[0061] Different approaches have been developed to humanize murine Abs in
order to reduce
16 the antigenicity of murine Abs in humans [9,10]. One widely used approach
is CDR-grafting, which
17 involves the grafting of all murine CDRs onto a human Ab frameworks. The
human Ab frameworlcs
18 are chosen based on their similarities to the frameworks of the murine Ab
to be humanized. The
19 CDR-grafting approach has been proven successful in some cases. Mowever, in
many more
instances, this humanization process could result in CDR conformabion changes,
which affect the
21 antigen-binding affinity. To restore the affinity, additional work for back-
mutation of several murine
22 framework amino acids, whlch are deemed to be critical for CDR loop
conformation, have to be
23 done.
24 [0062] Recently, Hwang et al. [121 employed an approach which consisted of
grafting CDRs
onto human germline Ab frameworks based on the CDR sequence similarities
between the murine
26 and human Abs while basically ignoring the frameworks. Because the
selection of the human
27 frameworks is driven by the sequence of the CDRs, this strategy minimizes
the differences between
28 the murine and human CDRs. This approach has the potential to generate
humanized Abs that
29 retain their binding affinity to their cognate ant{gen. Further, since all
residues in frameworks are
from human Ab germline sequences, the potential immunogenicity of non-human
Abs is highly
31 reduced.
32 [0063] Using the above approach, and as disclosed herein, the present
inventors humanized an
33 anti-VEEV murine mAb 1A4A1. The amino acid sequences of VH and VL from
murine 1A4A1 were
34 first aligned with human lg germline V and J genes. As shown in Figure 5,
the human heavy chain V
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CA 02607771 2007-11-01
1 gene segment H5-51 and J gene segment JH4 were selected to provide the
frameworks for the
2 murine 1A4A9 VH. Similariy, as shown in Figure 6, for the murine 1A4A1 VL,
the human light chain
3 V gene segment L15 and J gene segment Jk3 were selected.
4 [0064] The identities of the CDRI and CDR2 amino acid sequences between
murine 1A4A1 VH
and the human H5-51 gene segment were 20% and 47%, respectively, while the
identity of the
6 CDR3 between murine 1A4A1 VH and the JH4 gene segment was 33%. For the light
chain, the
7 identities of the CDR1 and CDR2 between mudne 1A4A1 VL and the human L15
gene segment
8 were 27% and 14%, respectively, while the identity of the CDR3 between
murine 1A4A1 VL and
9 human Jk3 gene segment was 22%. The CDRs of murine 1A4A1 VH were then
grafted onto the
frameworks of selected human ig germline H5-51 and JH4 gene segments, while
the CDRs of
11 murine 1A4A1 VL were grafted onto human L15 and Jk3 gene segments. The
hu1A4A1 VH was
12 further grafted onto the human gamma 1 heavy chain CHs to form a complete
heavy chain, while the
13 VL was grafted onto the human kappa I light chain CL to form a whole
humanized light chain. This
14 procedure is schematically illustrated in Figures 5 and 6 with the end
structure being illustrated in
Figure 7.
16 [0065] As shown in Figure 5, the murine 1A4A1 VH CDRs grafted onto the
human framework
17 comprised the following amino acid sequences:
18 VH CDRI: DYHVH (SEQ ID NO: 1)
19 VH CDR2: MTYPGFDNTNYSETFKG (SEQ ID NO: 2)
VH CDR3: GVGLDY (SEQ ID NO: 3)
21 [0066] As shown in Figure 6, the murine 1A4A1 VL CDRs grafted onto the
human framework
22 comprised the foliowing amino acid sequences:
23 VL CDR1: KASQDVDTAVG (SEQ ID NO: 4)
24 VL CDR2: WSSTRHT (SEQ ID NO: 5)
VL CDR3: HQYSSYPFT (SEQ ID NO: 6)
26 [0067] As shown in Figure 5, the VH of the humanized Ab according to the
present invention
27 comprises the following amino acid sequence:
28 Hu-VH:
29 EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYHVHWVRQMPGKGLEWMGMTYPGFDNTNYSETF
KGQVTISADKSiSTAYLQWSSLKASDTAMYYCARGVGLDYWGQGTLVTVSS (SEQ ID NO: 7).
31 [0068] Thus, as shown in Figure 6, the VL of the humanized Ab according to
the present
32 invention comprises the following amino acid sequence:
33 Hu-VL:
34 DIQMTQSPSSLSASVGDRVTITCKASQDVDTAVGWYQQKPEKAPKSLIYWSSTRHT
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSY'PFTFGPGTKVDIKR (SEQ ID NO: 8).
2i694804_1 g
CA 02607771 2007-11-01
1 [0069] In order to express heavy and light chains in a monocistronic
construct, a six-residue
2 peptide, RGRKRR (SEQ ID NO: 9) containing the recognition site for the
protease furin, designated
3 as "fur3n linker", or a twenty-four-residue peptide of the foot-and-mouth-
disease virus (FMDV)-
4 derived 2A self-processing sequence, APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:
10),
designated as "2A linker", was incorporated between the two chains. The
location of the furin or 2A
6 linker within the nucleic acid constructs of the Abs is illustrated in
Figure 7. Furin is a ubiquitous
7 subtilisin-like proprotein convertase, which is the major processing enzyme
of the secretory pathway
8 [131. The furin minimal cleavage site is R-X-X-R; however, the enzyme
prefers the site R-X-
9 (K/R}-R. An additional R at the P6 position appears to enhance cleavage. The
FMDV-derived 2A
linker is able to cleave at its own C terminus between the last two residues
through an enzyme-
11 independent but undefined mechanism, probably by ribosomal skip, during
protein translation. To
12 get the expressed Ab to be secreted to culture media, a leader sequence was
added upstream to
13 the Ab gene. Figure 7 illustrates the synthesized DNA sequence, of
approximately 2 kb, including
14 the human Ab kappa light chain L15 leader sequence, the humanized light
chain (VL + CL), the furin
or 2A linker, and the humanized heavy chain (VH + CHI + CH2 + CH3). This
sequence was then
16 cloned into an adenoviral vector. The unique restriction sites, as also
shown in Figure 7, ftanking the
17 V regions, which allow for efficient V region replacement and at the heavy
chain V-C region junction
18 for generation of fragment antigen-binding portion of Ab (Fab), were also
designed.
19 [0070] Protein G and A columns are widely used for a quick purification for
Abs because of
protein G and A binding to the Fc portion of [g. However, protein G and A
cannot only bind to
21 human Ig, but also bind to bovine lg, therefore they cannot be used for
puMcation of Hu1A4A1IgG1-
22 furin or Hu1A4A11gG1-2A in our study since pAd-Hu1A4A11gG1-furin or pAd-
Hu1A4A11gG1-2A-
23 infected HEK 293 cells were cultured in the medium with 5% FBS containing a
high percentage of
24 bovine Ig. Unlike protein G and A, protein L binds Ig through interactions
with the light chains.
Protein L only binds to Ig containing light chains of type kappa 1, 3 and 4 in
human and kappa I in
26 mouse. Most importantly, protein L does not bind to bovine Ig. Since our
humanized Ab has human
27 kappa 1 chain, we chose a protein L column to purify Hu1A4A11gG1-furin or
Hu1A4A1 fgG1-2A to
28 eliminate co-purification of bovine Ig. In this way, the purity of
Hu1A4A11gG1 -furin or Hu1A4A11gG1-
29 2A was relatively high in SDS-PAGE as shown in Figures 10, 11 and 12.
[0071] When the purffied product was subjected to 10% SDS-PAGE, HuIA4AIIgGI-
furin and
31 Hu1A4AIIgGI-2A showed up in a differentway. As illustrated in Figure 12,
Hu1A4A11gG1-2A
32 showed the same pa:ttems as a control human IgG1, one band of -150kDa in
non-reducing condition
33 (intact disulfide bridges) and two bands, 5OkDa for heavy chains and 25kDa
for light chains (broken
34 disulfide bridges) In reducing condition, indicating that the 2A linker
underwent self-processing
peri`ectty. On the other hand, Hu1A4A1fgG1-furin showed only one clear band of
--75kDa in
36 reducing condition observed as illustrated in Figures 10 and 11, indicating
that the furfn finker was
21694804.1 10
CA 02607771 2007-11-01
1 not cleaved. However, in another study (data not shown), the same furin
linker sequence was
2 cleaved in another Fab construct expressed in a mammalian system. This
indicated the
3 conformation of expressed Hu1A4A11gG1-furin probably rendered the furin
linker inaccessible to
4 furin or that the sequence surrounding the furin linker influenced furPn
cleavage.
[0072] The specific binding reactivities of purified Hu1A4A1igG1-furin and
Hu1A4A11gG1-2Ato
6 VEEV E2 antigen were examined by EUSA. As illustrated in Figure 13, both
versions of the
7 Hu1A4A1 IgG1 were found to bind to VEEV E2 In a dose-dependent manner,
similar to the binding to
8 VEEV E2 of its parental murine 1A4A1, indicating this non-cleaved Ab was
still reactive to VEEV E2
9 antigen in ELISA. Furthermore, both versions were evaluated for their
ability to block VEEV infection
in Vero cells using a standard plaque-reduction assay. The Hu1A4A1IgG1-fruin
showed a
11 neutralizing activity with 50% plaque reduction neutralization titer at
0.78 lag/ml, whereas
12 Hu1A4A1 igG1-2A showed a much higher neutralization titre at 0.1 isg/mi.
13 [0073] From the above results, it is concluded that the murine 1A4A1 Ab was
successfully
14 humanized. As illustrated in figure 14, the expressed and purified Ab of
Hu1A4A11gG1-2A was
cleaved between the heavy and -ight chains as expected;; however, Hu1A4A1 IgG1-
furin was not
16 cleaved. Nevertheless, the present inventors have exhibited that both
versions of the Hu1A4A91gG1
17 retained the anttgen binding specificity and virus neutralizing activity.
Thus, the Hu1A4A11gG1-furin
18 or Hu1A4A1igG1-2A discussed and characterized herein would serve as an
effective prophylactic
19 and therapeutic agent against VEEV infecdon.
[0074] Example 2: In vivo study - Protection of mice from VEEV chaiienae by
passive
21 immuniration with Hu1A4A1iaG1 furin orHu1A4A1iaG1-2A
22 [00751 Materials and methods
23 [0076] Passive immunization
24 [0077) Balb/c mice aged 6-8 weeks were injected intraperitoneally (i.p)
with 50 Ng of
Hu1A4A1(gG1-furin or Hu1A4A1IgG1-2A in 100 pl PBS, human anti-VEEV lgG in 100
lai PBS
26 (positive control) or 100 ul PBS alone (negative control) 24 h priorto VEEV
challenge.
27 [0078] VEEV chailensEe
28 [0079] Each mouse was challenged subcutaneously (s.c.) with 30-50 plaque
forming units (pfu)
29 of virulent VEEV (Trinidad donkey, TRD) in 50 pI of Leibovitz L15
maintenance medium (L15MM) 24
h after passive immunization. The challenge dose approximated to 100 x 50%
lethal dose (LD50).
31 Mice were examined frequently for signs of illness for 14 days, and humane
endpoints were used.
21694ao4.1 11
CA 02607771 2007-11-01
1 [0080] Results
2 [0081] Hu1A4A11aG1-furin or Hu1A4A1 iqG1-2A clearance in mice
3 100821 To determine the haif-Ãife of Hu1A4A1IgG1 fttrin or Hu1A4A11gG1-2A in
mouse serum,
4 groups of 4 mice, were injected i.p. with 50 pg, each mouse, of either
Hu1A4A1 IgG1 -furin or
Hu1A4A1IgG1-2A, or human anti-VEEV IgG and bled from the vein at increasing
time intervals after
6 injection. The quantity of Ab present in serum samples was estimated by
imrnunoassay.
7 Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A had a similar haif-life as human anti-
Vl'*EV igG, around 10
8 days.
9 [0083] Protection of mice from VEEV challenge by passive immunization with
HuiA4A11gG1-
furin or Hu1A4A1IgG1-2A
11 [0084] Groups of 8 mice were injected i.p. with the Hu1A4A1IgG1-furin,
Hu1A4A1IgG1-2A,
12 human anti-VEEV IgG or PBS alone and 24h later challenged s.c. with 100 x
LD50 of VEEV. None
13 of the PBS alone treated mice survived. All the Hu1A4A1IgG1-furin or
Hu1A4A1IgG1-2A treated
14 mice survived the VEEV challenge without any clinical signs at 14 days post-
challenge.
[0085] Discussion
16 [0086] Passive immunization of the Hu1A4A1 igGl-furin or Hu1A4A1 IgG1-2A in
mice (50
17 pg/mouse) 24 h before virulent VEEV challenge provided 100% protection
against 100xLD50
18 challenge of VEEV when mice were treated with 50 pg/each mouse of Hu1A4A1
IgG1 furin or
19 Hu1A4A1IyG1-2A. The mice were also found to be asymptomatic throughout the
14 day
observation period. These results indicate that the humanized anti-VEEV rAbs
of the present
21 invention has prophylactic capacity against VEEV infections. The half-lives
of the humanized anti-
22 VEEV rAbs in mice was around 10 days suggesting that the humanized anti-
VEEV rAbs of the
23 invention would be an effective prophylactic agairtst VEEV for at Ãeast
several weeks.
24 [0087] Bibliography
[0088] One or more of the following documents have been referred to in the
present disclosure.
26 The following documents are incorporated herein by reference in their
entirety.
27 [0089] [17 Weaver SC, Ferro C, Barrera R, Boshell ,J, Navarro JC.
Venezuelan equine
28 encephaÃitis. Annu Rev Entomol 2004;49:141-74.
29 [0090] [2] Rivas F, Diaz LA, Cardenas VM, Daza E. Bruzon L, Alcaia A, et
al. Epidemic
Venezuelan equine encephalitis in La Guajira, CoiombÃa, 1995. J Infect Dis
1997;175:828-32.
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CA 02607771 2007-11-01
1 [0091] [3] Pittman PR, Makuch RS, Mangiafico JA, Cannon TL, Gibbs PH, Peters
CJ. Long-term
2 duration of detectable neutralizing antibodies after adm[nistration of live-
attenuated VEE vaccine and
3 following booster vaccination with inactivated VEE vaccine. Vaccine 1996;
14:337-43.
4 [0092] [4] Jahr[ing PB, Stephenson EH. Protective efficacies of live
attenuated and
formaidehyde-inactivated Venezuelan equine encephalitis virus vaccines against
aerosol challenge
6 in hamsters. J Glin Microbiol 1984; 19:429-31.
7 [0093] [5] France JK, Wyrick BC, Trent DW. Biochemical and antigenic
comparison of the
8 envelope glycoproteins of Venezuelan equine encephalomyelitis virus strains.
J Gen Virol 1979;
9 44:725-4fl.
[0094] [6] Roehrig JT, Day JW, Kinney RM. Antigenic analysis of the surface
glycoproteins of a
11 Venezuelan equine encephaiomyeiitis virus (TC-83) using monoclonal
antibodies. Virology
12 1982;118:269-78.
13 [0095] [7] Roehrig JT, Mathews JH. The neutralization site on the E2
glycoprotein of
14 Venezuelan equine encephalomyelitis (TC-83) virus is composed of multiple
conformationally stable
epitopes. Virology 1985; 142:347-56.
16 [0096] [8] Schroff RW, Foon KA, Beatty SM, Oldham RK, Morgan Jr AC. Human
anti-murine
17 immunoglobulin responses in patients receiving monoclonal antibody therapy.
Cancer Res 1985;
18 45:879-85.
19 [0097] [9] Verhoeyen M, Milstein C, Winter G. Reshaping human ant[bodies:
grafting an
antilysozyme activity. Science 1988; 239:1534-6.
21 [0098] [101 DalE'Acqua WF, Damschroder MM, Zhang J, Woods RM, Widjaja L, Yu
J, et al.
22 Antibody humanization by framework shuffling. Methods 2005; 36:43-60.
23 [0099] [111 Hu WG, Alvi AZ, Fulton RE, Suresh MR, Nagata LE. Genettc
engineering of
24 streptavidin-binding peptide tagged single-chain variabie fragment antibody
to Venezuelan equine
encepha[itis virus. Hybrid Hybridomics 2002; 21:415-20.
26 [00100] [12] Hwang WY, Almagro JC, Buss TN, Tan P, Foote J. Use of human
gerrn[ine genes in
27 a CDR homology-based approach to antibody humanization. Methods 2005; 36:35-
42.
28 [00101] [13] van den Ouweland AM, van Duijnhoven HL, Keizer GD, Dorssers
LC, Van de Ven
29 WJ. Structural homology between the human fur gene product and the
subtilisin-iike protease
encoded by yeast KEX2. Nucleic Acids Res 1990; 18:664.
31 [00102] [141 Fulton RE, Nagata, L, Alvi, A; US Patent No. 6,818,748, Nov.
16, 2004.
32 [00103] [151 Johnson KM, Martin DH. Venezuelan equine encephalitis. Adv.
Vet Sci Comp Med.
33 1974; 18(0):79-116.
34 [00104] [161 Groot H, (1972) The health and economic importance of
Venezuelan equine
encepha[[tis (VEE) in Venezuelan encephalitis, Scientific pubiication no. 243,
pp. 7-16, Pan
36 American Health Organization, Washington DC.
21694804.1 13
CA 02607771 2007-11-01
1 [00105] [17] PhiNpotts RJ, Jones LD, Howard SC, Monoclonal antibody protects
mice against
2 infection and disease when given either before or up to 24h after airbome
chailenge with virulent
3 Venezuelan equine encephalitis virus. Vaccine, 2002 Feb 22; 20 (11-12): 1497-
504.
4
[00106j Although the invention has been described with reference to certain
specific
6 embodiments, various modifications thereof will be apparent to those skilled
in the art without
7 departing from the purpose and scope of the invention as outtined in the
cfaims appended hereto.
8 Any examples provided herein are included solely for the purpose of
illustrating the invention and are
9 not intended to limit the Invention in any way. Any drawings provided herein
are solely for the
purpose of illustrating various aspects of the invention and are not intended
to be drawn to scale or
11 to limit the invention in any way. The disclosures of all prior art recited
herein are incorporated
12 herein by reference in their entirety.
13
14
21694804.1 14
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CA 02607771 2007-11-01
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