U.S. patent application number 10/985581 was filed with the patent office on 2005-06-16 for interferon-gamma-binding molecules for treating septic shock, cachexia, immune diseases and skin disorders.
This patent application is currently assigned to INNOGENETICS N.V.. Invention is credited to Buyse, Marie-Ange, Sablon, Erwin.
Application Number | 20050129693 10/985581 |
Document ID | / |
Family ID | 26148270 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050129693 |
Kind Code |
A1 |
Buyse, Marie-Ange ; et
al. |
June 16, 2005 |
Interferon-gamma-binding molecules for treating septic shock,
cachexia, immune diseases and skin disorders
Abstract
The present invention concerns molecules which bind and
neutralize the cytokine interferon-gamma. More specifically, the
present invention relates to sheep-derived antibodies and
engineered antibody constructs, such as humanized single-chain Fv
fragments, chimeric antibodies, diabodies, triabodies, tetravalent
antibodies, peptabodies and hexabodies which can be used to treat
diseases wherein interferon-gamma activity is pathogenic. Examples
of such diseases are: septic shock, cachexia, multiple sclerosis
and psoriasis.
Inventors: |
Buyse, Marie-Ange;
(Merelbeke, BE) ; Sablon, Erwin; (Merchtem,
BE) |
Correspondence
Address: |
HOWREY SIMON ARNOLD & WHITE LLP
c/o IP DOCKETING DEPARTMENT
2941 FAIRVIEW PARK DRIVE, SUITE 200
FALLS CHURCH
VA
22042-7195
US
|
Assignee: |
INNOGENETICS N.V.
|
Family ID: |
26148270 |
Appl. No.: |
10/985581 |
Filed: |
November 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10985581 |
Nov 10, 2004 |
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10071485 |
Feb 7, 2002 |
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6830752 |
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10071485 |
Feb 7, 2002 |
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09485737 |
Feb 14, 2000 |
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6350860 |
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09485737 |
Feb 14, 2000 |
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PCT/EP98/05165 |
Aug 14, 1998 |
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Current U.S.
Class: |
424/145.1 ;
530/388.23 |
Current CPC
Class: |
C07K 16/468 20130101;
C07K 2317/24 20130101; C07K 16/249 20130101; C07K 2319/00 20130101;
A61K 38/00 20130101; C07K 16/00 20130101 |
Class at
Publication: |
424/145.1 ;
530/388.23 |
International
Class: |
A61K 039/395; C07K
016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 18, 1997 |
EP |
97870122.5 |
Jun 18, 1998 |
EP |
98870139.7 |
Claims
What is claimed is:
1. A molecule which binds and neutralizes interferon-gamma and
which is chosen from the group consisting of: a scFv comprising the
humanized variable domain of the monoclonal antibody D9D10 a
chimeric antibody comprising the humanized variable domain of the
monoclonal antibody D9D10 a diabody comprising the humanized
variable domain of the monoclonal antibody D9D10 and a multivalent
antibody comprising the humanized variable domain of the monoclonal
antibody D9D10.
2. The molecule according to claim 1, wherein said multivalent
antibody is chosen from the group consisting of triabodies and
tetravalent antibodies.
3. The molecule according to claim 2, wherein said triabody or
tetravalent antibody, comprise 3 and 4 variable domains,
respectively, of several anti-interferon-gamma antibodies.
4. The molecule according to claim 2, wherein said triabody
comprises 3 identical variable domains of an anti-interferon-gamma
antibody.
5. The molecule according to claim 2, wherein said triabody
comprises 3 identical D9D10 scFv's.
6. The molecule according to claim 2, wherein said triabody
comprises 3 identical humanized D9D10 scFv's.
7. The molecule according to claim 2, wherein said tetravalent
antibody comprises 4 identical domains of an anti-interferon-gamma
antibody.
8. The molecule according to claim 2, wherein said tetravalent
antibody comprises 4 identical D9D10 scFv's.
9. The molecule according to claim 2, wherein said tetravalent
antibody comprises 4 identical humanized D9D10 scFv's.
10. The molecule according to claim 2, wherein said tetravalent
antibody comprises a full-size humanized D9D10 antibody.
11. A pharmaceutical composition comprising a molecule according to
claim 1 or a mixture of said molecules in a pharmaceutically
acceptable excipient.
12. A method for neutralizing interferon-gamma activity in a mammal
comprising administering to the mammal a pharmaceutically effective
amount of a molecule that binds and neutralizes interferon-gamma,
said molecule selected from the group consisting of: a scFv
comprising the humanized variable domain of the monoclonal antibody
D9D10 a chimeric antibody comprising the humanized variable domain
of the monoclonal antibody D9D10 a diabody comprising the humanized
variable domain of the monoclonal antibody D9D10 and a multivalent
antibody comprising the humanized variable domain of the monoclonal
antibody D9D10.
13. The method according to claim 12 wherein the pharmaceutically
effective amount of the molecule that binds and neutralizes
interferon-gamma is administered to prevent or treating septic
shock, cachexia, autoimmune disease(s), and/or skin
disorder(s).
14. The method of claim 13 wherin the autoimmune disease is
multiple sclerosis, Crohn's disease, or rheumatoid arthritis.
15. The method of claim 13 wherein the skin disorder is bullous,
inflammatory, or neoplastic dermatosis.
16. The method of claim 12 wherein said multivalent antibody is
chosen from the group consisting of triabodies and tetravalent
antibodies.
17. The method according to claim 12 wherein said triabody or
tetravalent antibody, comprise 3 and 4 variable domains,
respectively, of several anti-interferon-gamma antibodies.
18. The method according to claim 12 wherein said triabody
comprises 3 identical variable domains of an anti-interferon-gamma
antibody.
19. The method according to claim 12 wherein said triabody
comprises 3 identical D9D10 scFv's.
20. The method according to claim 12 wherein said triabody
comprises 3 identical humanized D9D10 scFv's.
21. The method according to claim 12 wherein said tetravalent
antibody comprises 4 identical domains of an anti-interferon-gamma
antibody.
22. The method according to claim 12 wherein said tetravalent
antibody comprises 4 identical D9D10 scFv's.
23. The method according to claim 12 wherein said tetravalent
antibody comprises 4 identical humanized D9D10 scFv's.
24. The method according to claim 12 wherein said tetravalent
antibody comprises a full-size humanized D9D10 antibody.
25. A method for determining interferon gamma levels in a sample
comprising: a) contacting the biological sample to be analysed for
the presence of interferon-gamma with a molecule according to claim
1; b) detecting the immunological complex formed between
interferon-gamma and said molecule.
Description
[0001] This application is a divisional of co-pending application
Ser. No. 10/071,485, filed Feb. 7, 2002, which was a divisional of
application Ser. No. 09/485,737, filed Feb. 14, 2000, now U.S. Pat.
No. 6,350,860, which was a section 371 national stage filing of
PCT/EP98/05165, Filed Aug. 14, 1998 (published in English on Feb.
25, 1999 As WO 99/09055) and claiming priority to EP 97870122.5
filed Aug. 18, 1997; and EP 98870139.7 filed Jun. 18, 1998, each of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention concerns molecules which bind and
neutralize the cytokine interferon-gamma. More specifically, the
present invention relates to sheep-derived antibodies and
engineered antibody constructs, such as humanized single-chain Fv
fragments, chimeric antibodies, diabodies, triabodies, tetravalent
antibodies and peptabodies which can be used to treat diseases
wherein interferon-gamma activity is pathogenic. Examples of such
diseases are: septic shock, cachexia, multiple sclerosis and
psoriasis.
BACKGROUND OF THE INVENTION
[0003] Interferon-gamma (IFN.gamma.) is a member of the interferon
family of immunomodulatory proteins and is produced by activated T
helper type-1 cells (Th1 cells) and natural killer cells (NK
cells). Apart from its potent antiviral activity, IFN.gamma. is
known to be involved in a variety of immune functions (for a
review, see Billiau, 1996) and inflammatory responses. Indeed,
IFN.gamma. is the primary inducer of the expression of the major
histocompatibility complex (MHC) class-II molecules (Steinman et
al., 1980) by macrophages and other cell types and stimulates the
production of inflammatory mediators such as tumor necrosis
factor-alpha (TNF.alpha.), interleukin-1 (IL-1) and nitric oxide
(NO) (Lorsbach et al., 1993). In this respect, IFN.gamma. is shown
to be important in the macrophage-mediated defence to various
bacterial pathogens. Furthermore, IFN.gamma. is also shown to be a
potent inducer of the expression of adhesion molecules, such as the
intercellular adhesion molecule-1 (ICAM-1, Dustin et al., 1988),
and of important costimulators such as the B7 molecules on
professional antigen presenting cells (Freedman et al., 1991).
Moreover, IFN.gamma. induces macrophages to become tumoricidal
(Pace et al., 1983) and provokes Ig isotype switching (Snapper and
Paul, 1987).
[0004] The anti-viral, tumoricidal, inflammatory- and
immunomodulatory activity of IFN.gamma. clearly has beneficial
effects in a number of clinical conditions. However, there are a
number of clinical situations in which IFN.gamma.-activity has
deleterious effects. These include cancer cachexia (Denz et al.,
1993; Iwagaki et al., 1995), septic shock (Doherty et al., 1992),
skin disorders such as psoriasis and bullous dermatoses (Van den
Oord et al., 1995), allograft rejection (Landolfo et al., 1985;
Gorczynski, 1995), chronic inflammations such as ulcerative colitis
and Crohn's disease (WO 94/14467 to Ashkenazi & Ward), and
autoimmune diseases such as multiple sclerosis (M S, Panitch et
al., 1986), experimental lupus (Ozmen et al., 1995), arthritis
(Jacob et al., 1989; Boissier et al., 1995) and autoimmune
encephalomyelitis (Waisman et al., 1996).
[0005] Cachexia is a phenomenon often seen in cancer patients and
is associated with losses of lean body mass, and altered
carbohydrate and lipid metabolism. This so called `chronic wasting
syndrome` is often the immediate cause of death. In recent years,
interest has focused on the role of proinflammatory cytokines in
cancer related cachexia. Current data support the concept that
cachexia is linked to the presence of certain cytokines among which
IFN.gamma. seems to play a central role. Denz et al. (1993)
reported that increased neopterin and decreased tryptophan
concentrations--which are closely related to
IFN.gamma.-activity--are detected in cachectic patients suffering
from hematological disorders. Neopterin is synthesized and secreted
by monocytes/macrophages upon stimulation by IFN.gamma. from
activated T cells. Tryptophan is an indispensable amino acid which
can be catabolized by indoleamine 2,3-dioxygenase, an enzyme
induced by IFN's, and which absence initiates mechanisms
responsible for cachexia (Brown et al., 1991). The correlation
between high neopterin levels, decreased tryptophan levels and
weight loss was confirmed by Iwagaki et al. (1995). In experimental
models, cancer-induced cachexia can be altered by the
administration of IFN.gamma. neutralizing antibodies (Matthys et
al., 1991; Langstein et al., 1991)
[0006] Septic shock is the result of a severe bacterial infection,
and remains a common cause of death among critically ill,
hospitalized patients despite improvements in supportive care (Bone
et al., 1992). Although septic shock may be associated with
gram-positive infections, attention has focused on the more common
pathogenesis of gram-negative sepsis and the toxic role of
endotoxin (=lipopolysaccharide or LPS), a component of the outer
membrane of gram-negative and some gram-positive bacteria. Many of
the effects of LPS are mediated through the release of cytokines
such as TNF.alpha. (Tracey, 1991), IL-1 (Wakabayashi et al., 1991)
and IFN.gamma. (Bucklin et al., 1994). Much of the evidence
supporting the role of these cytokines as mediators of septic shock
comes from lethality studies involving the blockade of individual
cytokines, resulting in protection of experimental animals from
otherwise lethal doses of endotoxin or gram-negative bacteria. One
of the first events in septic shock is the activation of T cells by
antigen presenting cells onto which bacterial superantigen is bound
(Miethke et al., 1993). Upon activation, for which co-stimulation
of CD28 is essential (Saha et al., 1996), these T cells proliferate
and produce a surge of proinflammatory cytokines such as IL-2,
TNF.alpha. and IFN.gamma. eventuating in the clinical syndrome.
Also, it is hypothesized that LPS induces the expression of the
.alpha.1/.beta.1 integrin (VLA-1) heterodimer on activated
monocytes which then display an increased capacity to adhere to the
endothelial basement membrane. Similar effects can be induced by
incubation of monocytes with IFN.gamma. (Rubio et al., 1995). VLA-1
might also contribute to further monocyte activation and
potentiation of the production of monocyte-derived pro-inflammatory
cytokines during sepsis (Rubio et al., 1995). Although very
promising results were obtained with fantibodies neutralising
TNF.alpha. in experimental animal models, clinical trials with
anti-TNF.alpha. antibodies revealed only a slight reduction or even
no reduction in mortality rate of patients with septic shock
(Wherry et al., 1993; Reinhart et al., 1996). A -fusion protein
containing the extracellular portion of the TNF receptor and the Fc
portion of IgG1 also did not affect mortality (Fisher et al.,
1996). Pentoxifylline (PTX), a methyl xanthine derivative, is
currently being tested for its effect on the outcome of septic
shock. PTX is known to lower the serum concentrations of at least
TNF.alpha., IL-1 and IFN.gamma. (Bienvenu et al., 1995; Zeni et
al., 1996). Initial data reveal that PTX leads to an improvement of
the clinical status of septic patients (Mandi et al., 1995). There
is evidence that IFN.gamma. is a mediator of lethality during
sepsis. Antibodies that either neutralize IFN.gamma. or block the
IFN.gamma.-receptor are protecting against lethality (Bucklin et
al., 1994; Doherty et al., 1992). A synergistic effect between
IFN.gamma. and TNF.alpha. has also been suggested (Doherty et al.,
1992; Ozmen et al., 1994). Although not in itself lethal,
IFN.gamma. has been shown to be essential for the manifestation of
TNF-induced lethality in the generalized Shwartzman reaction (Ozmen
et al., 1994).
[0007] Bullous, inflammatory and neoplastic dermatoses are a
heterogenous group of skin disorders during which IFN.gamma. may
play a pathogenic role. Bullous dermatoses encompass epidermolysis
bullosa acquisita, bullous pemhigoid, dermatitis herpetiformes
During, linear IgA disease, herpes gestationis, cicatricial
pemhigoid, bullous systemic lupus erythematosis, epidermolysis
bullosa junctionalis, epidermolysis bullosa dystrophicans,
porphyria cutanea tarda and Lyell-Syndrome (Megahed, 1996). Also
erythema exsudativum multiform major (Kreutzer et al., 1996),
IgG-mediated subepidermal bullous dermatosis (Chan & Cooper,
1994), bullous lichen planus (Willsteed et al., 1991) and
paraneoplastic bullous dermatosis (Pantaleeva, 1990) can be
classified among the bullous dermatoses. A pathogenic role of
IFN.gamma. during bullous dermatoses has been suggested by Van den
Oord et al. (1995). The role of IFN.gamma. during inflammatory and
neoplastic dermatoses, compared to bullous dermatoses, has been
more extensively investigated. Indeed, it has been demonstrated
that IFN.gamma. is involved during the pathogenesis of verrucosis
(Asadullah et al., 1997), eosinophilic pustular folliculitis
(Teraki et al., 1996), cutaneous T cell lymphoma (Wood et al.,
1994), granuloma faciale (Smoller & Bortz, 1993), Sweet's
syndrome (Reuss-Borst et al., 1993), atopic eczema (Arenberger et
al., 1991), follicular mucinosis (Meisnerr et al., 1991),
lichen-planus and psoriasis (Vowels et al., 1994). One of the most
extensively studied inflammatory dermatoses is psoriasis. Psoriasis
is a hyperproliferative skin disorder affecting approximately 2% of
the population. Evidence is accumulating that the disease has a
T-cell mediated autoimmune etiology. The role of T-cells in
psoriasis has been demonstrated by Gottlieb et al. (1995). The
latter authors suggested that, in most of the patients, clinical
and histopathological features of psoriasis are primarily linked to
skin infiltration by IL-2 receptor-positive leukocytes. Disease
improvement can be induced by the administration of a fusion
protein composed of human interleukin-2 and fragments of diphteria
toxin, which selectively blocks the growth of activated
lymphocytes. Other effective anti-psoriatic, T-cell suppressing
agents include the immunosuppressive drugs cyclosporin and FK506
(Griffiths, 1986) and anti-CD4 monoclonal antibodies (Morel et al.,
1992). More direct evidence for the role of T cells in the
induction of the complex tissue alterations seen in psoriasis has
been generated by Schon et al. (1997) using a model with scid/scid
mice in which they transferred naive, minor histocompatibility
mismatched CD4.sup.+ T-cells, resulting in the development of a
skin disorder that resembles psoriasis. The autoimmune character of
the disease has been proposed by Valdimarsson et al. (1995) who
stated that products of activated T-cells can induce keratinocytes
of individuals with psoriatic predisposition to express
determinants that are recognized by T cells specific for epitopes
on .beta.-haemolytic streptococci. Several data suggest that
IFN.gamma. may play a crucial role in the pathogenesis of
psoriasis. IFN.gamma., produced by activated T cells would be
involved in the recruitment of lymphocytes (Nickoloff, 1988), in
the induction of activation and adhesion molecules on epidermal
keratinocytes (Dustin et al., 1988), as well as in the abnormal
keratinocyte proliferation (Barker et al., 1993). Not only enhanced
levels of IFN.gamma. has been detected in psoriatic epidermis
(Kaneko et al., 1990), also de novo suprabasal expression of
IFN.gamma. receptor in psoriasis has been demonstrated (Van den
Oord et al., 1995).
[0008] Inflammatory bowel disease (IBD), which encompasses
ulcerative colitis and Crohn's disease, is characterized by the
appearance of lesions of unknown aetiology in most parts of the
gut. IBD is rather common, with a prevalence in the range of 70-170
in a population of 100,000. The current therapy of IBD involves the
administration of anti-inflammatory or immunosuppressive agents,
which usually bring only partial results, and surgery. In view of
the apparent shortcomings of the present treatment, Ashkenazi and
Ward (WO 94/14467) suggested the usage of a bispecific antibody
construct targeting IFN.gamma. and another molecule, such as IL-1
and TNF.alpha., to treat IBD. However, the exact role of IFN.gamma.
during IBD is not well understood.
[0009] MS is a severely disabling progressive neurological disease
of unknown aetiology, but probably involving autoimmune responses
and resulting in the appearance of focal areas of demyelinisation
(Williams et al., 1994). MS affects 1 in 1000 persons in the USA
and Europe, but due to improved diagnosis that number is
increasing. Onset of disease is usually around 30 years of age and,
on average, patients are in need of treatment for another 28 years.
MS is among the most expensive chronic diseases of western society,
based on duration and intensity of care. However, diagnosis of
exacerbations and early identification of onset of exacerbations
has improved greatly, allowing design of novel treatment
strategies. Active multiple sclerosis lesions feature T-lymphocyte
and monocyte-macrophage accumulations at plaque margins where
myelin is being destroyed. The inflammatory cells that invade the
white matter and the soluble mediators that they release are held
primarily responsible for myelin breakdown. Population-based
studies indicate that certain HLA-antigens occur with higher
frequency in patients with MS (with predominant MHC being the
Dw2(DR2)DQ1.2 haplotype (Olerup et al., 1991). Similar associations
of class I and class II haplotypes have also been detected in other
autoimmune disorders such as rheumatoid arthritis and insulin
dependent diabetes (Nepom, 1993). The lesions of MS are comparable
to those found in chronic relapsing experimental allergic
encephalitis (EAE), an autoimmune disease that can be induced in
animals by immunization with e.g. whole myelin (Allen et al., 1993)
or with the myelin/oligodendrocyte glycoprotein (Genain et al.,
1995b). The lesions associated with EAE are similar in appearance
as the ones occurring in MS and also contain inflammatory
infiltrates of T-cells and macrophages (Genain, et al., 1995b).
Furthermore, in adoptive transfer experiments, T cells sensitized
to specific myelin antigens can transfer the disease state of EAE
(Genain et al., 1995b; Waldburger et al., 1996). A few years ago,
the American FDA approved the use of the immunosuppressive drug
interferon.beta. (trade name Betaseron) for treatment of chronic
relapsing MS. The effect of this drug--although modest--clearly
demonstrates the involvement of the cytokine network in the
pathophysiology of MS. In the last few years, a large number of
studies have addressed the molecular mechanism by which Betaseron
exerts its beneficial effects. Lately, it was shown that IFN.beta.
dose-dependently inhibited T-cell proliferation, expression of IL-2
receptors and secretion of IFN.gamma., TNF.alpha. and IL-13 (Rep et
al., 1996). Furthermore, it was demonstrated that IFN.beta. could
specifically prevent the IFN.gamma.-induced up regulation of MHC
class II antigens and adhesion molecules on antigen-presenting
cells (Jiang et al., 1995) and human brain microvessel endothelial
cells (Huynh et al., 1995).
[0010] One of the earliest events in MS is damage of the blood
brain barrier (BBB) by activated, encephalitogenic T-cells (Tsukada
et al., 1993). The mechanism by which these cells destruct locally
the BBB, which is mainly constituted of endothelial cells, is not
elucidated, but it is known that at the systemic level, local
production of certain cytokines such as IFN.gamma. enhance the
capability of lymphocytes to adhere to endothelial cells (Yu et
al., 1985; Tsukada et al., 1993). Also, on choroid plexus
epithelial cells of EAE animals, an increased expression of ICAM-1
and VCAM-1 (Steffen et al., 1994), for which LFA-1 and VLA-4 are
the natural ligands on lymphocytes, has been observed. Mc Carron et
al. (1993) reported that adhesion of MBP-specific T lymphocytes was
significantly up regulated when cerebral endothelial cells were
treated with IL-1, TNF.alpha. or IFN.gamma.. That the adhesion of
encephalitogenic T-cells to the endothelium is an early and very
important event in the onset of MS is shown by the finding that
anti LFA-1 therapy can completely block the induction of EAE
(Gordon et al., 1995). Additional circumstantial evidence for a
stimulatory role of IFN.gamma. in the pathophysiology of MS comes
from observations that disease exacerbations are induced by viral
upper respiratory infections, known to stimulate the secretion of
IFN.gamma. by type-2 helper T cells (Panitch, 1994). The
proinflammatory role of IFN.gamma. in autoimmune disease is
strengthened by an earlier finding that treatment of MS patients
with hIFN.gamma. resulted in an aggravation of the symptoms
(Panitch et al., 1986). The role of IFN.gamma. as proinflammatory
cytokine in autoimmune disorders has been studied in several
experimentally induced forms of autoimmunity. In experimental
neuritis, induced by myelin or antigen-specific T cells in rat,
IFN.gamma. clearly acted as pro-inflammatory cytokine and
administration of a monoclonal antibody to IFN.gamma. suppressed
the disease (Hartung et al., 1990). In the case of experimental
autoimmune thyroiditis (EAT) in mice, induced by the injection of
thyroglobulin, treatment of the animals with anti-IFN.gamma. at 4
weeks after induction of EAT proved to be beneficial, since
characteristic features of EAT such as the lymphocytic
infiltrations of the thyroid glands and the serum levels of
autoantibodies to thyroglobulin, were significantly reduced (Tang
et al., 1993).
[0011] In the mouse EAE model for MS, where the disease can be
induced by injection of either spinal cord homogenate or myelin
basic protein, elevated concentrations of several cytokines,
including IFN.gamma. were observed both in serum and in the lesions
in the CNS (Willenborg et al., 1995). However, administration of
anti-IFN.gamma. at the initiation of the disease, resulted in an
exacerbation of the disease (Billiau et al., 1988; Duong et al.,
1994; Willenborg et al., 1995). It must be noted, however, that in
these experiments the effect of anti-IFN.gamma. was determined at
the onset of acute EAE rather than at the time of chronic relapse
of the disease, which in fact is the only relevant situation for
MS. Pathologically, typical acute EAE differs substantially from MS
in that prominent inflammation occurs in gray, white and meningeal
structures, but demyelisation is scant or absent (Genain et al.,
1995b). In order to explain the findings with anti-IFN.gamma.
antibodies, the authors suggest a different action of IFN.gamma. at
the systemic level (anti-inflammatory action) compared to the local
level (inflammatory action) (Billiau et al., 1988), or suggest an
early role (within 24 h after immunization) of IFN.gamma. in
disease resistance (Duong et al., 1994). Willenborg et al. (1995)
conclude that the time of treatment plays a critical role on the
outcome and suggest this to be the explanation for conflicting
results in different autoimmune processes. Recently, Heremans et
al. (1996) described facilitation of spontaneous relapses in
chronic relapsing EAE in Biozzi ABH mice by administration of
anti-IFN.gamma. during the remission phase. The onset of relapses
was delayed when animals were treated with IFN.gamma. during the
remission phase, results which are in contradiction to the
excacerbation seen in humans who were treated with hIFN.gamma..
[0012] An experimental EAE model that more closely resembles the
disease course and symptomatology of MS in humans can be found in
marmosets. Indeed, in these animals a chronic relapsing-remitting
form of EAE can be induced which is characterized by an initial,
acute phase with clinically mild neurological signs, followed by
recovery. A late spontaneous relapse occurs in these animals and
chronic lesions resemble active plaques of chronic MS (Massacesi et
al., 1995). This unique model can efficiently be employed to
evaluate a prospective therapy for MS. In this model, a critical
role for TNF.alpha. in demyelisation is suggested by the
observation that rolipram, a selective inhibitor of the type IV
phosphodiesterase, suppressed TNF.alpha. secretion and
demyelisation (Genain et al., 1995a; Sommer et al., 1995) when
administered shortly after immunization, thus interfering with
acute EAE. The effect of anti-IFN.gamma. on acute EAE or on disease
relapse has to our knowledge never been investigated in
marmoset.
[0013] Taken together, it is well established that there are a
number of clinical situations in which IFN.gamma.-activity has
deleterious effects. Consequently, several potential therapies to
neutralize IFN.gamma.-activity have been proposed. Among the latter
proposals are the use of: anti-IFN.gamma. antibodies (Ozmen et al.,
1995; Bucklin et al., 1994), recombinant anti-IFN.gamma. Fv
fragments (EP 0528469 to Billiau & Froyen), bispecific
molecules (WO 94/14467 to Ashkenazi and Ward), drugs such as
pentoxifylline (Bienvenu et al., 1995), synthetic polypeptides
which inhibit binding of IFN.gamma. to its receptor (U.S. Pat. No.
5,451,658 to Seelig; U.S. Pat. No. 5,632,988 to Ingram et al.),
Epstein-Barr virus derived proteins (U.S. Pat. No. 5,627,155 to
Moore & Kastelein), soluble IFN.gamma. receptors (EP 0393502 to
Fountoulakis et al.; U.S. Pat. No. 5,578,707 to Novick &
Rubinstein) and oligonucleotides which bind to IFN.gamma.
(WO95/00529 to Coppola et al.). However, these compounds are faced
with problems such as suboptimal stability, affinity and clearance
rates, lack of specificity, efficacy and tissue penetrance, toxic
side effects and unwanted carrier effects. Indeed, the carrier
effect of antibodies can limit their efficiency to block the target
cytokine. For example, Montero-Julian et al. (1995) showed that
during treatment of myeloma patients with anti-IL-6, accumulation
of IL-6 in the serum in the form of monomeric immune complexes
occurred, hereby stabilizing the cytokine. Furthermore, it has also
been shown that the therapeutic efficacy of a cytokine can be
prolonged by the formation of cytokine/antibody complexes, since
the efficacy of recombinant human IL-2 treatment could be increased
by prolonging its in vivo half-life by complexing with an anti-IL-2
antibody (Courtney et al., 1994). The carrier-effect of
anti-cytokine antibodies can be overcome by the construction of
monovalent scFv fragments, although their low MW
(.A-inverted.30.000) and the associated fast clearance rate, make
them less suitable candidates for long-term treatment. However, the
undesirable carrier effect can be avoided by the formation of
higher immune complexes, as such increasing the clearance of the
cytokine-antibody complexes (Montero-Julian et al., 1995). The use
of monoclonal antibodies for diagnostic or therapeutic purposes in
vivo is, besides the carrier effect, also limited because of their
nature (i.e. the majority are murine mAb's and administration of
antibodies of mouse origin inevitably results in a human anti-mouse
antibody [HAMA] response), their suboptimal efficacy, stability and
affinity and their large molecular size. Proposed solutions to some
of these problems involve the use of F(ab')2, F(ab) and scFv
derivatives or of humanized versions of the parent antibody, either
by CDR grafting (Kettleborough et al., 1991) or by resurfacing of
the antibodies (Roguska et al., 1994). Another proposed solution is
the development of several modified antibodies or antibody
constructs by bioengineering or chemical methods. Indeed, some
mAb's were made more effective by conjugating chemotherapeutic
drugs and other toxins to the antibodies (Ghetie and Vitetta, 1994)
or by developing bispecific and/or multivalent antibody constructs
capable of simultaneously binding several -or two different
epitopes on the same- or different antigens. These antibody
constructs have been produced using a variety of methods: a)
antibodies of different specificities or univalent fragments of
pepsin-treated antibodies of different specificities have been
chemically linked (Fanger et al., 1992); b) two hybridomas
secreting antibodies of different specificity have been fused and
the resulting bispecific antibodies from the mixture of antibodies
were subsequently isolated; c) genitically engineered single chain
antibodies have been used to produce non-covalently linked
bispecific antibodies (e.g. diabodies (Holliger et al., 1993),
minibodies (Kostelny et al., 1992) and tetravalent antibodies (Pack
et al; 1995; WO 96/13583 to Pack) or covalently-linked bispecific
antibodies (e.g. chelating recombinant antibodies (Kranz et al.,
1995), single chain antibodies fused to protein A or Streptavidin
(Ito and Kurosawa, 1993; Kipriyanov et al., 1996) and bispecific
tetravalent antibodies (EP 0517024 to Bosslet and Deeman).
Recently, also trivalent antibody constructs, named triabodies
(Kortt et al., 1997), and pentavalent constructs, named peptabodies
(Terskikh et al., 1997), have been described. These constructs may
have a higher avidity in comparison to bivalent constructs and may
be useful for diagnostic or therapeutic purposes in vivo.
[0014] However, and despite the fact that several potential
therapies to neutralize IFN.gamma.-activity have been proposed, no
prior art exists regarding the production and existence of
engineered antibody constructs, such as humanized single-chain Fv
fragments, diabodies, triabodies, tetravalent antibodies,
peptabodies and hexabodies, and ruminant-derived antibodies such as
sheep antibodies which overcome the above-indicated problems and
which can efficiently be used to treat diseases wherein
interferon-gamma activity is pathogenic.
SUMMARY OF THE INVENTION
[0015] It is clear from the prior art as cited above that problems
such as suboptimal stability, affinity, clearance rate,
specificity, efficacy, and an unwanted carrier effect and HAMA
response hamper the successful usage of several therapeutics which,
potentially, could neutralize the activity of IFN.gamma.. Also
suggested solutions to overcome some of these problems did not
result in the development of effective products. Thus,
unpredictable and unknown factors still appear to determine the
success of these biologicals. Despite these unknown factors, the
present inventors have been able to design and develop useful
constructs which effectively neutralize IFN.gamma.-activity.
Indeed, the constructs have all a surprisingly high affinity for
IFN.gamma., they do not provoke a HAMA or related response, and
they do not result in a carrier effect. In addition, some of the
constructs pass the blood brain barrier, whereas others have a very
good clearance rate. Therefore, the present invention aims at
providing a molecule which binds and neutralizes interferon-gamma
and which is chosen from the group consisting of:
[0016] a scFv comprising the humanized variable domain of the
monoclonal antibody D9D 10
[0017] a chimeric antibody comprising the humanized variable domain
of the monoclonal antibody D9D10
[0018] a diabody comprising the humanized variable domain of the
monoclonal antibody D9D10
[0019] a multivalent antibody
[0020] a ruminant antibody.
[0021] The present invention further aims at providing a
multivalent antibody chosen from the group consisting of
triabodies, tetravalent antibodies, peptabodies and hexabodies.
[0022] The present invention also aims at providing a triabody,
tetravalent antibody, peptabody and hexabody which comprise 3, 4, 5
and 6 variable domains, respectively, of different
anti-interferon-gamma antibodies.
[0023] The present invention further aims at providing a triabody
as described above which comprises 3 identical variable domains of
an anti-interferon-gamma antibody. A preferred variable domain used
in the latter constructs is derived from the mouse
anti-interferon-gamma antibody D9D10 which is described by Sandvig
et al. (1987) and Froyen et al. (1993) or from the sheep
anti-interferon-gamma antibody described in the present
application. Therefore, the present invention aims at providing a
triabody as described above which comprises 3 identical D9D10
scFv's, 3 identical humanized D9D10 scFv's, 3 identical
sheep-derived anti-interferon-gamma scFv's or 3 identical humanized
sheep-derived anti-interferon-gamma scFv's.
[0024] The present invention further aims at providing a
tetravalent antibody (called MoTAb I) as described above which
comprises 4 identical domains of an anti-interferon-gamma antibody.
More specifically, the present invention aims at providing a
tetravalent antibody as described above which comprises either 4
identical D9D10 scFv's or 4 identical sheep-derived
anti-interferon-gamma scFv's in the format of a homodimer of 2
identical molecules, each containing 2 D9D10 scFv's or 2 humanized
D9D10 scFv's or 2 sheep-derived anti-interferon-gamma scFv's or 2
humanized sheep-derived anti-interferon-gamma scFv's, and a
dimerization domain, or, a full-size humanized D9D10 antibody or
sheep-derived anti-interferon-gamma antibody to which 2 humanized
D9D10 scFv's or 2 humanized sheep-derived anti-interferon-gamma
scFv's, respectively, are attached at the carboxyterminus (called
MoTAb II) (see FIG. 1).
[0025] The present invention further aims at providing a peptabody
and hexabody as described above which comprise 5 and 6 identical
variable domains of an anti-interferon-gamma antibody,
respectively. A preferred variable domain used in the latter
constructs is derived from the mouse anti-interferon-gamma antibody
D9D10 which is described above or from the sheep
anti-interferon-gamma antibody described in the present
application. Therefore, the present invention aims at providing a
peptabody and hexabody as described above which comprises 5 or 6
identical D9D10 scFv's, 5 or 6 identical humanized D9D10 scFv's, 5
or 6 identical sheep-derived anti-interferon-gamma scFv's, or, 5 or
6 identical humanized sheep-derived anti-interferon-gamma scFv's,
respectively.
[0026] The present invention further aims at providing a molecule
as described above, wherein said ruminant antibody is a sheep
antibody.
[0027] The present invention also aims at providing a molecule as
described above, wherein said sheep antibody is a monoclonal
antibody. Furthermore, the present invention aims at providing a
humanized antibody, a single-chain fragment or any other fragment
which is derived from said monoclonal antibody and which has
largely retained the specificity of said monoclonal antibody.
[0028] Moreover, the present invention aims at providing methods
for producing the above-described molecules.
[0029] The present invention further aims at providing a
pharmaceutical composition comprising a molecule as described
above, or a mixture of said molecules, in a pharmaceutically
acceptable excipient.
[0030] The present invention also aims at providing a molecule or a
composition as described above for use as a medicament.
[0031] Furthermore, the present invention aims at providing a
molecule or a composition as described above for preventing or
treating septic shock, cachexia, immune diseases such as multiple
sclerosis and Crohn's disease and skin disorders such as bullous,
inflammatory and neoplastic dermatosis.
[0032] Finally, the present invention aims at providing a molecule
as described above for determining interferon gamma levels in a
sample.
[0033] All the aims of the present invention are considered to have
been met by the embodiments as set out below.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1 schematically shows 2 different tetravalent antibody
constructs (MoTAB I and MotabII). MoTAb I represents a molecule
which consists of 4 identical scFv's in the format of a homodimer
of 2 identical molecules, each containing 2 scFv's. MoTAb II
represents a full-size antibody molecule to which 2 scFv's with the
same specificity are attached at the carboxyterminus. Optionally,
these constructs contain a purification/detection tag.
[0035] See also further Example 4.
[0036] FIG. 2 shows the coding (SEQ ID NO 1) and amino acid
sequence (SEQ ID NO 2) of humanized D9D10 scFv (containing a
C-terminal 6-histidine tag (bold)). CDR regions are underlined.
Mutations (murine-->human) are bold and underlined. The
N-terminal pelB signal sequence is put in bold.
[0037] FIGS. 3 and 4 shows the binding of different concentrations
of murine scFvD9D10 (FIG. 3) and humanized scFvD9D10 (FIG. 4) to
human IFN.gamma.. Human IFN.gamma. is immobilized indirectly to the
CM5 sensorchip via the murine D9D10 full size antibody as described
in example 1. Association rate constants derived from these binding
curves are shown. Dissociation rate constants could not be measured
accurately as dissociation is hardly detectable
(<5.times.10.sup.4 s.sup.-1) in this experimental setup.
[0038] FIG. 5 shows a schematic representation of the mammalian
expression plasmid pEE12hD9D10 used for expression of humanized
D9D10 whole antibody in (1) COS cells (2) stable recombinant Ns0
cell lines.
[0039] Major Plasmid Building Blocks:
[0040] prokaryotic sequences for plasmid DNA preparation in E.coli
(ori of replication and amp.sup.R ampicilline resistance expression
unit)
[0041] SV40 origin of replication (part of SV40E, SV40 early
promoter) allowing transient expression in SV40 permissive,
T-antigen producing cell lines (e.g. COS)
[0042] human Cytomegalovirus major immediate early
promoter/enhancer (hCMVprom+intron) controlled expression casette
for hD9D10 heavy chain protein (hD9D10-H)
[0043] human Cytomegalovirus major immediate early
promoter/enhancer (hCMVprom+intron) controlled expression casette
for hD9D10 light chain protein (hD9D10-L)
[0044] SV40 early promoter (SV40E) controlled glutamine synthetase
cDNA (GS) expression unit for selection/amplification
[0045] polyA=SV40 early region poly-adenylation signal
[0046] intron +polyA=SV40 t-antigen intron+SV40 early region
poly-adenylation signal
[0047] FIG. 6 shows a schematic representation of the mammalian
expression plasmid pEE14hD9D10 used for expression of humanized
D9D10 whole antibody in (1) COS cells (2) stable recombinant CHO-K1
cell lines.
[0048] Major Plasmid Building Blocks:
[0049] prokaryotic sequences for plasmid DNA preparation in E.coli
(on of replication and amp.sup.R ampicilline resistance expression
unit)
[0050] SV40 origin of replication (part of SV40E, SV40 early
promoter) allowing transient expression in SV40 permissive,
T-antigen producing cell lines (e.g. COS)
[0051] human Cytomegalovirus major immediate early
promoter/enhancer (hCMVprom+intron) controlled expression casette
for hD9D10 heavy chain protein (hD9D10-H)
[0052] human Cytomegalovirus major immediate early
promoter/enhancer (hCMVprom+intron) controlled expression casette
for hD9D10 light chain protein (hD9D10-L)
[0053] SV40 late promoter (SV40L) controlled glutamine synthetase
mini gene (GS+intron) expression unit for
selection/amplification
[0054] polyA=SV40 early region poly-adenylation signal
[0055] intron+polyA=SV40 t-antigen intron+SV40 early region
poly-adenylation signal
[0056] FIG. 7 shows the cDNA sequence encoding the humanized D9D 10
heavy chain fusion protein.
[0057] bp 1-60 : D9D10 Kappa-light chain signal sequence
[0058] bp 61-411: humanized D9D10 heavy chain variable domain
[0059] bp 412-1401: human IgG1 heavy chain constant domain
(C.sub.H1-Hinge-C.sub.H2-C.sub.H3)
[0060] bp 1402-1404: leu codon added by PCR cloning strategy (SEQ
ID NO 66)
[0061] FIG. 8 shows the cDNA sequence encoding the humanized D9D10
and MoTAbII light chain fusion protein.
[0062] bp 1-60 : D9D10 Kappa-light chain signal sequence
[0063] bp 61-381: humanized D9D10 light chain variable domain
[0064] bp 382-699: human kappa light chain constant domain (SEQ ID
NO 68)
[0065] FIG. 9 shows the amino acid sequence of the humanized D9D10
heavy chain fusion protein.
[0066] Aa 1-20: D9D10 light chain signal sequence
[0067] Aa 21-137: humanized heavy chain variable domain of
D9D10
[0068] Aa138-467: human IgG1 heavy chain constant domain
(C.sub.H1-hinge-C.sub.H2-C.sub.H3)
[0069] Aa 468: leu added by PCR cloning strategy
[0070] Aa 351: pro was mutated to ser: inactivation C1q complement
binding
[0071] Number of residues: 468.
[0072] Molecular weight (MW): 51413 (SEQ ID NO 67)
[0073] FIG. 10 shows the amino acid sequence of the humanized D9D10
and MoTAbII light chain fusion protein.
[0074] Aa 1-20: D9D10 light chain signal sequence
[0075] Aa 21 -127: humanized light chain variable domain of
D9D10
[0076] Aa 128-233: human kappa light chain constant domain
[0077] Number of residues: 233.
[0078] Molecular weight (MW): 25582 (SEQ ID NO 69)
[0079] FIG. 11 shows the binding in ELISA of different
concentrations of humanized D9D10 and humanized D9D10 MoTAbII
(=different dilutions of crude COS supernatant containing humanized
D9D10 or humanized D9D10 MoTAbII) to immobilized human IFN.gamma..
The assay is performed as described in example 2.
[0080] FIG. 12 shows the interaction of humanized D9D10 (=crude COS
supernatant containing humanized D9D10) with IFN.gamma. using SPR
analysis. The assay is performed as described in example 2.
[0081] FIG. 13 shows the binding in ELISA of different
concentrations of purified humanized D9D10 and MoTAbII to
immobilized human IFN.gamma.. The assay is performed as described
in example 2.
[0082] FIG. 14 shows a schematic representation of the
expressionplasmid pMoTAbIH6 used for the expression of MoTAbI in E.
coli.
[0083] FIG. 15 shows the cDNA sequence of MoTAbI
[0084] bp 1-351:V.sub.H D9D10
[0085] bp 352 -396: (G.sub.4S).sub.3 linker
[0086] bp 397 -717: V.sub.L D9D10
[0087] bp 718 -750: human IgG3 upper hinge
[0088] bp 751 -855: helix-turn-helix dimerisation domain
[0089] bp 856 -888: human IgG3 upper hinge
[0090] bp 889 -1239 V.sub.H D9D10
[0091] bp 1240 -1284: (G.sub.4S).sub.3 linker
[0092] bp 1285 -1605: V.sub.L D9D10
[0093] bp 1606 -1623: His6 tag (SEQ ID NO 84)
[0094] FIG. 16 shows the AA sequence of MoTAbI
[0095] aa 1-117: V.sub.H D9D10
[0096] aa 118 -132: (G.sub.4S).sub.3 linker
[0097] aa 133 -239: V.sub.L D9D10
[0098] aa 240 -250: human IgG3 upper hinge
[0099] aa 251 -285: helix-turn-helix dimerisation domain
[0100] aa 286 -296: human IgG3 upper hinge
[0101] aa 297 -413: V.sub.H D9D10
[0102] aa 414 -428: (G.sub.4S).sub.3 linker
[0103] aa 429 -525: V.sub.L D9D10
[0104] aa 526 -531: His6 tag (SEQ ID NO 85)
[0105] FIG. 17 shows a schematic representation of the mammalian
expression plasmid pEE12MoTAbII used for expression of D9D10MoTAbII
recombinant antibody in (1) COS cells (2) stable recombinant Ns0
cell lines.
[0106] Major Plasmid Building Blocks:
[0107] prokaryotic sequences for plasmid DNA preparation in E.coli
(ori of replication and amp.sup.R ampicilline resistance expression
unit)
[0108] SV40 origin of replication (part of SV40E, SV40 early
promoter) allowing transient expression in SV40 permissive,
T-antigen producing cell lines (e.g. COS)
[0109] human Cytomegalovirus major immediate early
promoter/enhancer (hCMVprom+intron) controlled expression casette
for D9D10MoTAbII heavy chain protein (MoTAbII-H)
[0110] human Cytomegalovirus major immediate early
promoter/enhancer (hCMVprom+intron) controlled expression casette
for D9D10MoTAbII light chain protein (MoTAbII-L)
[0111] SV40 early promoter (SV40E) controlled glutamine synthetase
cDNA (a) expression unit for selection/amplification
[0112] polyA=SV40 early region poly-adenylation signal
[0113] intron+polyA=SV40 t-antigen intron+SV40 early region
poly-adenylation signal
[0114] FIG. 18 shows a schematic representation of the mammalian
expression plasmid pEE14MoTAbII used for expression of D9D10MoTAbII
recombinant antibody in (1) COS cells (2) stable recombinant CHO-K1
cell lines.
[0115] Major Plasmid Building Blocks:
[0116] prokaryotic sequences for plasmid DNA preparation in E.coli
(ori of replication and amp.sup.R ampicilline resistance expression
unit)
[0117] SV40 origin of replication (part of SV40E, SV40 early
promoter) allowing transient expression in SV40 permissive,
T-antigen producing cell lines (e.g. COS)
[0118] human Cytomegalovirus major immediate early
promoter/enhancer (hCMVprom+intron) controlled expression casette
for D9D10MoTAbII heavy chain protein (MoTAbII-H)
[0119] human Cytomegalovirus major immediate early
promoter/enhancer (hCMVprom+intron) controlled expression casette
for D9D10MoTAbII light chain protein (MoTAbII-L)
[0120] SV40 late promoter (SV40L) controlled glutamine synthetase
mini gene (GS+intron) expression unit for
selection/amplification
[0121] polyA=SV40 early region poly-adenylation signal
[0122] intron+polyA=SV40 t-antigen intron+SV40 early region
poly-adenylation signal
[0123] FIG. 19 shows the cDNA sequence encoding the MoTABII fusion
protein
[0124] bp 1-60: D9D10 Kappa-light chain signal sequence
[0125] bp 61-411: humanized D9D10 heavy chain variable domain
[0126] bp 412-1401: human IgG1 heavy chain constant domain
(C.sub.H1-Hinge-C.sub.H2-C.sub.H3)
[0127] bp 1402-1404: leu codon added by PCR cloning strategy
[0128] bp 1405-1416: gly(3)-ser codon
[0129] bp 1417-2133: humanized D9D10 ScFv (SEQ ID NO 89)
[0130] FIG. 20 shows the amino acid sequence of MoTABII fusion
protein
[0131] Aa 1-20: mouse D9D10 light chain signal sequence
[0132] Aa 21-137: humanized heavy chain variable domain of
D9D10
[0133] Aa 138-467: human IgG1 heavy chain constant
domain(C.sub.H1-hinge-C- .sub.H2-C.sub.H3)
[0134] Aa 351: pro mutated to ser: inactivation C1q complement
binding
[0135] Aa 468: leu added by cloning strategy
[0136] Aa 469-472: gly(3)-ser linker
[0137] Aa 473-711: humanized D9D10 ScFv (V.sub.H473-490/gly-ser
linker/V.sub.L605-711) (SEQ ID NO 90)
[0138] FIG. 21 shows the interaction of MoTAbII (=crude COS
supernatant containing MoTAbII) with IFN.gamma. using SPR analysis.
The assay is performed as described in example 4.
[0139] FIG. 22 shows the amino acid sequence of the D9D10L10
diabody
[0140] aa 1-117: V.sub.H D9D10
[0141] aa 118 -127: (G.sub.4S).sub.2 linker
[0142] aa128-234: V.sub.L D9D10
[0143] aa 235 -240: His6-tag (SEQ ID NO 91)
[0144] FIG. 23 shows the coding sequence of the D9D10L10
diabody
[0145] bp 1-351: V.sub.H D9D10
[0146] bp 352 -381: (G.sub.4S).sub.2 linker
[0147] bp 382 -702: V.sub.L D9D10 (SEQ ID NO 92)
[0148] FIG. 24 shows the amino acid sequence of the D9D10 L5
diabody
[0149] aa 1-117: V.sub.H D9D10
[0150] aa 118 -122: G.sub.4S linker
[0151] aa 123 -229: V.sub.L D9D10
[0152] aa 230 -235: His6-tag 5 (SEQ ID NO 93)
[0153] FIG. 25 shows the coding sequence of the D9D10 L5
diabody
[0154] bp 1-351: V.sub.H D9D10
[0155] bp 352 -366: G.sub.4S linker
[0156] bp 367 -687: V.sub.L D9D10 (SEQ ID NO 94)
[0157] FIG. 26 shows the interaction of humanized L5 D9D10 diabody
(=crude lysate from E. Coli) with IFN.gamma. using SPR analysis.
The assay is performed as described in example 5.
[0158] FIG. 27 shows the coding sequence of the D9D10 L0
triabody
[0159] bp 1-351: V.sub.H D9D10
[0160] bp352-672: V.sub.L D9D10 (SEQ ID NO 101)
[0161] FIG. 28 shows the amino acid sequence of the D9D10 L0
triabody
[0162] aa 1-117: V.sub.H D9D10
[0163] aa 118 -224: V.sub.L D9D10
[0164] aa 225 -230: His6-tag (SEQ ID NO 102)
[0165] FIG. 29 shows the interaction of humanized L0 D9D10 triabody
(=crude lysate from E. Coli) with IFN.gamma. using SPR analysis.
The assay is performed as described in example 6.
[0166] FIG. 30 shows the neutralization of IFN-gamma-induced MHC
class II upregulation on human primary keratinocytes by D9D10 or
D9D10 scFv. Human keratinocytes were cultured for 24 h with or
without (not shown) 100 U/ml huIFN-gamma in the absence or the
presence of D9D10 (2 .mu.g/ml). Resting human keratinocytes do not
express MHC class II. IFN-gamma induces expression of MHC class II
in the keratinocytes and D9D10 (upper panel) or scFv D9D10 (lower
panel) inhibit this IFN-gamma-induced MHC class II expression. See
also further Example 7.1.
[0167] FIG. 31 shows the neutralization of IFN-gamma-induced MHC
class II upregulation on human primary keratinocytes by crude COS
supernatant containing either humanized D9D10 or MoTAbII. The
experiment was performed as described in FIG. 30 thin line: human
keratinocytes treated with human IFN.gamma.
[0168] bold line: A: human keratinocytes not treated with human
IFN.gamma.
[0169] B: effect of 400 ng/ml murine D9D10
[0170] C: effect of humanized D9D10 (crude COS supernatant)
[0171] D: effect of MoTAbII (crude COS supernatant)
[0172] FIG. 32 shows the effect of the anti-IFN-gamma antibody F3
and scFvF3 on the survival of mice in which the lethal shock
syndrome called "Shwartzman reaction" is induced. See also further
Example 7.3.
[0173] FIG. 33 shows the effect of the anti-IFN-gamma antibody F3
and scFvF3 on body weight of mice exhibiting IFN-gamma induced
cachexia. Mortality (number of dead mice/total number of mice) is
shown between brackets and the symbol "+". See also further Example
7.4.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0174] The invention described herein draws on previously published
work and pending patent applications. By way of example, such work
consists of scientific papers, patents or pending patent
applications. All of these publications and applications, cited
previously or below are hereby incorporated by reference.
[0175] The present invention is based on the finding that a
molecule which binds and neutralizes human interferon-gamma and
which is chosen from the group consisting of:
[0176] a scFv comprising the humanized variable domain of the
monoclonal antibody D9D10
[0177] a chimeric antibody comprising the humanized variable domain
of the monoclonal antibody D9D10
[0178] a diabody comprising the humanized variable domain of the
monoclonal antibody D9D10
[0179] a multivalent antibody
[0180] a ruminant antibody
[0181] is useful to treat diseases where IFN.gamma. activity is
pathogenic.
[0182] As used herein the terms "molecule which binds and
neutralizes IFN.gamma." refer to a molecule which recognizes and
binds any particular epitope of IFN.gamma. resulting in the
neutralization of any bioactivity of IFN.gamma.. Particular
epitopes of IFN.gamma. relate to the so-called E2 epitope
recognized and bound by the mAb D9D10, the so-called E1 epitope
(Kwok et al., 1993) or any other epitope. IFN.gamma. specifically
relates to human IFN.gamma. but may also relate to non-human
primate, mouse, rat, sheep, goat, camel, cow, llama or any other
IFN.gamma.. Furthermore, the term "bioactivity of IFN.gamma."
relates to the antiviral activity (Billiau, 1996), the induction of
the expression of MHC-class-II molecules by macrophages and other
cell types (Steinman et al., 1980), the stimulation of the
production of inflammatory mediators such as TNF.alpha., IL-1 and
NO (Lorsbach et al., 1993), the induction of the expression of
adhesion molecules such as ICAM-1 (Dustin et al., 1988) and of
important costimulators such as the B7 molecules on professional
antigen presenting cells (Freedman et al., 1991), the induction of
macrophages to become tumoricidal (Pace et al., 1983), the
induction of Ig isotype switching (Snapper and Paul, 1987), any
pathological and/or clinical activity during diseases where
IFN.gamma. is pathogenic (Billiau, 1996) or any other known
bioactivity of IFN.gamma.. In this regard, it should be clear that
any assay system demonstrating the IFN.gamma.-neutralizing capacity
of a molecule, such as the ones described by Novelli et al. (1991),
Lewis (1995) and Turano et al. (1992) can be used. Some of these
assays are also described in the subsection Evaluation of
anti-IFN.gamma. neutralizing molecules in the Examples section of
the present application (see further). It should be noted that the
molecules which bind and neutralize IFN-.gamma. as described above
neutralize at least one bioactivity, but not necessarily all
bioactivities, of IFN-.gamma..
[0183] The present invention further relates to a scFv comprising
the humanized variable domain of the monoclonal antibody D9D10. As
used herein, the term single-chain Fv, also termed single-chain
antibody, refers to engineered antibody constructs prepared by
isolating the binding domains (both heavy and light chain) of a
binding antibody, and supplying a linking moiety which permits
preservation of the binding function. This forms, in essence, a
radically abbreviated antibody, having only the variable domain
necessary for binding the antigen. Determination and construction
of single chain antibodies are described in U.S. Pat. No. 4,946,778
to Ladner et al. and in the Examples section of the present
application (see further). The term "humanized" means that at least
a portion of the framework regions of an immunoglobulin or
engineered antibody construct is derived from human immunoglobulin
sequences. It should be clear that any method to humanize
antibodies or antibody constructs, as for example by variable
domain resurfacing as described by Roguska et al. (1994) or CDR
grafting or reshaping as reviewed by Hurle and Gross (1994), can be
used. The humanization of the scFv comprising the variable domain
of the monoclonal antibody D9D10 is described further in the
Examples section of the present application. The monoclonal
antibody D9D10 was prepared essentially as described by Sandvig et
al. (1987) and Froyen et al. (1993). It should also be noted that
the process of humanization of an antibody or antibody construct is
regularly accompanied by a significant loss in binding affinity of
this antibody or antibody construct (Kettleborough et al., 1991;
Park et al., 1996 and Mateo et al., 1997). In contrast, and
surprisingly, the constructs humanized by the present inventors
were not characterized by a significant loss in binding affinity in
comparison to their non-humanized counterparts.
[0184] The present invention also relates to a chimeric antibody
comprising the humanized variable domain of the monoclonal antibody
D9D10. The term "chimeric antibody" refers to an engineered
antibody construct comprising variable domains of one species (such
as mouse, rat, goat, sheep, cow, llama or camel variable domains),
which may be humanized or not, and constant domains of another
species (such as non-human primate or human constant domains) (for
review see Hurle and Gross (1994)). It should be clear that any
method known in the art to develop chimeric antibodies or antibody
constructs can be used. The generation of a chimeric antibody
comprising the humanized variable domain of the monoclonal antibody
D9D10 is described further in the Examples section of the present
application.
[0185] The present invention also concerns a diabody comprising the
humanized variable domain of the monoclonal antibody D9D10. The
term "diabody" relates to two non-covalently-linked scFv's, which
then form a so-called diabody, as described in detail by Holliger
et al. (1993) and reviewed by Poljak (1994). It should be clear
that any method to generate diabodies, as for example described by
Holliger et al. (1993), Poljak (1994) and Zhu et al. (1996), can be
used. The generation of diabodies comprising the variable domain of
the monoclonal antibody D9D10 is described further in the Examples
section of the present application.
[0186] It should also be clear that the scFv's, chimeric antibodies
and diabodies described above are not limited to comprise the
variable domain of the monoclonal antibody D9D10 but may also
comprise variable domains of other anti-IFN.gamma. antibodies, such
as the sheep anti-IFN.gamma. antibody described further in the
present application, which efficiently neutralize the bioactivity
of IFN.gamma..
[0187] Furthermore, the diabodies described above may also comprise
two scFv's of different specificities. For example, the latter
diabodies may simultaneously neutralize IFN.gamma. on the one hand
and may target another molecule, such as TNF-.alpha., IL-1, IL-2,
B7.1 or CD80, B7.2 or CD86, IL-12, IL-4, IL-10, CD40, CD40L, IL-6,
tumour growth factor-beta (TGF-.beta.), transferrin receptor,
insulin receptor and prostaglandin E2 or any other molecule, on the
other hand.
[0188] The present invention also concerns multivalent antibodies
which bind and neutralize IFN.gamma.. As used herein, the term
multivalent antibody refers to any IFN.gamma.-binding and
IFN.gamma.-neutralizing molecule which has more than two
IFN.gamma.-binding regions. Examples of such multivalent antibodies
are triabodies, tetravalent antibodies, peptabodies and hexabodies
which bind and neutralize IFN.gamma. and which have three, four,
five and six IFN.gamma.-binding regions, respectively.
[0189] The present invention thus relates, as indicated above, to
triabodies which bind and neutralize IFN.gamma.. As used herein,
the term "triabody" relates to trivalent constructs comprising 3
scFv's, and thus comprising 3 variable domains, as described by
Kortt et al. (1997) and Iliades et al. (1997). A method to generate
triabodies is described by Kortt et al. (1997) and the generation
of triabodies comprising the variable domain of the monoclonal
antibody D9D10 is described further in the Examples section of the
present application. It should be noted that the triabodies of the
present invention may comprise: 3 variable domains of 3 different
anti-IFN.gamma. Ab's (i.e. 3 anti-IFN.gamma. Ab's which recognize
and bind a different epitope on IFN.gamma. [see also above]), 3
variable domains of 3 identical anti-IFN.gamma. Ab's such as 3
variable domains of D9D10 or 3 variable domains of humanized D9D10
or 3 variable domains of sheep anti-IFN.gamma. Ab's or 3 humanized
variable domains of sheep anti-IFN.gamma. Ab's, 1 or 2 variable
domain(s) of anti-IFN.gamma. Ab's in combination with 2 or 1
variable domain(s) of an Ab which binds to any other molecule than
IFN.gamma., respectively. Examples of such other molecules comprise
TNF-.alpha., IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-12, IL-4,
IL-10, CD40, CD40L, IL-6, tumour growth factor-beta (TGF-.beta.),
transferrin receptor, insulin receptor and prostaglandin E2.
[0190] The present invention further relates to tetravalent
antibodies which bind and neutralize IFN.gamma.. As used herein,
the term "tetravalent antibody" refers to engineered antibody
constructs comprising 4 antigen-binding regions as described by
Pack et al. (1995) and Coloma & Morrison (1997). Methods to
generate these tetravalent antibody constructs are also described
by the latter authors. The generation of the following 2 different
tetravalent antibodies comprising the variable domain of the
monoclonal antibody D9D10 are described further in the Examples
section of the present application: MoTabI which consists of 4
identical humanized D9D10 scFv's in the format of a homodimer of
two identical molecules each containing two D9D10 scFv's which are
linked together using a dimerization domain; the latter domain also
drives the homodimerization of the molecule, and, MoTab II which
consists of a full-size humanized D9D10 molecule to which two
humanized D9D10 scFv's are attached at the carboxyterminus
(CH3-domain). It should be noted that the tetravalent antibodies of
the present invention may comprise: 4 variable domains of 4
different anti-IFN.gamma. Ab's (i.e. anti-IFN.gamma. Ab's which
recognize and bind to a different epitope on IFN.gamma.), 4
variable domains of 4 identical anti-IFN.gamma. Ab's such as 4
variable domains of D9D10 or 4 variable domains of humanized D9D10
or 4 variable domains of sheep anti-IFN.gamma. Ab's or 4 humanized
variable domains of sheep anti-IFN.gamma. Ab's, 2 variable
domain(s) of one anti-IFN.gamma. Ab in combination with 2 variable
domain(s) of another anti-IFN.gamma. Ab, 2 variable domain(s) of
anti-IFN.gamma. Ab's in combination with 2 variable domain(s) which
binds to any other molecule than IFN.gamma.. Examples of such other
molecules comprise TNF-.alpha., IL-1, IL-2, B7.1 or CD80, B7.2 or
CD86, IL-12, IL-4, IL-10, CD40, CD40L, IL-6, TGF-.beta.,
transferrin receptor, insulin receptor and prostaglandin E2.
[0191] Furthermore, the term "dimerization domain" of MoTab I
refers to any molecule known in the art which is capable of
coupling the two identical molecules. Examples of such domains are
the leucine zipper domain (de Kruif & Logtenberg, 1996), the
helix-turn-helix motif described by Pack et al. (1993), the
max-interacting proteins and related molecules as described in U.S.
Pat. No. 5,512,473 to Brent & Zervos and the polyglutamic
acid-polylysine domains as described in U.S. Pat. No. 5,582,996 to
Curtis.
[0192] The present invention thus relates, as indicated above, to
peptabodies and hexabodies which bind and neutralize IFN.gamma.. As
used herein, the term "peptabodies" relates to pentavalent
constructs as described in detail by Terskikh et al. (1997). The
term "hexabodies" relates to hexavalent constructs which are
similar to the pentavalent constructs as described in detail by
Terskikh et al. (1997) but wherein the pentamerization domain is
replaced by any hexamerization domain known in the art. A method to
generate peptabodies is also described by Terskikh et al. (1997)
and a method to generate hexabodies can be derived from the
description by the latter authors. It should be noted that the
peptabodies and hexabodies of the present invention may comprise: 5
(relating to the peptabodies) or 6 (relating to the hexabodies)
variable domains of 5 or 6 different anti-IFN.gamma. Ab's (i.e. 5
or 6 anti-IFN.gamma. Ab's which recognize and bind a different
epitope on IFN.gamma. [see also above]), 5 or 6 variable domains of
identical anti-IFN.gamma. Ab's such as 5 or 6 variable domains of
D9D10, or, 5 or 6 variable domains of humanized D9D10, or, 5 or 6
variable domains of sheep anti-IFN.gamma. Ab's, or, 5 or 6
humanized variable domains of sheep anti-IFN.gamma. Ab's, less than
5 or 6 variable domain(s) of any anti-IFN.gamma. Ab's in
combination with less than 5 or 6 variable domain(s) of an Ab which
binds to any other molecule than IFN.gamma., respectively. Examples
of such other molecules comprise TNF-.alpha., IL-1, IL-2, B7.1 or
CD80, B7.2 or CD86, IL-12, IL-4, IL-10, CD40, CD40L, IL-6,
TGF-.beta., transferrin receptor, insulin receptor and
prostaglandin E2.
[0193] The present in invention further relates to ruminant
antibodies which bind and neutralize IFN.gamma.. The term
"ruminant" relates to animals belonging to the suborder Ruminantia
of even-toed hoofed mammals (as sheep, goats, cows, giraffes, deer,
llama, vicunas and camels) that chew the cud and have a complex 3-
or 4-chambered stomach.
[0194] More specifically, the present invention relates to sheep
antibodies which bind and neutralize IFN.gamma.. The term "sheep"
relates to any of numerous ruminant mammals belonging to the genus
Ovis. The generation of sheep anti-IFN.gamma. antibodies is
described in the Examples section of the present application. The
present invention also relates to sheep monoclonal antibodies. As
used herein, the term "monoclonal antibody" refers to an antibody
composition having a homogeneous antibody population. The term is
not limited regarding the species or source of the antibody, nor is
it intended to be limited by the manner in which it is made.
Indeed, the monoclonal sheep antibodies of the present invention
can be generated by any method known in the art. It should be noted
that also humanized antibodies, scFv's or any other fragment
thereof which has largely retained the specificity of said sheep
antibody or sheep monoclonal antibody are covered by the present
invention. As used herein, the term "fragment" refers to F(ab),
F(ab')2, Fv, and other fragments which retain the antigen binding
function and specificity of the parent antibody. It should also be
understood that the variable domains of the sheep anti-IFN.gamma.
(monoclonal) antibodies or scFv of the sheep anti-IFN.gamma.
(monoclonal) antibodies may be part of the chimeric antibodies,
diabodies, triabodies, tetravalent antibodies, peptabodies and
hexabodies as described above.
[0195] The present invention further relates to scFv's, chimeric
antibodies, diabodies, triabodies, tetravalent antibodies,
peptabodies, hexabodies and sheep antibodies which bind and
neutralize IFN.gamma. and which are produced by the methods as
described above and in the Examples section of the present
application.
[0196] The present invention further relates to a composition
comprising scFv's and/or chimeric antibodies and/or diabodies
and/or triabodies and/or tetravalent antibodies and/or peptabodies
and/or hexabodies and/or sheep antibodies which bind and neutralize
IFN.gamma. in a pharmaceutically acceptable excipient, possibly in
combination with other drugs or other antibodies, antibody
derivatives or constructs for use as a medicament to prevent or
treat septic shock, cachexia, immune diseases such as multiple
sclerosis and Crohn's disease and skin disorders such as bullous,
inflammatory and neoplastic dermatoses. Examples of such other
drugs or other antibodies, antibody derivatives or constructs are,
with regard to septic shock: an isotonic crystalloid solution such
as saline, dopamine, adrenaline and antibiotics; with regard to
cachexia: anti-TNF-alpha antibodies; with regard to multiple
sclerosis: ACTH and corticosteroids, interferon beta-1b
(Betaseron), interferon beta-1a (Avonex), immunosuppressive drugs
such as azathioprine, methotrexate, cyclophosphamide, cyclosporin A
and cladribine (2-CdA), copolymer 1 (composed of 4 amino acids
common to myelin basic proteins), myelin antigens, roquinimex A,
the mAb CAMPATH-1H and potassium channel blockers; with regard to
Crohn's disease: sulfasalazine, corticosteroids, 6
mercaptopurine/azathioprine and cyclosporin A; with regard to
psoriasis: cyclosporin A, methotrexate, calcipotriene (Dovonex),
zidovudine (Retrovir), histamine2 receptor antagonists such as
ranitidine (Zantac) and cimetidine (Tagamet), propylthiouracil,
acitretin (Soriatane), fumaric acid, vitamin D derivates,
tazarotene (Tazorac), IL-2 fusion toxin, tacrolimus (Prograf),
CTLA4Ig, anti-CD4 mAb's and T-cell receptor peptide vaccines. It
should also be clear that any possible mixture of the
above-indicated IFN-.gamma.-binding molecules may be part of the
above-indicated pharmaceutical composition.
[0197] As used herein, the term "composition" refers to any
composition comprising as an active ingredient scFv's and/or
chimeric antibodies and/or diabodies and/or triabodies and/or
tetravalent antibodies and/or peptabodies and/or hexabodies and/or
sheep antibodies which bind and neutralize IFN.gamma. according to
the present invention possibly in the presence of suitable
excipients known to the skilled man. The scFv's and/or chimeric
antibodies and/or diabodies and/or triabodies and/or tetravalent
antibodies and/or peptabodies and/or hexabodies and/or sheep
antibodies which bind and neutralize IFN.gamma. of the invention
may thus be administered in the form of any suitable composition as
detailed below by any suitable method of administration within the
knowledge of a skilled man. The preferred route of administration
is parenterally. In parenteral administration, the compositions of
this invention will be formulated in a unit dosage injectable form
such as a solution, suspension or emulsion, in association with a
pharmaceutically acceptable excipient. Such excipients are
inherently nontoxic and nontherapeutic. Examples of such excipients
are saline, Ringer's solution, dextrose solution and Hank's
solution. Nonaqueous excipients such as fixed oils and ethyl oleate
may also be used. A preferred excipient is 5% dextrose in saline.
The excipient may contain minor amounts of additives such as
substances that enhance isotonicity and chemical stability,
including buffers and preservatives.
[0198] The scFv's and/or chimeric antibodies and/or diabodies
and/or triabodies and/or tetravalent antibodies and/or peptabodies
and/or hexabodies and/or sheep antibodies which bind and neutralize
IFN.gamma. of the invention are administered at a concentration
that is therapeutically effective to treat or prevent septic shock,
cachexia, immune diseases such as multiple sclerosis and Crohn's
disease and skin disorders such as bullous, inflammatory and
neoplastic dermatoses. The dosage and mode of administration will
depend on the individual. Generally, the compositions are
administered so that the scFv's and/or chimeric antibodies and/or
diabodies and/or triabodies and/or tetravalent antibodies and/or
peptabodies and/or hexabodies and/or sheep antibodies which bind
and neutralize IFN.gamma. are given at a dose between 1 .mu.g/kg
and 10 mg/kg, more preferably between 10 .mu.g/kg and 5 mg/kg, most
preferably between 0.1 and 2 mg/kg for each IFN-.gamma.-binding
molecule. Preferably, they are given as a bolus dose. Continuous
short time infusion (during 30 minutes) may also be used. If so,
the scFv's and/or chimeric antibodies and/or diabodies and/or
triabodies and/or tetravalent antibodies and/or peptabodies and/or
hexabodies and/or sheep antibodies which bind and neutralize
IFN.gamma. or compositions comprising the same may be infused at a
dose between 5 and 20 .mu.g/kg/minute, more preferably between 7
and 15 .mu.g/kg/minute (for each IFN-.gamma.-binding molecule).
[0199] According to the specific case, the "therapeutically
effective amount" of a scFv's and/or chimeric antibodies and/or
diabodies and/or triabodies and/or tetravalent antibodies and/or
peptabodies and/or hexabodies and/or sheep antibodies which bind
and neutralize IFN.gamma. needed should be determined as being the
amount sufficient to cure the patient in need of treatment or at
least to partially arrest the disease and its complications.
Amounts effective for such use will depend on the severity of the
disease and the general state of the patient's health. Single or
multiple administrations may be required depending on the dosage
and frequency as required and tolerated by the patient.
[0200] The present invention further relates to scFv's and/or
chimeric antibodies and/or diabodies and/or triabodies and/or
tetravalent antibodies and/or peptabodies and/or hexabodies and/or
sheep antibodies which bind and neutralize IFN.gamma. for
determining IFN.gamma. levels in a biological sample,
comprising:
[0201] 1) contacting the biological sample to be analysed for the
presence of IFN.gamma. with a scFv and/or chimeric antibody and/or
diabody and/or triabody and/or tetravalent antibody and/or
peptabodies and/or hexabodies and/or sheep antibody as defined
above,
[0202] 2) detecting the immunological complex formed between
IFN.gamma. and said scFv and/or chimeric antibody and/or diabody
and/or triabody and/or tetravalent antibody and/or peptabodies
and/or hexabodies and/or sheep antibody.
[0203] As used herein, the term "a method to detect" refers to any
immunoassay known in the art such as assays which utilize biotin
and avidin or streptavidin, ELISA's and immunoprecipitation,
immunohistochemical techniques and agglutination assays. A detailed
description of these assays is given in WO 96/13590 to Maertens
& Stuyver. The immunohistochemical detection of IFN.gamma. in
cryosections of spinal cord and brain of non-human primates
suffering from experimental autoimmune encephalomyelitis is
described in detail in the Examples section of the present
application. The term "biological sample" relates to any possible
sample taken from a mammal including humans, such as blood (which
also encompasses serum and plasma samples), sputum, cerebrospinal
fluid, urine, lymph or any possible histological section, wherein
IFN.gamma. might be present.
[0204] The present invention will now be illustrated by reference
to the following examples which set forth particularly advantageous
embodiments. However, it should be noted that these embodiments are
illustrative and are not to be construed as restricting the
invention in any way.
EXAMPLES
[0205] 1. Generation of Humanized scFvD9D10
[0206] As the use of mouse monoclonals in humans induces a HAMA
response, a humanized antibody or antibody derivative is the
alternative. Humanized scFvD9D10 need to have similar binding and
neutralization properties as their original mouse counterparts, but
will elicit hardly any immune response in humans as compared to the
parent mouse scFv.
[0207] 1.1. Modelling
[0208] We used computer modelling techniques for the construction
of a humanized scFvD9D10 in order to develop an active scFv with
retained structure and affinity. The scFv was humanized using a
resurfacing strategy which includes the replacement of `non-human`
residues without significant structural changes of the scFv
molecule. This work consisted of 2 main parts. In the first part, a
3D-structure of the mouse scFv was constructed. For this purpose,
we have homology-modeled D9D10 using Ig V.sub.L and V.sub.H domains
with a similar sequence and a known structure. In the second part
(the actual humanization step), we have aligned D9D10 with similar
human sequences to identify `typically human residues`. After
verifying their structural compatibility with the D9D10model, they
have been proposed as residues-to-be-humanized.
[0209] PART 1: 3D-structure of scFvD9D10
[0210] Identification of Known Structures with the Most Resembling
Sequence
[0211] Different BLAST-searches were performed by entering the
D9D10 sequence of either V.sub.K or V.sub.H, by using the `BLASTP`
search program and by selecting the Brookhaven Protein Data Bank as
the database to be searched. This search was performed 4 times,
namely for V.sub.K with and without CDR-loops and for V.sub.H with
and without CDR-loops. The obtained data are summarized in Table
1.
1TABLE 1 Summary of BLAST-search results PDB score + CDR score -
CDR rank rank Code ident./sim. ident./sim. for V.sub.H source I.D.
A) BLAST-search using D9D10-V.sub.K sequence 1 1BAF 87%/92% 90%/95%
>50 mouse Fab frag. mAb An02 compl. w. its hapten
(2,2,6,6-Tetramethyl-1- Piperidinyloxy-Dinitrophenyl) 2 1FOR
80%/90% 85%/93% 16 mouse Igg2a Fab frag. (Fab17-Ia) 3 2IFF 78%/86%
84%/90% 15 mouse Igg1 Fab Frag. (Hyhel-5) compl. w. Chicken
Lysozyme mutant R68K 4 1FIG 75%/86% 80%/90% 28 mouse Chain L,
Immunogl G1 (Kappa Light Chain) Fab' frag, Mouse 5 1FVB 80%/87%
83%/89% >50 mouse IgA Fv frag. (Anti-Alpha(1->6) Dextran)
(Theoret. Model) 6 2HFL 77%/85% 83%/89% 14 mouse IgGl Fab frag.
(HyHEL-5) compl. w. Chicken Lysozyme . . . . . . . . . . . . . . .
. . . . . . 19 1NCA 60%/73% 70%/84% 1 mouse N9 neuraminidase-NC41
compl. w. Influenza Virus . . . . . . . . . . . . . . . . . . . . .
B) BLAST-search using D9D10-V.sub.H sequence 1 1NCA 83%/89% 91%/95%
19 mouse? N9 neuraminidase-NC41 compl. w. Influenza Virus 2 1NCB
80%/88% 87%/94% >50 mouse? N9 Neuraminidase-Nc41 Mut. N329D
compl. w. Fab, Influenza Virus 3 1TET 80%/86% 87%/92% 38 mouse Igg1
Monocl. Fab frag (Te33) compl. w. Cholera Toxin Peptide 3 4 1DBA
80%/87% 86%/92% >50 mouse Fab' frag. of the Db3 Anti- Steroid
Monocl. Ab . . . . . . . . . . . . . . . . . . . . . 16 1FOR
58%/76% 63%/83% 2 mouse Igg2a Fab frag. (Fab17-Ia) . . . . . . . .
. . . . . . . . . . . . .
[0212] A sequence similarity of more than 70% guarantees a strong
structural similarity. For V.sub.K, at least 6 very good matching
structures (all murine proteins) could be identified: 1BAF, 1FOR,
2IFF, 1FIG, 1FVB and 2HFL. The scores for the search with CDR-loops
varied from 87% to 77% for identical residues, and from 92% to 85%
for chemically similar residues. The scores for the search without
CDR-loops ranged from 90% to 83% identical residues and from 95% to
89% similar residues. The small difference in homology between the
searches with and without CDR-loops suggests that even some of the
CDR-loops are structurally similar. For V.sub.H, analogous results
were obtained. Four very well matching structures could be
identified: 1NCA, 1NCB, 1TET and 1DBA with scores varying from 83%
to 80% identical residues and from 89% to 87% similar residues when
CDR-loops are included. If CDR-loops were not taken into account,
significantly higher scores were obtained: from 91% to 86% for
identical residues and 95% to 92% for similar residues. The latter
was due to the fact that CDR-H3 from D9D10 was not matching well
with any sequence.
[0213] Three-Dimensional Fitting of the Best Candidates
[0214] From these scores, it was clear that the V.sub.K-fragment
from 1FOR resembled very well V.sub.K from D9D10 (rank nr 2). A
reasonably well homology was also found for its V.sub.H counterpart
(rank nr 16). For the heavy domain, 1NCA had a very high score for
V.sub.H (rank nr 1) and an acceptable score for its V.sub.K-domain
(rank nr 19). Since the .beta.-barrels of Fv fragments are well
conserved, and since for both V.sub.K and V.sub.H we dispose of two
very good resembling fragments with fairly well matching
counterparts, we had enough information to start the construction
of the D9D10 model.
[0215] When superimposing (fitting) the complete main chain of 1FOR
and 1NCA we obtained a root-mean-square (rms) deviation of 1.1
.ANG. (values around. or less than 1 .ANG. indicate a strong
structural similarity). Fitting on V.sub.K alone gave 1.0 .ANG. and
on V.sub.H we obtained 0.8 .ANG.. This means that both the complete
structures and the separate V-domains are nearly identical. In
order to obtain an even smaller rms-deviation, we fitted all
.beta.-strands of the central .beta.-barrel, giving an
rms-deviation of 0.52 .ANG.. When the C-terminal strands and
certain diverging residues were not taken into account, an
rms-deviation as low as 0.37 .ANG. was obtained. The high
structural resemblance of the central .beta.-barrel of both 1FOR
and 1NCA ensures us that we have correctly positioned the two
domains relative to each other.
[0216] In the next step, only the V.sub.K fragment of 1FOR and the
V.sub.H of 1NCA were retained and CDR-loops of 1FOR and 1NCA were
adopted without further modeling.
[0217] Modeling of the D9D10 Sequence Onto the Constructed
Framework
[0218] When the sequences of D9D10 were compared with those of
1FOR-V.sub.K and 1NCA-V.sub.H, 21 and 20 mutations were necessary
to mutate 1FOR and 1NCA into D9D10, respectively. These mutations
were done simultaneously using the Dead-End Elimination method
(Desmet et al., 1992) which found the globally best conformation
for all 41 mutations. For both V.sub.K and V.sub.H, the mutations
could be done without inducing sterical or energetical conflicts.
As a consequence, we have obtained a very reliable 3D-model for the
variable domains of D9D10 (except for CDR-H3).
[0219] PART 2: Humanization of D9D10
[0220] Identification of Residues to be Humanized
[0221] In order to identify typical D9D10 `murine` residues,
V.sub.K and V.sub.H sequences were again subjected to a
BLASTP-search, but this time the entire `non-redundant Genbank`
database (PDB+SwissProt+SPupdate+PIR) was searched for similar
sequences. Out of the resulting matches, only human and humanized
sequences were retained and aligned with D9D10.
[0222] The alignment revealed several systematic differences in
sequence between the murine D9D10 molecule and the best matching
human V.sub.K and V.sub.H fragments. From this comparison, we have
derived a consensus list of human residues.
[0223] Each of these residues was then placed onto the D9D10 model
and the following properties were examined: (i) the compatibility
with the framework and with neighboring residues, (ii) the solvent
accessibility and (iii) the proximity to the CDR-loops. In general,
only D9D10 residues which were not found in any human sequence,
which were structurally compatible with the D9D10 framework (and
CDR's), and which were clearly solvent exposed, were selected for
humanization.
[0224] For the V.sub.K domain we proposed 8 mutations, which were
spatially clustered into 2 surface patches of 3 residues each plus
two isolated residues. For the V.sub.H domain we pinpointed 9
residues to be humanized. The latter residues formed a surface
cluster of 5 residues, one of 2 residues and 2 additional isolated
residues. For neither of the two domains, buried residues were
retained in the mutation list. The reason for this is that we
explicitly wanted to preserve the D9D10 framework structure and,
also, that buried residues are not `visible` to the immune system
anyway.
[0225] Finally, the side-chain conformation of the 8+9 mutations
was modeled using the Dead-End Elimination algorithm. We found that
all mutations were energetically favorable. This strengthened the
hypothesis that the humanization procedure would not affect the
antigen binding properties of D9D10.
[0226] 1.2. Construction, Expression, Purification and Evaluation
of Humanized scFvD9D10
[0227] Eight substitutions in V.sub.HD9D10 and 9 in V.sub.LD9D10
had to be carried out as shown in FIG. 2. Since the different
mutations were spread among the whole V.sub.H and V.sub.L
sequences, it was decided to assemble the whole V.sub.H and V.sub.L
sequences out of synthetic oligonucleotides, hereby including all
necessary substitutions during the oligonucleotide synthesis as an
alternative to mutagenesis. During the oligonucleotide synthesis,
non-optimal E.coli codons were substituted for more optimal ones
coding for the same amino acid. Both V.sub.H and V.sub.L regions
were assembled separately according to the PCR assembly method
described by Stemmer et al. (1995). The assembled V.sub.H and
V.sub.L regions were first subcloned in pGEM-T vectors (PROMEGA
Corp., Madison Wis., US) and their correct sequence was confirmed
by DNA sequencing. Both humanised regions were subsequently
introduced into the pscFvD9D10H6 expression vector (Froyen et al.,
1993). For the assembly of the heavy chain, we synthesized 18
oligo's, 40 nucleotides in length, which collectively encode both
strands of the V.sub.H region from the AlwNI site to the Styl site.
The plus strand as well as the minus strand consist of 9 oligo's
configured in such a way that, upon assembly, complimentary oligo's
will overlap by 20 nucleotides. In these oligo's we included
mutations both leading to "humanised" amino acids at the
predetermined sites and to "optimised" E. coli codons.
2!Oligo? ? ? ? !No.? Oligo Seq. 1s 5'-CGCGCAGCCGCTGGATTGTTATTACT
(SEQ ID NO 3) CGCTGCCCAACCAG-3' 2as 5'-CAGCTGCACCTGGGCCATCGCTGGTT
(SEQ ID NO 4) GGGCAGCGAGTAAT-3' 3s 5'-CGATGGCCCAGGTGCAGCTGGTGCAG
(SEQ ID NO 5) AGCGGTAGCGAACT-3' 4as 5'-CGCTCGCACCCGGTTTTTTCAGTTCG
(SEQ ID NO 6) CTACCGCTCTGCAC-3' 5s 5'-GAAAAAACCGGGTGCGAGCGTTAAGA
(SEQ ID NO 7) TCAGCTGCAAAGCG-3' 6as 5'-TCGGTGAAGGTATAACCGCTCGCTTT
(SEQ ID NO 8) GCAGCTGATCTTAA-3' 7s 5'-AGCGGTTATACCTTCACCGATTACGG
(SEQ ID NO 9) TATGAACTGGGTTA-3' 8as 5'-ACCTTGACCCGGCGCCTGTTTAACCC
(SEQ ID NO 10) AGTTCATACCGTAA-3' 9s 5'-AACAGGCGCCGGGTCAAGGTCTGAAA
(SEQ ID NO 11) TGGATGGGTTGGAT-3' 10as 5'-TTTCACCGGTGTAGGTGTTGATCCAA
(SEQ ID NO 12) CCCATCCATTTCAG-3' 11s 5'-CAACACCTACACCGGTGAAAGCACCT
(SEQ ID NO 13) ACGTTGACGATTTC-3' 12as 5'-CTGAAAACGAAACGACCTTTGAAATC
(SEQ ID NO 14) GTCAACGTAGGTGC-3' 13s 5'-AAAGGTCGTTTCGTTTTCAGCCTGGA
(SEQ ID NO 15) TACCAGCGTTAGCG-3' 14as 5'-GCTGATCTGCAGGTAGGCCGCGCTAA
(SEQ ID NO 16) CGCTGGTATCCAGG-3' 15s 5'-CGGCCTACCTGCAGATCAGCTCTCTG
(SEQ ID NO 17) AAAGCGGAAGACAC-3' 16as 5'-GCGCGCAGAAGTAGGTCGCGGTGTCT
(SEQ ID NO 18) TCCGCTTTCAGAGA-3' 17s 5'-CGCGACCTACTTCTGCGCGCGTCGCG
(SEQ ID NO 19) GTTTCTACGCGATG-3' 18as 5'-GCGCCCTTGGCCCCAGTAATCCATCG
(SEQ ID NO 20) CGTAGAAACCGCGAC-3'
[0228] After assembly of the 18 40-mer oligonucleotides, the
desired fragment was PCR amplified using 2 oligonucleotides
complementary to the 5' and 3' end of the fragment
respectively.
3 Oligo No. Oligo Seq. 1s 5'-CGCGCAGCCGCTGGATTGTTATTAC- (SEQ ID NO
21) 3' 2as 5'-GCGCCCTTGGCCCCAGTAATC-3' (SEQ ID NO 22)
[0229] The resulting 381 bp fragment was cloned into a pGEM-T
vector, resulting in PGEM-TV.sub.HH and several clones were
sequenced. A similar approach was followed for the light chain.
Hereby 14 oligos were synthesized, 2 48-mers and 12 40-mers, which
collectively encode both strands of the V.sub.L region from the
SacI site to the XhoI site. However, since the Sacd site was
present exactly on an amino acid substitution site, this
restriction site could not be retained in the synthetic V.sub.L
gene. As an alternative, a Bst1107I site was created which will,
after ligation with the blunted Sacd site, restore the exact
V.sub.L reading frame.
4 Oligo No. Oligo Seq. 1s 5'-GCGGTATACTGACCCAGAGCCCGGCG (SEQ ID NO
23) ACCATGAGCGCGAGCCCGGGT-3' 2as 5'-CAGGTCAGGGTAACACGTTCACCC- GG
(SEQ ID NO 24) GCTCGCGCTCATGG-3' 3s 5'-GAACGTGTTACCCTGACCTGCAGCGC
(SEQ ID NO 25) GAGCTCTAGCATCA-3' 4as 5'-ATGATACCAGAACATATAGCTGATGC
(SEQ ID NO 26) TAGAGCTCGCGCTG-3' 5s 5'-GCTATATGTTCTGGTATCATCAGCGT
(SEQ ID NO 27) CCGGGTCAGAGCCC-3' 6as 5'-TATCATAGATCAACAGACGCGGGCTC
(SEQ ID NO 28) TGACCCGGACGCTG-3' 7s 5'-GCGTCTGTTGATCTATGATACCAGCA
(SEQ ID NO 29) ACCTGGCGAGCGGT-3' 8as 5'-CCGCTGAAACGCGCCGGAACACCGCT
(SEQ ID NO 30) CGCCAGGTTGCTGG-3' 9s 5'-GTTCCGGCGCGTTTCAGCGGTAGCGG
(SEQ ID NO 31) TAGCGGTACCAGCT-3' 10as 5'-ACGGCTGATGGTCAGGCTATAGCTGG
(SEQ ID NO 32) TACCGCTACCGCTA-3' 11s 5'-ATAGCCTGACCATCAGCCGTATGGAA
(SEQ ID NO 33) CCGGAAGATTTCGC-3' 12as 5'-TCTGATGGCAGAAATAGGTCGCGAAA
(SEQ ID NO 34) TCTTCCGGTTCCAT-3' 13s 5'-GACCTATTTCTGCCATCAGAGCTCTA
(SEQ ID NO 35) GCTATCCGTTCACC-3' 14as 5'-CGCGCTCGAGTTTGGTACCCTGACCG
(SEQ ID NO 36) AAGGTGAACGGATAGCTAGAGC-3'
[0230] After assembly of the 2 48-mer and 12 40-mer
oligonucleotides, the desired fragment was again PCR amplified
using 2 oligonucleotides complementary to the 5' and 3' end of the
fragment respectively.
5 Oligo No. Oligo Seq. 1s 5'-CGCGGTATACTGACCCAGAGC-3' (SEQ ID NO
37) 2as 5'-CGCGCTCGAGTTTGGTACCCTG-3' (SEQ ID NO 38)
[0231] The resulting 316 bp fragment was cloned into a pGEM-T
vector, resulting in PGEM-TV.sub.LH and several clones were
sequenced. The assembly PCR protocol (Stemmer et al., 1995)
consisted of 3 steps: gene assembly, gene amplification and
cloning. Since single-stranded ends of complementary DNA fragments
were filled-in during the gene assembly process, cycling with Taq
DNA polymerase resulted in the formation of increasingly larger DNA
fragments until the full-length gene was obtained. It can be noted
that DNA ligase has not been used in the process. After assembly,
the desired fragments were amplified using 5' and 3' end
complementary primers. The resulting fragments were subsequently
cloned into a suitable cloning vector such as pGEM-T, giving
PGEM-TV.sub.LH and PGEM-TV.sub.HH. The final vector,
pscFvD9D10V.sub.Hum, was constructed by ligating a 310 bp
Bst1107I/XhoI fragment originating from vector pGEM-TV.sub.LH with
a 3180 bp SacIblunt/XhoI fragment originating from vector
pscFvD9D10H6V.sub.HH (=pscFvD9D10H6 in which V.sub.H was replaced
by the humanized V.sub.H obtained from pGEM-TV.sub.HH).
[0232] Induction of the humanised scFv D9D10 was carried out in
E.coli strain JM83. Detection of His6-tagged scFv's on western blot
was done with an anti D9D10 rabbit polyclonal antibody and an anti
His6 monoclonal antibody (Babco, Richmond, Calif., USA). Compared
to the non-humanized scFvD9D10 (Froyen et al., 1993), the humanized
scFvD9D10 was expressed at approximately 3-5 times higher levels
(30-40 mg/l). This increase in expression level can be due to the
fact that during assembly the humanized scFvD9D10 coding sequence
was codon-optimised for E. coli expression. Alternatively, one or
several of the humanized amino acids can have a beneficial effect
on the expression level; or the increase in expression level can be
caused by a combination of the two. As with the non-humanized scFv,
most of the expressed protein was still present intracellularly
(70-80%), with 5-10% present in the periplasmic fraction and 10-20%
secreted to the medium.
[0233] The cells were harvested and lysed in the presence of
protease inhibitors at 4.degree. C. by the French press (2 passages
at 14.000 psi). The cell lysate was clarified by centrifugation and
the supernatant was used for purification. The supernatant was
loaded on Zn.sup.2+-IDA Sepharose FF and the resin was washed by
applying an imidazole step gradient. The different pools were
analysed by SDS-PAGE under reducing and non reducing
conditions.
[0234] The humanized scFv bound and eluted as expected in the 150
mM imidazole elution pool and SDS-PAGE showed that the recovered
scFv was >90% pure in a single step. The shift in relative
migration under reducing conditions showed that the scFv was
purified in an oxidized form. However, in contrast to the mouse
scFv, the humanized scFv showed a high tendency for non specific
adsorption, because only 40-50% of the initial product was
recovered after dialysis.
[0235] The humanized scFvD9D10 was shown to have the same
biological activity as the mouse scFvD9D10 for neutralizing the
antiviral activity of human IFN.gamma. (described in example
7).
[0236] Affinity could be calculated for murine and humanized scFv
using Surface Plasmon Resonance(SPR)-analysis with the BIACORE.RTM.
(Biacore AB, Uppsala, Sweden). This technology permits real-time
mass measurements using surface plasmon resonance. SPR is an
optical phenomenon, seen as a sharp dip in the intensity of light
reflected from a thin metal film coated onto a glass support. The
position of this dip depends on the concentration of solutes close
to the metal surface. In general, a protein (e.g. antibody) is
coupled to the dextran layer (covering the gold film) of a sensor
chip and solutions containing different concentrations of a binding
protein (e.g. antigen) are allowed to flow across the chip. Binding
(association and dissociation) is monitored with mass sensitive
detection.
[0237] In order to determine the affinity of the D9D10 derivatives
for hIFN.gamma., BIACORE.RTM. experiments were performed in which
the murine D9D10 was immobilized onto a CM5 sensorchip (Biacore
AB). D9D10 was immobilized using amine coupling according to the
manufacturer's procedure. To decrease the non specific interaction
of human IFN.gamma. with the carboxylic groups of the dextran
layer, the sensorchip was pretreated with 4 cycles of EDC/NHS--thus
reducing the amount of unblocked carboxylic groups remaining on the
sensor surface--before immobilizing D9D10. Then, immobilization of
D9D10 was carried out using a continuous flow of 5 .mu.l/min on a
sensor chip surface initially activated with 17 .mu.l of an 0.05M
NHS/ 0.2M EDC mixture. 35 .mu.l of typically 3 .mu.g/ml D9D10 was
injected over the activated surface. Residual unreacted ester
groups were blocked by injecting 17 .mu.l of 0.1M ethanolamine pH
8.5. D9D10 was immobilised directly on a CM5 chip at an optimal
concentration of 3 .mu.g/ml in an acetate buffer pH 5.4 resulting
in an immobilization level of about 600 RU. Most accurate affinity
data were obtained by injecting human IFN.gamma. and monitoring the
subsequent binding of scFvD9D10; the latter interacting with
remaining free epitopes on human IFN.gamma.. On and off rates were
calculated using the BIAevaluation software (Biacore AB).
[0238] Results of a typical experiment are shown in FIG. 3 for
murine scFvD9D10 and in FIG. 4 for humanized scFvD9D10 (These data
were generated in separate experiments). Calculated data were in
good agreement. As off rates were hardly detectable for both
constructs in most experiments, only on rates are shown for the
concentrations tested. These data clearly indicated that the
humanization did not hamper the binding characteristics of the scFv
fragment.
[0239] Monoclonal antibodies were generated against the humanized
scFvD9D10. A female BALB/c mouse was immunized (injected
intraperitoneally) 3 times with humanized scFvD9D10 (i.e., at days
0(50 .mu.g), 32(25 .mu.g) and 56(25 .mu.g)). Three months after, a
final boost of 25 .mu.g was given. Three days after this last
injection, spleen cells were retrieved from the immunized mouse and
used for cell fusion. Dissociated splenocytes from the immunized
mouse were fused with murine myeloma cells SP2/0-Ag14 (ATCC,
CRL-1581) at a ratio of 10:3 using a polyethylene glycol/DMSO
solution mainly according the procedure-as described by Kohler and
Milstein (1975). The fused cells were mixed up and resuspended in
DMEM medium supplemented with hypoxanthine, sodium pyruvate,
glutamine, a non-essential amino acid solution, 20%
heat-inactivated fetalclone (Hyclone Lab., Utah) and 10%
BM-Condimed (Boehringer Mannheim). The cells were then distributed
to 96 well plates to which aminopterin was added 24 hours after the
cell fusion. Each well contained between 1 to 5 growing hybridoma
clones at the average. After 8 days supernatant of the wells was
collected and screened in an ELISA for binding to humanized
scFvD9D10. The antibodies of the hybridomas thus generated were
further tested for their binding capacity to murine and humanized
scFvD9D10 and human IgG. Certain monoclonal antibodies derived from
this hyper immune mouse did recognize not only humanized scFvD9D10
but also human IgG, indicating the quality of the humanization
strategy. Using the antibodies which specifically interact with
humanized scFvD9D10 (1D5C5; 11E2G6; 10F12A2 available at
Innogenetics N.V., Industriepark Zwijnaarde 7, Box 4, B-9052 Ghent,
Belgium) and do not cross react with the yet tested human IgG
preparations, an ELISA is generated for detecting and quantifying
D9D10 derived constructs in human and primate serum.
[0240] Immunization experiments in rabbit and mouse with his-tagged
proteins including the humanized scFvD9D10 revealed weak to fairly
high immunogenic responses of the his tail. Consequently, we made a
new construct and removed the C-terminal hexahistidinetag from the
scFvD9D10 (humanized scFvD9D10H6.sup.-). This was done by cutting
vector pscFvD9D10V.sub.Hum with XhoI and EcoRI and substituting the
His6-tail with a tandem stop codon and a unique NcoI site for easy
identification. This was accomplished using two synthetic oligo's
(oligo 1: 5'-TCGAGATCAAACGGTAATAGCCATGG-3' (SEQ ID NO 39); oligo 2:
5'-AATTCCATGGCTATTACCGTTTGATC-3' (SEQ ID NO 40)) which, when
annealed, reconstitute the D9D10 V.sub.L coding sequence, followed
by tandem stop codons and a unique NcoI site for identification.
The annealed double-stranded oligo has sticky ends corresponding to
a XhoI site at the 5' end and EcoRI site at the 3' end. The oligo
was ligated into the XhoI/EcoRI opened pscFvD9D10V.sub.Hum vector
resulting in pscFvD9D10V.sub.Hum[H6.sup.-]. Expression analysis
showed identical expression levels and localisation compared to the
His6-tagged D9D10 in E. coli.
[0241] 2. Generation of Humanized, Chimeric D9D10
[0242] Two fusion cDNA-genes respectively coding for the heavy and
light chain fusion-proteins of the humanized D9D10 whole antibody
were constructed. The light chain fusion cDNA consists of the cDNA
encoding the mouse D9D10 light chain leader sequence (Ldr), needed
for efficient transport of the fusion protein in the host cell, the
humanized D9D10 light chain variable domain cDNA (V.sub.Lh),
followed by a human immunoglobulin kappa-light chain constant
domain (C.sub.L).
[0243] The heavy chain fusion cDNA consists of the mouse D9D10
light chain leader cDNA-sequence (Ldr), followed by the humanized
D9D10 heavy chain variable domain cDNA (V.sub.Hh) and a human IgG1
heavy chain constant domain
(C.sub.H.dbd.C.sub.H1-Hinge-C.sub.H2-C.sub.H3) cDNA, in which the
C1q-complement binding site in the C.sub.H2 region, known to induce
complement activation upon injection of the recombinant antibody,
was mutated (Pro.sub.331.fwdarw.Ser) (Xu et al., 1994).
[0244] PCR Cloning of Human Immunoglobulin C.gamma.1 and C.sub.K
cDNA
[0245] Total RNA was isolated from human tonsil cells (frozen
pellet of .+-.10.sup.7 cells) following the Chomczynski GuSCN/acid
phenol isolation method (Chomczynski and Sacchi, 1987). 140 .mu.g
total RNA was obtained. cDNA was prepared by annealing 700 ng total
RNA to 300 ng random hexamers (Pharmacia, Upsala, Sweden) and
reverse transcription for 90 min at 42.degree. C. using AMV reverse
transcriptase (RT-Stratagene) in a final volume of 20 .mu.l (50 mM
Tris pH 8.3, 40 mM KCl, 6 mM MgCl2, 5 mM DTT). The reaction was
inactivated by heating at 90.degree. C. for 15 min.
[0246] Cloning of the Human C.sub.K cDNA:
[0247] The cDNA was used as template for PCR amplification of the
human C.sub.K cDNA using primer sequences based on the Genbank
database sequence, accession # V00557 and # J00241.
6 oligo #7061 (C.kappa. sense primer): ThrValAla...
5'-TCGAAGCTTAGTACTGTGGCTGCACCATCTGT-3' (SEQ ID NO 41) HindIII
ScaI
[0248]
7 oligo #7060 (C.sub.K antisense primer): CysGluGly...
5'-GTCGAATTCTGCGCACTCTCCCCTGTTGAAGC-3' (SEQ ID NO 42) EcoRI
FspI
[0249] PCR amplification using the 7060/7061 primers is expected to
yield a fragment of 342 basepairs. ScaI/FspI digestion of this
fragment should yield a blunt fragment starting at the first AA,
Thr of C.sub.K and ending at the last AA, Cys. A stop codon is not
present.
[0250] PCR reaction was carried out in a final volume of 50 .mu.l,
using 2 .mu.l of the RT reaction, 10 pmol of each primer and 5 U of
either Taq DNA polymerase (Stratagene, La Jolla, Calif., USA).
dNTPs were present at a final concentration of 200 .mu.M in
1.times.Taq buffer as provided by the supplier. Reactions were
overlaid with 75 .mu.l paraffin oil. Cycling conditions were as
follows. After an initial denaturation of 5 min at 95.degree. C. 40
PCR cycles (1 min 94.degree. C., 1 min at appropriate annealing
temperature of 60.degree. C. and 1 min at 72.degree. C.) were
carried out. There was a final extension phase of 10 min at
72.degree. C. 5 .mu.l amounts of the reaction were run on agarose
gels.
[0251] The PCR reaction with the 7060/7061 primer pair yielded a
single band of .+-.300 bases, which was purified using the
Geneclean.TM. kit (Bio 101, Vista, Calif., USA), digested with
EcoRI/HindIII, phenol:CHCl.sub.3 extracted and ligated into
EcoRI/HindIII digested pBSK(-) vector (Stratagene). The ligation
mix was electroporated into the DH5.alpha.F' bacterial strain.
Transformed bacteria were plated onto X-gal/IPTG LB agar plates for
blue/white selection of recombinants. Four white colonies were
selected for further analysis and plasmid DNA was prepared.
EcoRI/HindIII restriction analysis showed that all 4 C.sub.K
transformants contained an insert of the correct length. The 4
inserts were entirely sequenced. One clone was completely identical
to the database sequence (accession nrs V00557 and J00241). The
corresponding plasmid was named pBLSKIGkappaC.
[0252] Cloning of the Human C.gamma.1 Heavy Chain Constant Domain
cDNA:
[0253] The cDNA was used as template for PCR amplification using
primer sequences based on the Genbank database sequence: accession
# Z17370.
8 oligo #7601 (C.gamma.1 sense primer; 48-mer, should only be
C.gamma.1 specific) AlaSerThr...
5'-CTAGAATTCTGCGCATCCACCAAGGGCCCATCGGTCTTCCCCCTGGCA-3' (SEQ ID NO
43) EcoRI FspI
[0254]
9 oligo #7600 (C.gamma.1 antisense primer): LysGlyProSer...
5'-GTAAAGCTTGAGCTCTTACCCGGAGACAGGGAGAGG-3' (SEQ ID NO 44) HindIII
SacI
[0255] PCR amplification using the 7601/7600 primer couple is
expected to yield a fragment of 1016 basepairs. FspI/SacI cleavage
of this fragment followed by removal of the SacI 3' overhang should
yield a blunt fragment starting with the first AA, Ala of C.gamma.1
and ending with the last AA, Lys. A stop codon is not included. PCR
reactions were carried out in a final volume of 50 .mu.l, using 2
.mu.l of the RT reaction, 10 pmol of each primer and 5 U of Taq DNA
polymerase (Stratagene). dNTPs were present at a final
concentration of 200 .mu.M in 1.times.Taq buffer as provided by the
supplier. Reactions were overlaid with 75 .mu.l paraffin oil.
Cycling conditions were as follows: after an initial denaturation
of 5 min at 95.degree. C. 40 PCR cycles (1 min 94.degree. C., 1 min
at appropriate annealing temp. 55.degree. C. and 1 min at
72.degree. C.) were carried out. There was a final extension phase
of 10 min at 72.degree. C. 10 .mu.l amounts of the reaction were
run on agarose gels. A single band of around 1 kb was obtained. The
1 kb band, obtained with the 7601/7600 primer pair, was purified
using the Qiaquick.TM.-kit (Qiagen, Hilden, Germany) and ligated
into pGEM-T-vector. The ligation mix was transformed into the
DH5.alpha.F' bacterial strain. Transformed bacteria were plated
onto X-gal/IPTG LB agar plates for blue/ white selection of
recombinants.
[0256] Eight white colonies were selected for further analysis and
plasmid DNA was prepared. Restriction analysis with BstXI
(=specific for IgG-1; absent in IgG-2) showed that 6 transformants
contained an C.gamma.1 insert of the correct length. One clone was
entirely sequenced and was shown to be identical to the database
sequence, except for 3 codon switches, which correspond to a
described allotypic variant Gm(-1,4) of the human IgG1
(lys214->arg214, asp356->glu356 and leu358->met358
respectively). Since the Gm(-1) ("nonmarker"), glu356/met358, also
occurs on C.gamma.2, this marker will likely not be immunogenic
when introduced in humans. The cloned sequence also contained two
silent base switches in comparison to the database sequence Z17370.
The final construct was named pGEMThIGG1c.
[0257] The C1q-complement binding site present in the C.sub.H2
region of the human IgG1, known to induce complement activation
upon injection of the recombinant antibody (Xu et al., 1994), was
later mutated (Pro.sub.331.fwdarw.Ser) as described further during
the assembly of the humanized D9D10 fusion cDNA.
[0258] Construction of Fusion cDNAs
[0259] In order to assemble the light- and heavy chain fusion
genes, several intermediate cloning constructs, generated by
PCR-assembly and amplification, were needed.
[0260] Assembly of the Light Chain Fusion cDNA
[0261] The mouse D9D10 V.sub.K leader sequence cDNA was cloned by
PCR-assembly (Stemmer et al., 1995) of four partially overlapping
synthetic oligonucleotides [IG8180, IG8179,IG8178 and IG8176] of
each 40 bps, and subsequent PCR-amplification with two specific
outside primers [IG 8175 and 8174]. The resulting 100 bp PCR
fragment I, named Ldr, consist of a 5' untranslated region of 20
bp, including an XbaI cloning site, and the cDNA encoding the
complete D9D10 V.sub.K leader peptide (20 AA) and 20 bp of the
humanized D9D10 light chain variable domain cDNA encoding the first
6 AA.
10 Sense strand oligos: XbaI IG8180
5'-GTCCCCCGGGTACCTCTAGAATGGATTTTCAAGTGCAGAT-3' (SEQ ID NO 45)
IG8179 5'-TTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATACTCTCG-3' (SEQ ID NO
46)
[0262]
11 Antisense strand oligos: IG8178
5'-CTCTGGGTCAGCTCGATGTCCGAGAGTATGACTGAGGCAC-3' (SEQ ID NO 47)
IG8176 5'-TGATTAGCAGGAAGCTGAAAATCTGCACTTGAAAATCCAT-3' (SEQ ID NO
48) PCR amplification primers: XbaI IG8175 (sense)
5'-GTCCCCCGGGTACCTCTAGAATG-3' (SEQ ID NO 49) IG8174 (antisense)
5'-CTCTGGGTCAGCTCGATGTCC-3' (SEQ ID NO 50) IG8175.fwdarw. IG8180
IG8179 ------------------- ------------------ IG8176 IG8178
----------------- ----------------- IG8174
[0263] The humanised light chain variable domain as present in
pGEM-T-V.sub.LH, described earlier, was PCR-amplified using primers
[IG8172 and IG8171] designed to produce PCR fragment II containing
the complete variable domain cDNA with exception of the last 3
amino acids (IKR), and flanked at the 3'-terminus by an
XhoI-cloning site.
12 IG8172(sense) 5'-GACATCGAGCTGACCCAGAGCCCGGCG-3' (SEQ ID NO 51)
XhoI IG8171(antisense) 5'-CGCGCTCGAGTTTGGTACCCTG-3' (SEQ ID NO
52)
[0264] Fusion of the two DNA fragments PCR-I (Ldr) and PCR-II
(V.sub.Lh), having 20 bp overlap, was performed by overlap PCR
using primerset IG8175 and IG8171. The resulting PCR-III fragment
was directly cloned in pGEM-T resulting in the pGEMLdrV.sub.Lh
plasmid.
13 Xba I IG8175(sense) 5'-GTCCCCCGGGTACCTCTAGAATG-3' (SEQ ID NO 49)
XhoI IG8171(antisense) 5'-CGCGCTCGAGTTTGGTACCCTG-3' (SEQ ID NO
52)
[0265] The human .sub.K-light chain constant domain was cloned by
PCR-amplification using pBLSKIGkappaC as template with primers
IG8170 and IG8169. The resulting PCR-IV fragment consists of the
cDNA sequence encoding the last 3 AA of V.sub.Lh and the complete
human Ckappa constant domain, followed by a stop codon and an EcoRI
cloning site. The PCR-IV DNA was directly cloned in the pGEM-T
vector resulting in the PGEM-TC.sub.L plasmid.
14 XhoI IG8170(sense) 5'-GCGCCTCGAGATCAAACGGACTGT-
GGCTGCACCATCTG-3' (SEQ ID NO 53) EcoRI IG8169(antisense)
5'-GCCGGAATTCCTAGCACTCTCCCCTGTTGAAG-3' (SEQ ID NO 54)
[0266] Fusion of LdrV.sub.Lh and C.sub.L cDNA in the pGEM-T
backbone was realised by insertion of the C.sub.L-containing
XhoI-SpeI fragment, isolated from pGEM-TC.sub.L plasmid, in the
pGEMLdrV.sub.Lh plasmid. The resulting construct was named
pGEMhD9D10.sub.L.
[0267] Assembly of the Heavy Chain Fusion cDNA
[0268] The mouse D9D10 V.sub.K leader sequence cDNA was cloned by
PCR-assembly (Stemmer et al., 1995) of four partially overlapping
synthetic oligonucleotides [IG8180, IG8179,IG8176 and IG8177] of
each 40 bps, and subsequent PCR-amplification with two specific
outside primers [IG 8175 and 8173]. The resulting 100 bp PCR-V
fragment, named Ldr-2, consist of a 5' untranslated region of 20
bp, including an XbaI cloning site, and the cDNA encoding the
complete D9D10 V.sub.K leader peptide (20 AA) and 20 bp of the
humanized D9D10 heavy chain variable domain cDNA encoding the first
6 AA.
[0269] Sense Strand Oligos:
15 Sense strand oligos: XbaI IG8180
5'-GTCCCCCGGGTACCTCTAGAATGGATTTTCAAGTGCAGAT-3' (SEQ ID NO 45)
IG8179 5'-TTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATACTCTCG-3' (SEQ ID NO
46) Antisense strand oligos: IG8177
5'-CTCTGCACCAGCTGCACCTGCGAGAGTATGACTGAGGCAC-3' (SEQ ID NO 55)
IG8176 5'-TGATTAGCAGGAAGCTGAAAATCTGCACTTGAAAATCCAT-3' (SEQ ID NO
48)
[0270] PCR Amplification Primers:
16 PCR amplification primers: XbaI IG8175(sense)
5'-GTCCCCCGGGTACCTCTAGAATG-3' (SEQ ID NO 49) IG8173(antisense)
5'-CTCTGCACCAGCTGCACCTGC-3' (SEQ ID NO 56) IG8175.fwdarw. IG8180
IG8179 ------------------ ------------------ IG8176 IG8177
----------------- ----------------- IG8173
[0271] The humanised variable heavy chain domain as present in
pGEM-T-V.sub.HH, described earlier, was PCR-amplified using primers
(IG8168 and IG8167) designed to produce PCR-VI fragment containing
the complete variable domain cDNA, and flanked at the 3'-terminus
by an XhoI-cloning site.
17 IG8168(sense) 5'-CAGGTGCAGCTGGTGCAGAGCGGTAG-3' (SEQ ID NO 57)
XhoI IG8167(antisense)
5'-CGCCGGCTCGAGACGGTGACCGTGGTCCCTTGGCCCCAGTAATCC-3' (SEQ ID NO
58)
[0272] Fusion of Ldr-2 and V.sub.Hh was performed by overlap PCR on
a mixture of PCR-V and PCR-VI using sense primer IG 8175 and an
antisense primer IG 8166, resulted in a PCR fragment (LdrV.sub.Hh)
which was directly cloned in a pGEM-T vector, resulting in
pGEMLdrV.sub.Hh.
18 XbaI IG8175(sense) 5'-GTCCCCCGGGTACCTCTAGAATG-3' (SEQ ID NO 49)
XhoI IG8166(antisense) 5'-CGCCGGCTCGAGACGGTGACC-3' (SEQ ID NO
59)
[0273] The human heavy chain constant domain cDNA was produced by
PCR amplification on pGEMThIGG1c as template, using sense primer IG
8165, designed to introduce a XhoI restriction site and antisense
primer IG 8164 that added an extra leucine to the C.sub.H sequence
and introduced a STOP codon followed by an EcoRI cloning site. The
introduction of a codon for a leucine provided, together with the
codon for a lysine (normally the last amino acid), a HindIII
restriction site. This HindIII site was used to insert a
scFv-module (cfr MoTAbII expression plasmids, see below). The
resulting fragment PCR-VII was inserted in the pGEM-T vector
resulting in plasmid PGEM-TC.sub.H.
19 XhoI IG8165(sense) 5'-GCCGCTCGAGCGCATCCACCAAGGGC-3' (SEQ ID NO
60) EcoRI HindIII IG8164(antisense) 5'-GCCGGAATTCGCTAAAGCTTACC-
CGGAGACAGGGAGAGG-3' (SEQ ID NO 61)
[0274] The amino acid Pro at position 331 in the C.sub.H2 domain of
both IgG1 and IgG4 immunoglobulins is described to contribute to
their differential ability to bind and activate complement (Xu et
al., 1994). The Pro33 1-codon CCC was therefore mutated to a
Ser331-codon, TCC. Two specific primers IG 8460 and IG8459 were
designed, to introduce this mutation by PCR mutagenesis.
[0275] Two separate PCR-amplifications were performed on
PGEM-T-C.sub.H as template using (1) primers IG2617, matching with
the T7-promoter region in pGEM-T and IG8460, resulting in a 733 bp
PCR-VIII fragment, and (2) primers IG 8459 and IG3899, matching the
SP6-promoter in pGEM-T, resulting in a 473 bp PCR-IX fragment.
Overlap PCR was subsequently performed on a mixture of PCR-VIII and
PCR-IX, using again the primers IG2617 and IG3899, resulting in a
1178 bp PCR-X fragment. The amplified PCR-X fragment was eventually
inserted as an XhoI-SpeI fragment (1018 bp) in the pGEMLdrV.sub.Hh
plasmid. The resulting pGEMhD9D10.sub.H plasmid contains the
complete coding sequence of the humanized D9D10 heavy chain fusion
protein.
20 IG8459(sense) 5'-GCCCTCCCAGCCTCCATCGAGAAAAC-3' (SEQ ID NO 62)
Ser.sub.331 IG8460(antisense) 5'-GTTTTCTCGATGGAGGCTGGGAGGGC-3' (SEQ
ID NO 63) Ser.sub.331 IG2617(sense-T7) 5'-TAATACGACTCACTA-3' (SEQ
ID NO 64) IG3899(antisense-SP6) 5'-ATTTAGGTGACACTATAG-3' (SEQ ID NO
65) *Construction of mammalian expression plasmids
[0276] Successful high level expression of recombinant
immunoglobulins has been reported in both lymphoid and non-lymphoid
mammalian cell lines. Basically an expression plasmid(s),
containing the immunoglobulin genes coding for respectively heavy
and light chain proteins under transcriptional control of a
promoter/enhancer unit recognized in mammalian cells, is introduced
in the chosen host cells together with (as one plasmid or on
separate plasmids) a drug-resistance gene expression unit by
classical cell transfection techniques. Cells that have randomly
integrated the foreign expression units in their cell genome are
intially selected for their drug-resistant phenotype and secondly
for high level, stable expression of the protein of interest, the
immunoglobulin. After gene integration, an increase in the
immunoglobulin expression level can be obtained by coamplification
of the genes through further selection of isolated recombinant cell
lines for increased resistance to the drug resistance marker.
[0277] One possible example of a successful strategy for mammalian
cell expression is the glutamine synthetase based
selection/amplification method shown to result in high level
production of mammalian proteins in different cell types including
Chinese hamster ovary cells (CHO) (Cockett et al., 1990) and
myeloma cells, Ns0 (Bebbington et al., 1992). The use of the system
is covered by patents WO87/04462 and WO89/10404 (Lonza Biologicals,
Slough, UK).
[0278] Following the GS-expression method, the fusion genes coding
for respectively the heavy- and light chain of the recombinant
immunoglobulins were cloned in a mammalian expression plasmid
(pEE12 or pEE14) under transcriptional control of the strong
Cytomegalovirus major immediate early promoter/enhancer (CMV-MIE).
This plasmid also carries a cloned glutamine synthetase (GS) gene
expression element that can act as a dominant selectable marker in
a variety of cells. GS indeed provides the only pathway for
synthesis of glutamine using glutamate and ammonia as substrates.
The final fusion product LdrV.sub.LhC.sub.L or hD9D10.sub.L was
directly cloned as an XbaI-EcoRI fragment isolated from the plasmid
pGEMhD9D10.sub.L in the mammalian expression vectors pEE14 (for
CHO) and pEE12 (for Ns0) (Lonza biologicals) under transcriptional
control of the CMV promoter, resulting in the plasmids
pEE12hD9D10.sub.L and pEE14hD9D10.sub.L.
[0279] The cDNA encoding the heavy chain fusion protein
LdrV.sub.HhC.sub.H or hD9D10.sub.H was first transferred from the
pGEMhD9D10.sub.H construct as an XbaI-EcoRI fragment in the
intermediate vector pEE6hCMV-BglII (Lonza Biologicals), also behind
the CMV promoter. From the latter construct pEE6hD9D10.sub.H a
complete mammalian expression casette, consisting of CMV-promoter
followed by the fusion gene and a polyadenylation site, were
transferred as an BglII-BamHI DNA fragment in the BamHI opened
plasmids pEE12hD9D10.sub.L and pEE14hD9D10.sub.L expression
plasmids already available. The final expression plasmids, named
pEE12hD9D10 and pEE14hD9D10 then consists of the pEE-backbone
plasmid containing the GS-selection unit, carrying the light chain
fusion gene expression casette followed by a comparable heavy chain
fusion gene expression casette.
[0280] The approach of assembling a single expression plasmid
containing separate transcription units for both heavy and light
chains and the selectable marker is advised in order to ensure
coamplification with the marker gene.
[0281] A schematic representation of both plasmids is given in
FIGS. 5 and 6.
[0282] The cDNA sequence encoding the complete humanized D9D10
heavy chain fusion protein is given in FIG. 7. (SEQ ID NO 66)
[0283] The cDNA sequence encoding the humanized D9D10 light chain
fusion protein is given in FIG. 8. (SEQ ID NO 68)
[0284] The amino acid sequence of the humanized D9D10 heavy chain
fusion protein is given in FIG. 9. (SEQ ID NO 67)
[0285] The aminoacid sequence of the humanized D9D10 light chain
fusion protein is given in FIG. 10. (SEQ ID NO 69)
[0286] Small Scale Expression of Humanized D9D10 Chimeric Antibody
in COS Cells
[0287] A quick way to determine the feasibility of expressing a
recombinant protein in mammalian cells and to evaluate its
functionality is transient expression of the product in COS cells
(Gluzmann, 1981). COS cells are Simian Virus 40 (SV40)-permissive
CV1 cells (African monkey kidney) stably transformed with an
origin-defective SV40 genome, thereby constitutively producing the
SV40 T-antigen. In SV40-permissive cells, T-antigen initiates high
copy number transient episomal replication of any DNA-vector that
contains the SV40 origin of DNA replication. Both the pEE12 and
pEE14 expression vectors contain an SV40 origin of replication in
the SV40 early promoter region controlling the GS-selection gene,
and thus permits efficient transient expression in COS cells.
[0288] Small amounts of functionally active antibody were made by
transient expression in COS cells. COS7 cells (ATCC CRL 1651) were
routinely cultured in DMEM supplemented with 0.03% glutamine and
10% fetal calf serum. For preparative scale transfection, an
optimized DEAE-transfection protocol (McCutchan, 1968) was used.
Alternatively, other well known transfection methods such as
Ca-phosphate precipitation, electroporation, liposome-based
transfection can be used. Briefly, exponentially growing COS7 cells
were seeded in cell factories (Nunc, Rochester, N.Y., USA) at 3.5
10.sup.4 cells/cm.sup.2. about 18 h before transfection, after
which the cells were washed twice with MEM-Hepes pH 7.1 (Gibco,
Rockville, Md., USA) and allowed to cool to bench temperature. 0.5
.mu.g/cm.sup.2 cell surface of high quality plasmid DNA
(CsCl-density purification) of the mammalian expression plasmids
pEE12hD9D10 and pEE14hD9D10 was ethanol precipitated, redissolved
in 25 .mu.l/cm.sup.2 MEM-Hepes pH 7.1 and slowly added to the same
volume of 2 mg/ml DEAE-dextran MW 500.000 (Pharmacia) in MEM-Hepes
pH 7.1. The DNA-DEAE-dextran precipitate (50 .mu.l/cm.sup.2) was
allowed to form for 20-25 min, put on the cells for 25 min and
removed to be stored at -20.degree. C. (the same precipitate can be
reused in a second transfection experiment with the same
efficiency).
[0289] The cells were incubated during the next 3.5 hours in DMEM
growth medium (Gibco) containing 0.1 mM chloroquine (Sigma) (0.3 ml
/cm.sup.2) in a CO.sub.2-incubator at 37.degree. C., then washed
two times with growth medium and further incubated for 18 hrs in
complete culture medium enriched with 0.1 mM sodium butyrate
(Sigma) at 37.degree. C. (0.3 ml/cm.sup.2). The next day the cells
were washed twice with serum free DMEM medium supplemented with
0.03% glutamine (Merck) and then incubated for 48 h (determined in
analytical scale experiments as the optimal harvest time) in 150
.mu.l/cm.sup.2 cell surface of the same medium at 37.degree. C.,
after which conditioned medium was harvested and stored at
-70.degree. C. until purification. As negative control COS cells
were also transfected with the empty expression vectors pEE12 and
pEE14.
[0290] Quality control of the crude CM was performed by
IFN.gamma.-binding assay in ELISA format, by SPR-analysis and by
measuring the inhibition of IFN.gamma. mediated MHC class
II-induction.
[0291] Human Interferony-Coating Elisa
[0292] 96 well ELISA culture plates (Nunc 469914) were coated with
100 ng/well hIFN.gamma. (Genzyme 80-3348-01, 1 mg/ml) diluted in 50
mM TrisHCl pH8.5, 150 mM NaCl, by 18 h incubation at 4.degree. C.
Blocking of nonspecific binding was performed in PBS/0.1% caseine
(200 .mu.l/well, 1 h, 37.degree. C.). All washing steps were
performed with PBS/0.05% Tween-20 (3.times.200 .mu.l/well).
Purified mouse-human chimeric D9D10 whole antibody (EP 0 528 469 to
Billiau and Froyen), produced by transient expression in COS cells,
was used as positive control (concentration range 500 ng/well to 4
ng/well, 1/2 dilution series prepared in the sample diluent, 100
.mu.l/well). Samples were diluted in a 1/2 dilution series in
PBS/0.1% caseine, and incubated for 2 h at 37.degree. C. Detection
was performed using an alkaline-phophatase conjugated
goat-anti-human IgG.sub.H+L (PromegaS3821), diluted 1/2000 in
PBS/0.05% caseine, incubated for 2 h at 37.degree. C. AP-substrate
(SigmaN-2765) was used at a concentration of 1 mg/ml in 100 mM
TrisCl pH9.5, 100 mM NaCl, 5 mM MgCl.sub.2. Plates were analysed at
405/595 nm after resp. 15 and 30 min incubation at 37.degree.
C.
[0293] Results are shown in FIG. 11: humanized D9D10 clearly
interacts with human IFN.gamma. coated onto the wells.
[0294] SPR Analysis
[0295] A comparable set up was used as described for the evaluation
of the murine and humanized scFvD9D10 derivatives. Briefly, murine
D9D10 was immobilized directly onto a B1 sensorchip (BIACORE
AB)--containing less carboxylic groups and for which as such no
pretreatment is necessary--at a concentration of 10 .mu.g/ml D9D10
in an acetate buffer pH 4.8 using amine coupling. A fixed
concentration of 8 .mu.g/ml human IFN.gamma. was added, followed by
the injection of either murine D9D10 (10 .mu.g/ml; positive
control) or crude COS supernatant containing humanized D9D10.
Results are shown in FIG. 12. These data clearly illustrate the
presence of active, IFN.gamma. binding molecules in the COS
supernatant. As no exact concentrations were determined of the
humanized D9D10, no affinity data were calculated.
[0296] Inhibition of MHC Class II-induction See Example 8.1.
[0297] Purification of Humanized D9D10
[0298] Humanized D9D10 was purified using classical protein A
chromatography (Perry and Kirby, 1990; Page and Thorpe, 1996).
Quality control of the purified antibody construct was performed by
Western Blot (classical technology) and ELISA. The latter is done
as described above and results are shown in FIG. 13. From these
results it is clear that purified, humanized D9D10 is specifically
interacting with IFN.gamma. coated onto the wells.
[0299] Generation of Stable Mammalian Expression Cell Lines
[0300] For generation of stable mammalian expression cell line, two
host cell lines Ns0 (Galfre and Milstein, 1981; ECACC 85110503) and
CHO-K1 (ATCC CCL61) were used.
[0301] The glutamine-dependent NS0 cells were routinely cultured in
Lonza DME (JRH 51435)/200 mMglutamine/10% FCS. High quality plasmid
DNA pEE12hD9D10, prepared by CsCl-density purification, and
linearized by SalI digestion, was used for transfection of the NS0
cells by electroporation (40 .mu.g DNA/10.sup.7 cells). Transfected
cells were then selected for the glutamine-independent phenotype by
gradual reducing the glutamine concentration. Selection was
performed in Lonza DME (JRH51435)/GS supplement (JRH58672)/10%
dialysed FCS. Individual NSO clones were isolated after .+-.2 weeks
of selection. The clones were analysed for recombinant antibody
production and secretion by testing the cell conditioned medium in
the IFN.gamma.-coating ELISA described earlier.
[0302] Several positive cell lines were selected for subsequent
vector amplification by growth in the presence of the GS-inhibitor
MSX (methionine sulfoximine), resulting in increased humanized
D9D10 antibody expression levels.
[0303] Large scale production of the recombinant antibody using
high expressing NS0 recombinant cell lines is done in bioreactor
systems (e.g. hollow fibre systems)
[0304] CHO-K1 cells were routinely cultured in GMEM-S
(JRH51492)/200 mM glutamine/10% FCS. High quality plasmid DNA
pEE14hD9D10, prepared by CsCl-density purification, was directly
used for transfection of CHO-K1 cells by Ca.sup.2+-phosphate
transfection techniques (12 .mu.g/1.15 10.sup.6 cells seeded 18 h
before transfection on T-flasks). Selective medium,
GMEM-S(JR51492)/GS supplement (JRH58672)/10% dialysed FCS/25 .mu.M
MSX was added to the cells 24 h post-transfection. Individual
clones could be isolated .+-.2 weeks after transfection. Selected
clones were analysed for recombinant antibody expression and
secretion by testing the cell conditioned medium in the
IFN.gamma.-coating Elisa described earlier. Several positive cell
lines were selected for subsequent vector amplification by growth
in the presence of increased concentrations of the GS-inhibitor
MSX, resulting in increased antibody expression levels.
[0305] Large scale production of the recombinant antibody using
high expressing CHO-K1 recombinant cell lines is done in bioreactor
systems (e.g. hollow fibre or ceramic core systems).
[0306] 3. Generation of Humanized Sheep Anti-IFN.gamma.
Antibodies
[0307] Sheep antibodies were generated by immunizing sheeps
according to standard immunization protocols. Briefly, sheeps were
injected intradermally on multiple sites with the antigen
(recombinant human IFN.gamma.(procaryotic origin)) for several
times over a timeframe of several months (day 0, 14, 28, 56, extra
injections on a monthly basis). Serum is tested for its antiviral
activity and its affinity (using SPR analysis).
[0308] As elution conditions necessary to elute an antigen from its
antibody reflect the affinity of the antibody (McCloskey et al.,
1997), experiments are performed in which the elution conditions of
the sheep antibodies for human IFN.gamma. were compared with those
of the scFvD9D10 antibody.
[0309] Sheep monoclonal antibodies are generated by fusing
B-lymphocytes isolated from peripheral blood with murine Sp2/0
myeloma cells according to the protocol as described in example 1.
The affinity of the antibodies for human IFN.gamma. is determined
by SPR analysis as described in example 1.
[0310] 4. Generation of Anti-IFN.gamma. Tetravalent Antibody
Constructs
[0311] 4.1. Generation of MoTAb I
[0312] The MoTAb I (Monospecific Tetravalent Antibody) molecule is
defined as a molecule which consists of 4 identical scFv molecules
(e.g. humanized D9D10 scFv's) in the format of a homodimer of two
identical molecules, each containing two scFv's. Both scFv's are
linked together using a dimerisation domain, which drives the
homodimerisation of the molecule (see FIG. 1). Comparable
structures have already been described (Pack et al., 1995,
Pluckthun & Pack, 1997).
[0313] The humanized D9D10 scFv was used as a building block to
generate the MoTAbI molecule using standard recombinant DNA
techniques. A single MoTAb subunit started with a humanized D9D10
scFv followed by a dimerisation domain flanked by flexible linkers.
The dimerisation domain was in turn linked C-terminally to a second
D9D10 scFv. Finally a detection and purification tag was added to
the extreme C-terminus of the molecule. However, in order to
circumvent possible immunological reactions against the tag, MoTAb
I was also produced in an untagged version. The sequence coding for
the dimerisation domain and the flanking linkers were made
synthetically using the method described by Stemmer et al. (1995).
This synthetic domain was subsequently linked to both D9D10 scFv's.
As linkers between the dimerisation domain and the scFv's, we have
used the flexible and proteolysis-resistant truncated human IgG3
upper hinge region (Pack & Pluckthun, 1992). As dimerisation
domain we used either the helix-turn-helix motif described by Pack
et al. (1993) or the leucine-zipper dimerisation domain originating
from the human JEM-1 protein as described by Duprez et al. (1997).
Optionally, an additional cysteine residue is inserted next to the
dimerisation domain to provide extra stability. When applicable, a
C-terminal detection and purification tag e.g. a hexahistidine
sequence, is used. The sequences were assembled in such a way that
functional domains were easily replaceable using unique restriction
sites present in the molecule. For the construction of the
pGEM-THDH vector, we synthesized 10 oligo's which collectively
encode both strands of the HDH region (hinge region-dimerization
domain-hinge region) flanked by a XhoI and a SpeI restriction site.
The plus strand as well as the minus strand consist of 5 oligo's
configured in such a way that, upon assembly, complimentary oligo's
will overlap by 20 nucleotides. In these oligo's the codons where
optimised for optimal E.coli usage. The resulting 223 bp fragment
was cloned into a pGEM-T vector and several clones were
sequenced.
[0314] Assembly Oligonucleotides for the HDH-domain:
21 Oligo No. Oligo Seq. 1s 5'-CGCGCTCGAGATCAAACGGACCCCGC (SEQ ID NO
70) TGGGTGATACCACTC-3' 2as 5'-CAGTTCACCTCCGGAGGTATGAGTGG (SEQ ID NO
71) TATCACCCAGCGGG-3' 3s 5'-ATACCTCCGGAGGTGAACTGGAAGAG (SEQ ID NO
72) CTGTTGAAACATCT-3' 4as 5'-GACCTTTCAGCAGTTCTTTCAGATGT (SEQ ID NO
73) TTCAACAGCTCTTC-3' 5s 5'-GAAAGAACTGCTGAAAGGTCCGCGGA (SEQ ID NO
74) AAGGTGAACTGGAG-3' 6as 5'-TTCAGGTGCTTCAGCAATTCCTCCAG (SEQ ID NO
75) TTCACCTTTCCGCG-3' 7s 5'-GAATTGCTGAAGCACCTGAAAGAGCT (SEQ ID NO
76) GTTGAAAGGTACCC-3' 8as 5'-ATGGGTAGTATCACCTAGGGGGGTAC (SEQ ID NO
77) CTTTCAACAGCTCT-3' 9s 5'-CCCTAGGTGATACTACCCATACCAGC (SEQ ID NO
78) GGTCAGGTGCAACT-3' 10as 5'-CGCGGAATTCGCGTTCGCGACTAGTT (SEQ ID NO
79) GCACCTGACCGCTGGT-3'
[0315] Amplification Oligonucleotides for the HDH-domain:
22 Oligo No. Oligo Seq. 1s 5'-CGCGGTATACTGACCCAGAGC-3' (SEQ ID NO
80) 2as 5'-CGCGCTCGAGTTTGGTACCCTG-3' (SEQ ID NO 81)
[0316] The MoTAbI expressionplasmid was constructed as followed:
The scFvD9D10 coding sequence was amplified by PCR using the
pscFvD9D10V.sub.Hum plasmid as a template. The sense primer used in
this amplification carried a unique SpeI restriction site in such a
way that the resulting scFvD9D10 sequence could be fused in-frame
at the C-terminus of the dimerisation domain.
23 sense primer: 5'-CGCGACTAGTGCAGAGCGGTAGCGAACTG-3' (SEQ ID NO 82)
antisense primer: 5'-GCCAGTGAATTCTATTAGTGGTGATG-3' (SEQ ID NO
83)
[0317] The resulting PCR fragment was inserted into the pGEM-T
vector and verified by DNA sequence analysis. The resulting plasmid
was named pGEM-TscFvD9D10 f s/e. Subsequently, the MoTABI
expressionplamid was assembled in a three-point ligation using
following fragments: The N-terminal scFvD9D10 originating from
vector pscFvD9D10V.sub.Hum as a XhoI/BcoRI fragment. This fragment
also carried the antibiotic resistance gene (Amp), the origin of
replication and the expression- and secretion signals. A second
fragment, originating from pGEM-THDH cut with XhoI and SpeI,
carried the helix-turn-helix dimerisation domain already described
previously flanked by human IgG3 upper hinge regions. Finally, a
third fragment, originating from the SpeI/EcoRI cut pGEM-TscFvD9D10
f s/e plasmid, carried the C-terminal scFvD9D10 with the
hexahistidine tag. The final expressionplasmid was named pMoTAbIH6
(FIG. 14) and carried the MoTAbI molecule under control of the lac
promotor and the pelB signal sequence as the secretion signal
(FIGS. 15 and 16). (SEQ ID NO 84 and 85)
[0318] To reduce immunogenicity, the hexahistidine sequence was
removed using synthetic oligo's in a similar way as described
previously for the humanized scFvD9D10, resulting in MoTabI. The
MoTAb I expression plasmid was introduced into a suitable E.coli
expression strain, e.g . JM83 and BL21. Good expressionlevels could
be obtained in both strains. Detection of the MoTabI molecule (60
kDa) on western blot was done with an anti D9D10 rabbit polyclonal
antibody and/or an anti His6 monoclonal antibody (Babco). However,
only a minor amount of the MoTAbI molecule was present in a soluble
form in the bacterial periplasm. The majority of the MoTAbI
molecule was not able to traverse the bacterial membrane and was
present as cytoplasmic inclusion bodies. This was confirmed by
N-terminal amino acid sequencing which revealed still the presence
of the pelB signal sequence on the molecule. The functionality of
the minor amount of secreted MoTAbI could however be confirmed
using an ELISA. In this ELISA, recombinant human IFN.gamma. was
coated onto a polystyreneplate and incubated with periplasmic
fractions originating from E.coli cells expressing the MoTAbI
molecule. Bound MoTAbI molecules where then detected using a rabbit
polyclonal serum generated against the D9D10 scFv followed by a
peroxidase labeled goat anti rabbit secondary serum.
[0319] Since most MoTAbI molecules were present in cytoplasmic
inclusion bodies, the molecules were purified from this fraction
under denaturing conditions followed by refolding to functional
molecules. However, since the MoTAbI molecule has the pelB signal
sequence still attached, a new cytoplasmic expressionplasmid was
constructed. In this expressionplasmid, MoTAbI expression is under
control of the strong leftward promotor of phage lambda (P.sub.L).
Since no secretion to the periplasmic space is necessary, the
MoTAbI coding sequence was fused directly to an ATG startcodon.
This was accomplished by isolating the MoTAbI coding sequence
lacking the pelB signal sequence by PCR from the pMoTAbI
expressionplasmid and recloning it into the EcoRV opened pBSK(+)
vector (Stratagene). A SapI restriction site giving access to the
first mature codon was hereby generated. After DNA sequence
verification the MoTAbI coding sequence was inserted as a SapI
blunt/SalI fragment into the NcoI blunt/ SalI cut pIGRI2
vector.
24 *pIGRI2 expressionvector nucleotide sequence 1
TTCCGGGGATCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAAT (SEQ ID NO 86)
51 TCATATAAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGG 101
CGGTGTTGACATAAATACCACTGGCGGTGATACTGAGCACATCAGCAGGA 151
CGCACTGACCACCATGAAGGTGACGCTCTTAAAAATTAAGCCCTGAAGAA 201
GGGCAGGGGTACCAGGAGGTTTAAATCATGGTAAGATCAAGTAGTCAAAA 251
TTCGAGTGACAAGCCTGTAGCCCACGTCGTAGCAAACCACCAAGTGGAGG 301
AGCAGTAACCATGGTTACTGGAGAAGGGGGACCAACTCAGCGCTGAGGTC 351
AATCTGCCCAAGTCTAGAGTCGACCTGCAGCCCAAGCTTGGCTGTTTTGG 401
CGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAA 451
GCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCA 501
CCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAG 551
TGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAA 601
CGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTC 651
GGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACG 701
TTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACT 751
GCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGC 801
GTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTAT 851
CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAAAAGGATCT 901
AGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAG 951
TTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC 1001
TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAC 1051
CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT 1101
TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCT 1151
TCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGC 1201
CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC 1251
GATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA 1301
GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGG 1351
AGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAA 1401
AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG 1451
CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT 1501
GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGA 1551
TTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAA 1601
CGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT 1651
TCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT 1701
GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTC 1751
AGTGAGCGAGGAAGCGGAAGAGCGCTGACTTCCGCGTTTCCAGACTTTAC 1801
GAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGT 1851
TTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCT 1901
GCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGG 1951
AGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCG 2001
CCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAG 2051
GGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATT 2101
CTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTC 2151
GAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAG 2201
GTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCG 2251
AGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAAGTTAGG 2301
CTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATC 2351
TACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGG 2401
AAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAAC 2451
GCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGC 2501
CTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAG 2551
CGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTC 2601
GCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGG 2651
CACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGA 2701
CGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCT 2751
CTCAAGGGCATCGGTCGGCGCTCTCCCTTATGCGACTCCTGCATTAGGAA 2801
GCAGCCCAGTAGTAGGTTTGAGGCCGUGAGCACCGCCGCCGCAAGGAATG 2851
GTGCATGTAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCC 2901
ACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCG 2951
ATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG 3001
TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGAATCCACAGG 3051
ACGGGTGTGGTCGCCATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGA 3101
AGCGAGCAGGACTGGGCGGCGGCCAAAGCGGTCGGACAGTGCTCCGAGAA 3151
CGGGTGCGCATAGAAATTGCATCAACGCATATAGCGCTAGCAGCACGCCA 3201
TAGTGACTGGCGATGCTGTCGGAATGGACGATATCCGGCAAGAGGCCCGG 3251
CAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGC 3301
CGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTG 3351
CGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAATCGATGAT 3401
AAGCTGTCAAACATGAGAATTAA
[0320] The new vector is called pIGRI2MoTAbI. A version lacking the
hexahistidine tag as constructed in a similar way starting from the
previous MoTAbI expressionplasmid without hexahistidine tail. The
new MoTAbI expressionvectors were subsequently transferred to
E.coli expressionstrains MC1061(pAcI), SG4044(pcI857) and
UT5600(pAcI). As expected, most of the expressed MoTAbI was present
as cytoplasmic inclusionbodies. MoTAbI molecules were purified from
cytoplasmic inclusion bodies under denaturing conditions followed
by standard refolding procedures as described by De Bernardez Clark
(1998).
[0321] 4.2. Generation of MoTAb II
[0322] The D9D10 MoTAb II is defined as a humanized D9D10 whole
antibody molecule to which a humanized D9D10ScFv sequence was
attached at the carboxyterminus (CH3-domain) of the heavy chain
(see FIG. 1). A comparable type of molecule has already been
described in literature (Coloma and Morrison, 1997).
[0323] For the expression of the D9D10 MoTAbII protein two fusion
genes, respectively coding for heavy and light chain protein of the
assembled antibody, were constructed. The heavy chain fusion gene
consists of an immunoglobulin leader sequence (D9D10 V.sub.K leader
cDNA) followed by the humanized D9D10 heavy chain variable domain
cDNA, a human IgG1 heavy chain constant domain
(C.sub.H1-Hinge-C.sub.H2-C.sub.H3) cDNA, a short G.sub.3S linker
sequence (Coloma and Morrison, 1997) and the humanized D9D10 ScFv
sequence. Alternative linker sequences such as the (G.sub.4S).sub.3
sequence or the flexible and proteolysis-resistant truncated mouse
IgG3 upper hinge region (Pack & Pluckthun, 1992) can be
used.
[0324] The light chain fusion gene is identical to the humanized
D9D10 recombinant antibody light chain gene (2) and contains the
D9D10 V.sub.K leader, the humanized light chain variable domain
cDNA and the human IgG1 constant domain (kappa).
[0325] Construction of MoTAb II Heavy Chain cDNA
[0326] The basic constructs generated for expression of the
humanized D9D10 antibody could be used as backbone for the MoTAbII
constructs. As described several intermediate cloning constructs,
mainly generated by PCR-assembly and -amplification, eventually
resulted in two final constructs, named pGEMhD9D10.sub.L and
pGEMhD9D10.sub.H. The latter plasmid was used as acceptorfragment
after digestion with HindIII and EcoRI, which eliminates the STOP
codon for insertion of a HindIII-EcoRI donorfragment isolated from
a plasmid pGEM-T-D9D10HE, resulting in the in frame fusion of the
hD9D10.sub.H cDNA to a cDNA sequence encoding the Gly.sub.3Ser
linker followed by the humanizedScFv-module and a STOP codon. The
resulting plasmid was named pGEM-MoTAbII.sub.H.
[0327] pGEM-T-D9D10HE was constructed by PCR amplification using
pScFvD9D10V.sub.Hum as template with primers IG8078 and IG8077. The
resulting 755 bp PCR fragment, containing the Gly.sub.3Ser linker
followed by the humanized scFv-module and a STOP codon, was
directly cloned in the pGEM-T vector.
25 IG8078 (sense): HindIII (SEQ ID NO 87)
5'-CCCAAGCTTGGCGGAGGCTCACAGGTGCAGCTGGTGCAGAG-3' EcoRI IG8077
(antisense): 5'-CGGAATTCTACCGTTTGATCTCGAGTTTGG-3' (SEQ ID NO
88)
[0328] Construction of Mammalian Expression Plasmids
[0329] Expression in mammalian cell lines was performed completely
as described for the humanized D9D10 antibody (cf example 2). The
cDNA encoding the LdrV.sub.HhC.sub.HScFv or MoTAbII.sub.H fusion
protein was initially inserted in the pEE6hCMV-BglII (Lonza
biologicals) intermediate expression vector, under transcriptional
control of the hCMV promoter. This was performed by transfer of the
EcoRI-XbaI DNA insert from pGEMMoTAbII.sub.H into the
pEE6hCMV-BglII vector. From the pEE6MoTAbII.sub.H plasmid a
complete mammalian expression casette, consisting of CMV-promoter
followed by the fusion gene and a polyadenylation site, was then
transferred as a BglII/BamHI fragment to the BamHI opened
pEE12hD9D10.sub.L and pEE14hD9D10.sub.L expression plasmids already
available (construct was earlier described for the humanized D9D10
antibody construct in example 2). The final expression plasmids,
named pEE12MoTAbII and pEE14MoTAbII then consisted of the
pEE-backbone plasmid containing the GS-selection unit, carrying the
light chain fusion gene expression casette followed by a comparable
heavy chain fusion gene expression casette. A schematic
representation of both plasmids is given in FIGS. 17 and 18. The
approach of assembling a single expression plasmid containing
separate transcription units for both heavy and light chains and
the selectable marker, is adviced in order to ensure
coamplification with the marker gene. The cDNA sequence encoding
the complete MoTAbII heavy chain fusion protein is given in FIG. 19
(SEQ ID NO 89). The amino acid sequence of the MoTAbII heavy chain
fusion protein is given in FIG. 20 (SEQ ID NO 90).
[0330] Small Scale Expression of D9D10 MoTAbII in COS Cells
[0331] Transient expression in COS monkey kidney cells was
performed using both mammalian expression constructs pEE12MoTAbII
and pEE14MoTAbII completely as described for the humanized D9D10
antibody (cf example 2). Quality control was performed by
IFN.gamma.-binding ELISA and SPR-analysis.
[0332] ELISA
[0333] The same set up was used as described in example 2. Results
are shown in FIG. 11. Specific binding to IFN.gamma. is detected.
The signal is lower than the signal obtained with crude COS
supernatant of humanized D9D10. However, no concentrations were
determined of MoTAbII.
[0334] SPR Analysis
[0335] A similar set up was used as described for the evaluation of
the murine and humanized scFvD9D10 derivatives. Briefly, murine
D9D10 was immobilized directly onto a B1 sensorchip at a
concentration of 10 .mu.g/ml D9D10 in an acetate buffer pH 4.8
using amine coupling. A fixed concentration of 8 .mu.g/ml human
IFN.gamma. was added, followed by the injection of either murine
D9D10 (10 .mu.g/ml; positive control) or crude COS supernatant
containing MoTAb II. Results are shown in FIG. 21. These data
clearly illustrate the presence of active, IFN.gamma. binding
molecules in the COS supernatant. As no exact concentrations were
determined of the MoTAB II, no affinity data could be
calculated.
[0336] Inhibition of MHC Class I Induction cf Example 8.1.
[0337] Purification
[0338] MoTAbII was purified using classical protein A
chromatography (Perry and Kirby, 1990; Page and Thorpe, 1996).
Quality control of the purified construct was done by Western Blot
(classical technology) and ELISA. The latter was performed as
described in example 2 and results are shown in FIG. 13. From these
results we can conclude that MoTAbII is specifically interacting
with human IFN.gamma..
[0339] Generation of Stable Mammalian Expression Cell Lines
[0340] For generation of stable mammalian expression cell line, two
host cell lines Ns0 (Galfre and Milstein, 1981; ECACC 85110503) and
CHO-K1 (ATCC CCL61) were used. Transfection and selection
procedures were completely identical as described for the humanized
D9D10 whole antibody, using the plasmids pEE12MoTAbII for Ns0 and
pEE14MoTAbII for CHO-K1. For both NS0 and CHO-K1, several MoTAbII
producing cell lines (determined in IFN.gamma.-binding ELISA) were
initially isolated and used as parental clones for further
amplification of recombinant protein expression levels as described
earlier.
[0341] Production of large amounts of the recombinant protein is
performed on bioreactor systems optimal for the respective host
cells.
[0342] 5. Generation of Anti-IFN.gamma. Diabodies
[0343] Diabodies are dimeric antibody fragments. In each
polypeptide, a heavy-chain variable domain (V.sub.H) is linked to a
light-chain variable domain (V.sub.L) but unlike scFv's, each
antigen-binding site is formed by pairing of one V.sub.H and one
V.sub.L domain from two different polypeptides. This is achieved by
shortening the linker between the V.sub.H and V.sub.L domains in
each molecule (Holliger et al., 1993). Since diabodies have two
antigen-binding sites they can either be monospecific or
bispecific. Monospecific bivalent molecules are generated by the
shortening the flexible linker sequence of the scFv molecule to
between five and ten residues and by cross-pairing 2 scFv molecules
with shortened linker. In order to stabilize the molecule, an
optional cysteine residue can be inserted in the linker. As an
example for the different steps involved in such a construction we
have documented the construction of D9D10-derived monospecific,
humanized anti-IFN.gamma. diabodies. The 15 residue linker of the
His6-tagged, humanized scFvD9D10 was replaced by the 5 or 10
residue linker using overlap extension PCR. Shortly, both D9D10
V.sub.H and V.sub.L coding sequences were PCR amplified whereby the
V.sub.H antisense primer and the V.sub.L sense primer have
sequences coding for the 5- or 10-mer linker sequence. The
resulting V.sub.H and V.sub.L PCR fragments were subsequently mixed
and a second PCR with the V.sub.H sense and V.sub.L antisense
primers was performed. The resulting PCR fragment is cloned into
the pBSK(+) plasmid (Stratagene) en verified by DNA sequence
analysis (FIGS. 22-25) (SEQ ID NO 91-94). The D9D10 diabody coding
sequence was subsequently transferred as a SapI blunt/EcoRI
fragment and inserted into the NcoI blunt/EcoRI opened vector
pTrc99A (Amann et al., 1988). In this vector, expression of the
diabodies is under control of the IPTG inducible Trc promotor. The
diabodies were expressed in E. coli strains HB101 or JM83.
Periplasmic fractions were prepared following a modified protocol
described by Neu and Heppel (1965). Briefly, cells were harvested
by centrifugation and resuspended in ice cold shockbuffer (100 mM
Tris-HCl pH 7.4; 20% sucrose; 1 mM EDTA pH8). After incubation on
ice during 10 min. with occasional stirring, the mixture was
centrifuged at 10.000 rpm during 1,5 min. The supernatans was
removed and the pellet was immediately resuspended in ice cold
distilled water. After incubation on ice during 10 min. with
occasional stirring, the mixture was centrifuged at 14,000 rpm and
the obtained supernatans was the soluble periplasmic fraction. The
periplasmic fractions were tested for binding to IFN.gamma. using
SPR-analysis. The experimental set up was as described in example
2. The undiluted samples were injected onto the surface of a B1
sensorchip coated with murine D9D10 onto which IFN.gamma. was
injected. Results obtained with L5 D9D10 diabodies are shown in
FIG. 26. A clear, specific binding of the diabodies was detected.
Comparable results were obtained with the L10 D9D10 diabody.
[0344] The bivalent, monospecific diabody molecules are purified
from the periplasmic extract via IMAC or from periplasmic inclusion
bodies using denaturing conditions followed by refolding.
[0345] Overlap Extension PCR Primers for the L10 D9D10
Diabodies:
26 D9D10V.sub.H forward (sense) primer
5'-GGCCGCTCTTCGAAATACCTATTGCCTACGG (SEQ ID NO 95) CAG-3'
D9D10L10V.sub.H backward (antisense) primer
5'-CTGGGTCAGTACGATGTCAGAGCCACCTCCG (SEQ ID NO 96)
CCTGAACCGCCTCCACCTGAGGAGACGGTGACCG TGGTC-3' D9D10L10V.sub.L forward
(sense) primer 5'-GTCACCGTCTCCTCAGGTGGAGGC- GGTTCAG (SEQ ID NO 97)
GCGGAGGTGGCTCTGACATCGTACTGACCCAGAG CC-3' D9D10V.sub.L backward
(antisense) primer 5'-GCCAGTGAATTCTATTAGTGGTGATG-3' (SEQ ID NO
98)
[0346] Overlap Extension PCR Primers for the L5 D9D10
Diabodies:
27 D9D10V.sub.H forward (sense) primer
5'-GGCCGCTCTTCGAAATACCTATTGCCTACGG (SEQ ID NO 95) CAG-3'
D9D10L5V.sub.H backward (antisense) primer
5'-CTGGGTCAGTACGATGTCTGAACCGCCTCCA (SEQ ID NO 99)
CCTGAGGAGACGGTGACCGTGGTC-3' D9D10L5V.sub.L forward (sense) primer
5'-GTCACCGTCTCCTCAGGTGGAGGCGGTTCAG (SEQ ID NO 100)
ACATCGTACTGACCCAGAGCC-3' D9D10V.sub.L backward (antisense) primer
5'-GCCAGTGAATTCTATTAGTGGTGATG-3' (SEQ ID NO 98)
[0347] 6. Generation of Anti-IFN.gamma. Triabodies
[0348] The construction of triabody molecules was analogous to the
scheme described above for diabody molecules, except that the
(G.sub.4S).sub.3 linker between the humanized D9D10 VH and VL was
completely deleted (FIGS. 27 and 28) (SEQ ID NO 101-102)
(zero-residue linker or -1-residue linker according to the Kabat
numbering (Kortt et al., 1997; Iliades et al., 1997) ). The
humanized D9D10 triabody construct is a mono-specific molecule
resulting from the spontaneous association of three zero-residue
linker (or -1-residue) D9D10 scFv molecules in the bacterial
periplasm. A trimer was formed whereby three pairs of V.sub.H and
V.sub.L domains interact to form three active antigen combining
sites. If necessary, in order to drive triabody formation as well
as to maintain stability, we can explore the possibility of
introducing additional association domains or disulfide
bridges.
[0349] The produced triabodies were tested for IFN.gamma. binding
using SPR-analysis. Periplasmic fractions were prepared as
described in example 5. SPR-analysis was performed as described in
example 5. Results are shown in FIG. 29. A clear, specific binding
of the triabody was obtained.
[0350] The triabody molecules were purified from the periplasmic
extract, made from uninduced bacterial cultures, via IMAC and
further by gel filtration or alternatively by purification under
denaturing conditions from periplasmic inclusionbodies followed by
refolding. The multimeric behaviour of the purified molecules was
analysed. The ability of the purified triabody to bind human
interferon y was tested using SPR-analysis and ELISA experiments as
described earlier. For these tests we produced milligram amounts of
highly purified material in a suitable E. coli expression
system.
[0351] Overlap Extension PCR Primers for the L0 D9D10
Triabodies:
28 D9D10V.sub.H forward (sense) primer
5'-GGCCGCTCTTCGAAATACCTATTGCCTACGG (SEQ ID NO 95) CAG-3'
D9D10L0V.sub.H backward (antisense) primer
5'-CTGGGTCAGTACGATGTCTGAGGAGACGGTG (SEQ ID NO 103) ACCGTGGTC-3'
D9D10L0V.sub.L forward (sense) primer
5'-GTCACCGTCTCCTCAGACATCGTACTGACCC (SEQ ID NO 104) AGAGCC-3'
D9D10V.sub.L backward (antisense) primer
5'-GCCAGTGAATTCTATTAGTGGTGATG-3' (SEQ ID NO 98)
[0352] 7. Generation of MoTAb's (and BiTAb's) Originating From
Fusion Proteins, From Serum Multisubunit Proteins and From
scFv's
[0353] The multi subunit (oligomeric) structure of proteins may be
exploited to obtain multivalent antibodies, when they are used as
fusion partner with scFv antibodies. Either the whole polypetide
chain, or the association sequence domain may be used as fusion
partner.
[0354] For example, haemoglobin is a tetrameric serum protein,
consisting from 2 alpha and 2 beta globin subunits. The dimer
dissociation constant is estimated to be in the order of 1 nM (Pin
et al., 1990). The tetramer--dimer dissociation constant of
haemoglobin in oxy-conformation was studied by gel filtration on
Superose 12 and was calculated to be 1 .mu.M (Manning et al, 1996).
Although non-covalent associations are known to be susceptible to
equilibrium rules, it has been described that the subunit
interactions are favoured in concentrated protein solutions like
serum and also may be increased by the presence of other
stabilising compounds (Srere and Mathews, 1990).
[0355] Recombinant haemoglobin expression has been extensively
investigated as a possible blood substitute in order to circumvent
the transmission of infectious disease agents during blood
transfusion. The alpha- and beta-globin polypeptides have already
been expressed from a single operon in E. coli (Hoffman et al.,
1990). In this case, the recombinant haemoglobin was purified from
the soluble cytoplasmatic fraction and the tetrameric E. coli
product had essentially the same characteristics as the native
protein. Analogous results were obtained when recombinant
haemoglobin was expressed in S. cerevisiae (Pagnier et al., 1992;
Mould et al., 1994; Sutherland-Smith et al., 1998).
[0356] Protein engineering strategies (Olson et al., 1997) and
chemical modification by pegylation (Pettit and Gombotz, 1998) are
investigated to enhance the stability and the circulation half
times in vivo. So fusion of relevant scFv molecules to the
respective alpha and beta subunit of human haemoglobin and
expression of the fusion proteins from a single operon in either E.
coli or S. cerevisiae would yield a functional tetrameric
monospecific (if identical scFv's are used) or bispecific (when
different scFv's are used) molecules at high level.
[0357] 8. Evaluation of Anti-IFN.gamma. Neutralizing Molecules
[0358] 8.1. Inhibition of MHCII-induction
[0359] In the first experiments, the effect of IFN.gamma. on the
induction of MHC class II expression on human keratinocytes was
examined. For this, primary human keratinocytes (passage 1) were
cultured with two concentrations of human IFN.gamma. (100 U/ml and
200 U/ml) during 24 and 48 hours. After culture, cells were
collected and the expression of MHC class II antigen on the
activated keratinocytes was measured by FACS-scan after staining
(30 minutes at 4.degree. C.) of the cells with a PE-labelled
anti-MHC-class II mAb. The results showed that resting
keratinocytes do not express MHC class II molecules and that
IFN.gamma. induces the expression after 24 hours in a
dose-dependent way. The induction is still enhanced after 48 hours
of culture.
[0360] In the next study, the effect of anti-human IFN.gamma.
D9D10H3 full size antibody or scFvD9D10-cmyc on the
IFN.gamma.-induced MHC-Class II expression on human keratinocytes
was examined. In this experiment, human primary keratinocytes
(passage 1) were cultured with human IFN.gamma. (100 U/ml) in the
presence or absence of different concentrations (2-0.5-0.12-0.03)
D9D10 Ab or D9D10scFv for 48 hours. IFN.gamma. was preincubated
with D9D10H3 or scFvD9D10 during 1 hour at 37.degree. C. before
adding to the keratinocytes. After culture, cells were collected
and the expression of MHC-Class II on these activated keratinocytes
was measured. For this, keratinocytes were incubated (30 minutes at
4.degree. C.) with a PE-labelled anti-MHC-ClassII mAb (Becton
Dickinson), washed twice with PBS and fixed. The MHC-Class II
expression was further analysed on a FACS-scan. The results of
these experiments are represented in FIG. 30. It is shown that the
MHC class II antigen is not expressed on the membrane of resting
keratinocytes and that IFN.gamma. clearly induces this MHC class II
expression. This IFN.gamma. induced MHC class II expression is dose
dependently inhibited by D9D10H3 and to a lesser extent by
scFvD9D10. We can conclude that about 4 times more scFv (0.12
.mu.g/ml) than full size antibody (0.5 .mu.g/ml) is needed to
obtain a 50% inhibition of the IFN.gamma.-induced MHC classII
expression on keratinocytes.
[0361] Similar experiments were performed in order to evaluate the
neutralization capacity of humanized D9D10 and MoTAbII. Results are
summarized in FIG. 31. Although in this experiment, MHC class II
induction could be only induced to a lesser extent, both humanized
D9D10 and MoTAbII clearly inhibit the IFN.gamma.-induction.
[0362] 8.2. Inhibition of Anti-Viral Activity
[0363] For neutralization of the antiviral activity of hIFN.gamma.,
serial dilutions of samples (anti-IFN.gamma. constructs) were
prepared in microtiter plates. To each well, hIFN.gamma. was added
to a final concentration of 5 antiviral protection Units/ml, as
tested on A549 cells. The mixtures were incubated for 4 h at
37.degree. C. and 25000 A549 cells were added to each well. After
an incubation period of 24 at 37.degree. C. in a CO.sub.2
incubator, 25 .mu.l of 8.times.10.sup.5 PFU EMC virus/ml was added
to the cultures for at least 24 h. As soon as virus-infected
control cultures reached 100% cell destruction, a crystal violet
staining was performed in order to quantify surviving cells. The
neutralization capacity of the anti-IFN.gamma. constructs was
defined by the concentration of the construct needed to neutralize
95% of the antiviral activity of 5 U/mi human IFN.gamma.. The
neutralization potency of the scFvD9D10 and the humanized scFvD9D10
was determined and was 1.2 .mu.g/ml and 1.5 .mu.g/ml,
respectively.
[0364] 8.3. Beneficial Effects in Septic Shock in Mice
[0365] Septic shock has been demonstrated to be a complex human
disease manifestation that occurs after the release of
lipopolysaccharide (LPS) into the circulation. The subsequent
production of high cytokine levels in the serum are known to play a
crucial role in septic shock. We generated data in a mouse model
system using an anti-mouse IFN.gamma. called F3 (Froyen et al.,
1995).
[0366] The generalized Shwartzman model is a lethal shock syndrome
in experimental animals which is elicited by 2 consecutive
injections of LPS. In the laboratory of prof. Billiau (Rega
Institute, Catholic University Leuven, Belgium), such a model was
developed in mice (Billiau et al., 1987). At time 0, the mice were
injected with 5 .mu.g LPS into the footpad, followed 24 h later by
a second intravenous injection of 100 .mu.g. Morbidity and
mortality was scored for 5 days. Untreated animals normally died
within 2 days after the second injection. Mice pretreated with the
anti-muIFN.gamma. antibody F3 were completely protected against the
lethal effect and only showed moderate disease symptoms. This
protection could be achieved with as little as 2.4 .mu.g F3 given
24 h before the first injection. In order to score the severity of
the disease, the symptoms were classified in 5 groups:
[0367] Score 0: not sick or mild piloerection
[0368] Score 1: piloerection and diarrhoea
[0369] Score 2: hemorhagic conjunctivitis and bleeding at the mouth
and anus
[0370] Score 3: paralysis of the hind legs
[0371] Score 4: death
[0372] The highest score that could be obtained is 4. Since the
number of mice in each group was relatively low (5), we established
a limit of the disease score (=2) that had to be reached in the
saline group in order to be a representative experiment.
[0373] The schedule we used in order to compare F3 and its scFv in
this Shwartzman model was as follows: NMRI mice were given the
preparative dose of 5 .mu.g LPS at time 0. At the time points +6 h,
+12 h and +23 h the mice were injected ip with 190 .mu.g scFvF3
(Froyen et al., 1995) or 30 .mu.g F3. Control animals were given
saline at the same time points. Each group consisted of 5 mice. The
mice were given a score according to the above mentioned
classification.
[0374] In the first experiment, 40% more mice were protected in the
scFvF3 group when compared with the control group. A second
experiment was set up using a slightly adapted protocol: an
additional injection was given at timepoint +3 h. The result of
this experiment (shown in table) was similar to that of experiment
1 in that 40% more mice survived in the scFvF3 group in comparison
with the control group as can be seen in FIG. 32. In addition to
scFvF3, a Fab antibody fragment of F3 was included in the second
group. All these mice survived the experiment.
[0375] The mean disease scores of these experiments, demonstrate a
significant difference for both F3 and the scFv compared to the
control group. The mean disease scores of the 5 mice of each group
were as follows:
29 Saline scFvF 3F3 FabF3 exp. 1 3.2 1.8 0.0 ND exp. 2 2.6 0.8 0.6
0.6
[0376] 8.4. Beneficial Effects During Cachexia in Mice
[0377] In a model for cachexia developed at the Rega Institute
(Matthys et al., 1991), nude mice were injected intraperitoneally
(ip) with CHO cells producing mouse IFN.gamma. (Mick cells). Mice
receiving CHO-Mick cells will exhibit cachexia (including body
weight loss) within 48 hours. The cachectic effect is correlated
with the number of Mick cells. Thus with small tumor cell inocula
(0.8-3.0.times.10.sup.7 cells), cachexia is transient and mice will
completely recover. However, with high inocula
(>3.4.times.10.sup.7 cells), mice continue to loose weight and
will die within 7 days. It is shown that IFN.gamma. plays an
essential role in the pathogenesis of the Mick-induced cachexia as
monoclonals against IFN.gamma. can reverse the wasting effect:
pretreatment (day-1) with the anti-muIFN.gamma. antibody F3
inhibits cachexia.
[0378] In order to compare the effects of F3 and its scFv on the
established cachexia model, the following experiment has been set
up: mice were injected with 2-4.times.10.sup.7 Mick cells on day 0
and antibody preparations were administered ip at time points +1.5
h, +6 h, +22 h and +66 h relative to the time of Mick cell
inoculation. For scFvF3, a dose of 190 .mu.g was given each
injection while for F3, 40 .mu.g was given. Control animals were
injected with saline at the same time points. In each group, 3 or 4
mice were used. Mice were weighed for 10 consecutive days and
mortality was scored. The results of 2 independent experiments are
shown in FIG. 33. The mice treated with scFvF3 were better
protected against the cachectic effect than the control mice.
[0379] These results also indicate that scFvF3 antibody fragments
do have a protective effect of cachexia but to a lesser extent than
the parental F3 antibody. Although results were promising, it was
clear that the effect of the scFv fragment was limited either due
to its fast clearance or to lowered affinity. Optimization of the
injection schedule was needed to obtain comparable results.
[0380] 8.5. Beneficial Effects in Septic Shock in Non-Human
Primates
[0381] The best documented sepsis model in non-human primates is
the one in which baboons are given lethal infusions of E.coli. As
described by Creasey et al. (1991), response to lethal E.coli
challenge occurs in 3 stages: an inflammatory stage marked by a
fall in white blood cell count (0-2 hr) and the appearance in
plasma of TNF.alpha., IL-1.beta. and IL-6; a coagulant stage marked
by a fall in fibrinogen concentration (2-6 hr); and a hypoxic cell
injury stage marked by a rise in SGPT/BUN and by a gradual
cardiovascular collapse, and death (6-24 hr).
[0382] Since the baboon animal model was not readily available, we
are establishing a comparable rhesus monkey model. D9D10 and
derived constructs interacted well with rhesus IFN.gamma. as
determined in an antiviral bioassay (set up as described in example
8.2).
[0383] Septic shock can be induced by infusion either of life
bacteria or of endotoxin in sedated monkeys. After administration
of different concentrations of the D9D10 anti-hIFN.gamma.
derivatives, several parameters are monitored including:
[0384] mortality (should be 100% in control (non-treated)
group)
[0385] pathophysiology
[0386] serum concentration of cytokines such as TNF.alpha., IL-1
and IL-6 using ELISA or bioassay (Villinger et al., 1993)
[0387] endotoxin profile using the limulus amoebocyte lysate
assay
[0388] 8.6. Beneficial Effects During Experimental Autoimmune
Encephalomyelitis in Non-human Primates
[0389] A. Pharmacokinetics of D9D10 and Derivatives in Monkey and
Effect on hIFN.gamma. Clearance
[0390] The clearance of the antibody derivatives is of importance
as molecules with a slow clearance have a prolonged efficacy. This
implicates that less material has to be injected which is better
for the patient and which is cost effective, especially when a
longer treatment period is advisable. Therefore, complexes of
IFN.gamma. and D9D10 derivatives are used in clearance studies in
non-human primates as a prerequisite to guide further in vivo
studies in these animals.
[0391] The clearance of D9D10, scFvD9D10H6.sup.-, D9D10 MOTAB I and
D9D10 MOTAB II, is monitored after a bolus injection in healthy
marmoset. Specific ELISA's are used for monitoring; no labelling of
the antibody constructs is required.
[0392] Blood clearance of radiolabelled marmoset IFN.gamma. after a
bolus intravenous injection alone or in combination with one of the
antibody constructs are also performed.
[0393] B. Beneficial Effects of the D9D10 Antibody Constructs on
EAE in Non-human Primates
[0394] In order to evaluate the therapeutic potential of the
anti-IFN.gamma. Mab D9D10 and derivatives, we are testing this
antibody in a relevant non-human primate model for MS as the final
step in our preclinical research. This model is required since the
antibody is not cross-reacting with IFN.gamma. from rodents and the
biological activity of IFN.gamma. is very species specific
(huIFN.gamma. is not active on cells other than human or non-human
primates (Terrell and Green, 1993)). D9D10 and derived constructs
interact well with marmoset IFN.gamma. as determined in an
antiviral bioassay (set up as described in example 8.2) and using
surface plasmon resonance (set up as described in example 1).
[0395] The EAE model is chosen as it is a generally accepted model
for Multiple Sclerosis. We opt for the EAE model in common marmoset
(Callithrix jacchus) as it is well developed (Massacesi et al.,
1995; Genain et al., 1995), it has a pathology of MR-detectable
lesions which reflects those in MS and the model shows a high
incidence of EAE induction with a chronic
progressive/relapsing-remitting course.
[0396] Acute PK-Tox
[0397] A limited PK-Tox study required by ethical prescriptions in
all research involving non-human primates, is set up in order to
test the toxicity of the substances administered either
intravenously or in the lumbar cerebrospinal fluid (CSF), as the
contribution of systemic and/or local IFN.gamma. to the development
of the disease is still unclear. Relatively high concentrations of
the antibody preparations, especially for the scFv, are injected
intravenously as one of our goals is to reach therapeutical
concentrations in the CNS. Although it is known that BBB leakage
occurs at the site of inflammation ('t Hart, personal
communication), a positive concentration gradient will be
beneficial.
[0398] Timing of the Study
[0399] Determination of the baseline parameters is done 1 week
prior to administration of the study drug. Animals are observed for
signs of toxicity for 30 days. During this period pharmacokinetic
parameters are monitored. Six weeks after the administration of the
study drug an additional blood sample is collected to determine
whether or not the animals mounted an immune response to any of the
D9D10 constructs or to recombinant marmoset IFN.gamma..
[0400] Parameters
[0401] During this study the following parameters are
determined:
[0402] Clinical Monitoring
30 Daily: Food consumption Weekly: Body weights Day 14, 28:
Haematology Clinical chemistry Urine analysis
[0403] Immunological Monitoring
[0404] Serum and CSF levels of humanized D9D10, MoTAbI and II or
IFN.gamma. are measured at different time points. When severe
toxicity occurs in one of the animals, the animal are sacrificed
and subjected to a detailed necropsy, in order to determine whether
this toxicity is drug related.
[0405] Diffusion of D9D10 Derivatives Into the Lesions
[0406] As both systemically and locally (in the brain) produced
IFN.gamma. can have a disease promoting role in EAE, antibody
derivatives must be able to neutralize both. Consequently,
transudation of the D9D10 derivatives into the lesions in brain and
spinal cord is necessary for a local effect on IFN.gamma.. However,
it is known that in MS the blood brain barrier is impaired in a
subset of the active brain lesions for a limited period of time.
More specifically, BBB breakdown is reflecting the state of
inflammation (Hawkins et al., 1990).
[0407] The differential ability of the anti-IFN.gamma. constructs
to enter the brain is crucial for the choice of the component(s)
which will be used for evaluation of the therapeutic efficacy of an
anti-IFN.gamma. treatment in EAE.
[0408] The entry of the constructs into the brain compartment is
measured by post-mortem magnetic resonance imaging (MRI)-scan of
the brain and the spinal cord of a relapsing monkey, injected
intravenously with a gadolinium-diethylene-triamine pentaacetic
acid (Gd-DTPA)-labelled D9D10 construct 1 hour prior to sacrifice.
MRI-scans are compared and are related to an MRI-scan taken just
before death after an injection with a small gadolinium salt that
easily enters through leakages in the BBB (Gonzalez-Scarano et al.,
1987; Hawkins et al., 1990; Youl et al, 1991).
[0409] These results reveal which D9D10 construct most easily
enters the brain and which molecule eventually enters the active
lesions where the BBB is already restituted.
[0410] Therapeutic Treatment of Marmoset Monkeys Undergoing EAE
Disease Relapse
[0411] The therapeutic effect of either systemically or locally
administered anti-IFN.gamma. on the outcome of EAE in marmoset is
evaluated. The start of the treatment of the monkeys is situated at
the beginning of the first relapse of EAE, which usually occurs
several months after the initial immunization. During the
experiment the following observations, analysis and measurements
are carried out as of the time of relapse.
[0412] Clinical Monitoring
[0413] The severity of EAE is scored daily on an arbitrary scale
modified from Massacesi et al. (1995)
[0414] Body weight and body temperature (at time of blood
sampling)
[0415] Behavioural tests for monitoring the failure of neurological
functions
[0416] Magnetic resonance imaging (MRI) of the CNS
[0417] Biochemical parameters: neopterin (specifically formed in
activated macrophages) is measured in urine
[0418] Immunological Monitoring
[0419] At several indicated time points serum is taken to monitor
the blood levels of the antibody constructs or IFN.gamma. and to
monitor the marmoset anti-mouse or anti-IFN response.
[0420] Pathology
[0421] MRI-guided histopathology analysis has proven a powerful
tool for detailed analysis of MR-detectable lesions with
histological methods. Briefly, at a chosen moment but preferably
shortly after in vivo MR-images have been recorded, the monkey are
euthanised. The brain and spinal cord is carefully excised and
fixed in toto for 3 days in 4% buffered formaldehyde. Then a
T2-weighted scan is made in axial and coronal direction, with a
slice thickness of 1 mm covering the whole brain. For orientation
of the axial slices of in vivo and in vitro images the anterior and
posterior tips of the corpus callosum are used as internal
reference points.
[0422] The excellent structural conservation and the high
resolution of the MR-image make accurate three-dimensional
localisation of potential lesions possible. Regions of interest are
subsequently excised and histologically analysed for infiltrating
cells (Haematoxylin-eosin), demyelisation (KLB staining of myelin
lipids) and axonal structure (silver impregnation acc. to
Boielschowsky).
[0423] One half of an excised brain and spinal cord is snap-frozen
in liquid nitrogen. Thin cryosections are made and processed for
immunohistology staining, such as for visualisation of cytokine
secreting cells (especially IFN.gamma.) or for phenotyping of
infiltrated or tissue cells.
[0424] 8.7. Beneficial Effects of Anti-IFN.gamma. Antibody
Constructs in Crohn's Disease
[0425] A. In Vitro Assay Using Patient-Derived Lymphocytes and
Antigen Presenting Cells
[0426] Lymphocytes isolated from either peripheral blood or
surgical specimen (lamina propria or ileum E) from patients with
Crohn's disease, are used for assessment of cytokine profile,
lymphotyping, and functional cytotoxicity. The latter is performed
by adding patient-derived antigen presenting cells and measuring
the cytokine profile. The effect of anti-IFN.gamma. derived
antibody constructs on cytokine production is measured.
[0427] B Anti-IFN.gamma. Treatment of Crohn's Disease
[0428] Patients with active Crohn's disease are infused with
anti-IFN.gamma. in a dose ranging from 1 to 20 mg/kg. Responders in
the study may continue to receive repeated doses of
anti-IFN.gamma.. In all patients, clinical responses are observed
and Crohn's disease activity index (CDAI) is determined.
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Sequence CWU 1
1
104 1 804 DNA Unknown Genomic 1 atgaaatacc tattgcctac ggcagccgct
ggattgttat tactcgctgc ccaaccagcg 60 atggcccagg tgcagctggt
gcagagcggt agcgaactga aaaaaccggg tgcgagcgtt 120 aagatcagct
gcaaagcgag cggttatacc ttcaccgatt acggtatgaa ctgggttaaa 180
caggcgccgg gtcaaggtct gaaatggatg ggttggatca acacctacac cggtgaaagc
240 acctacgttg acgatttcaa aggtcgtttc gttttcagcc tggataccag
cgttagcgcg 300 gcctacctgc agatcagctc tctgaaagcg gaagacaccg
cgacctactt ctgcgcgcgt 360 cgcggtttct acgcgatgga ttactggggc
caagggacca cggtcaccgt ctcctcaggt 420 ggaggcggtt caggcggagg
tggctctggc ggtggcggat cggacatcgt actgacccag 480 agcccggcga
ccatgagcgc gagcccgggt gaacgtgtta ccctgacctg cagcgcgagc 540
tctagcatca gctatatgtt ctggtatcat cagcgtccgg gtcagagccc gcgtctgttg
600 atctatgata ccagcaacct ggcgagcggt gttccggcgc gtttcagcgg
tagcggtagc 660 ggtaccagct atagcctgac catcagccgt atggaaccgg
aagatttcgc gacctatttc 720 tgccatcaga gctctagcta tccgttcacc
ttcggtcagg gtaccaaact cgagatcaaa 780 cggcaccatc accatcacca ctaa 804
2 267 PRT Artificial Sequence Synthetic 2 Met Lys Tyr Leu Leu Pro
Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala
Met Ala Gln Val Gln Leu Val Gln Ser Gly Ser Glu 20 25 30 Leu Lys
Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly 35 40 45
Tyr Thr Phe Thr Asp Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly 50
55 60 Gln Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu
Ser 65 70 75 80 Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser
Leu Asp Thr 85 90 95 Ser Val Ser Ala Ala Tyr Leu Gln Ile Ser Ser
Leu Lys Ala Glu Asp 100 105 110 Thr Ala Thr Tyr Phe Cys Ala Arg Arg
Gly Phe Tyr Ala Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln 145 150 155 160 Ser Pro
Ala Thr Met Ser Ala Ser Pro Gly Glu Arg Val Thr Leu Thr 165 170 175
Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met Phe Trp Tyr His Gln Arg 180
185 190 Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu
Ala 195 200 205 Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly
Thr Ser Tyr 210 215 220 Ser Leu Thr Ile Ser Arg Met Glu Pro Glu Asp
Phe Ala Thr Tyr Phe 225 230 235 240 Cys His Gln Ser Ser Ser Tyr Pro
Phe Thr Phe Gly Gln Gly Thr Lys 245 250 255 Leu Glu Ile Lys Arg His
His His His His His 260 265 3 40 DNA Unknown Genomic 3 cgcgcagccg
ctggattgtt attactcgct gcccaaccag 40 4 40 DNA Unknown Genomic 4
cagctgcacc tgggccatcg ctggttgggc agcgagtaat 40 5 40 DNA Unknown
Genomic 5 cgatggccca ggtgcagctg gtgcagagcg gtagcgaact 40 6 40 DNA
Unknown Genomic 6 cgctcgcacc cggttttttc agttcgctac cgctctgcac 40 7
40 DNA Unknown Genomic 7 gaaaaaaccg ggtgcgagcg ttaagatcag
ctgcaaagcg 40 8 40 DNA Unknown Genomic 8 tcggtgaagg tataaccgct
cgctttgcag ctgatcttaa 40 9 40 DNA Unknown Genomic 9 agcggttata
ccttcaccga ttacggtatg aactgggtta 40 10 40 DNA Unknown Genomic 10
accttgaccc ggcgcctgtt taacccagtt cataccgtaa 40 11 40 DNA Unknown
Genomic 11 aacaggcgcc gggtcaaggt ctgaaatgga tgggttggat 40 12 40 DNA
Unknown Genomic 12 tttcaccggt gtaggtgttg atccaaccca tccatttcag 40
13 40 DNA Unknown Genomic 13 caacacctac accggtgaaa gcacctacgt
tgacgatttc 40 14 40 DNA Unknown Genomic 14 ctgaaaacga aacgaccttt
gaaatcgtca acgtaggtgc 40 15 40 DNA Unknown Genomic 15 aaaggtcgtt
tcgttttcag cctggatacc agcgttagcg 40 16 40 DNA Unknown Genomic 16
gctgatctgc aggtaggccg cgctaacgct ggtatccagg 40 17 40 DNA Unknown
Genomic 17 cggcctacct gcagatcagc tctctgaaag cggaagacac 40 18 40 DNA
Unknown Genomic 18 gcgcgcagaa gtaggtcgcg gtgtcttccg ctttcagaga 40
19 40 DNA Unknown Genomic 19 cgcgacctac ttctgcgcgc gtcgcggttt
ctacgcgatg 40 20 41 DNA Unknown Genomic 20 gcgcccttgg ccccagtaat
ccatcgcgta gaaaccgcga c 41 21 25 DNA Unknown Genomic 21 cgcgcagccg
ctggattgtt attac 25 22 21 DNA Unknown Genomic 22 gcgcccttgg
ccccagtaat c 21 23 48 DNA Unknown Genomic 23 cgcggtatac tgacccagag
cccggcgacc atgagcgcga gcccgggt 48 24 40 DNA Unknown Genomic 24
caggtcaggg taacacgttc acccgggctc gcgctcatgg 40 25 40 DNA Unknown
Genomic 25 gaacgtgtta ccctgacctg cagcgcgagc tctagcatca 40 26 40 DNA
Unknown Genomic 26 atgataccag aacatatagc tgatgctaga gctcgcgctg 40
27 40 DNA Unknown Genomic 27 gctatatgtt ctggtatcat cagcgtccgg
gtcagagccc 40 28 40 DNA Unknown Genomic 28 tatcatagat caacagacgc
gggctctgac ccggacgctg 40 29 40 DNA Unknown Genomic 29 gcgtctgttg
atctatgata ccagcaacct ggcgagcggt 40 30 40 DNA Unknown Genomic 30
ccgctgaaac gcgccggaac accgctcgcc aggttgctgg 40 31 40 DNA Unknown
Genomic 31 gttccggcgc gtttcagcgg tagcggtagc ggtaccagct 40 32 40 DNA
Unknown Genomic 32 acggctgatg gtcaggctat agctggtacc gctaccgcta 40
33 40 DNA Unknown Genomic 33 atagcctgac catcagccgt atggaaccgg
aagatttcgc 40 34 40 DNA Unknown Genomic 34 tctgatggca gaaataggtc
gcgaaatctt ccggttccat 40 35 40 DNA Unknown Genomic 35 gacctatttc
tgccatcaga gctctagcta tccgttcacc 40 36 48 DNA Unknown Genomic 36
cgcgctcgag tttggtaccc tgaccgaagg tgaacggata gctagagc 48 37 21 DNA
Unknown Genomic 37 cgcggtatac tgacccagag c 21 38 22 DNA Unknown
Genomic 38 cgcgctcgag tttggtaccc tg 22 39 26 DNA Unknown Genomic 39
tcgagatcaa acggtaatag ccatgg 26 40 26 DNA Unknown Genomic 40
aattccatgg ctattaccgt ttgatc 26 41 32 DNA Unknown Genomic 41
tcgaagctta gtactgtggc tgcaccatct gt 32 42 32 DNA Unknown Genomic 42
gtcgaattct gcgcactctc ccctgttgaa gc 32 43 48 DNA Unknown Genomic 43
ctagaattct gcgcatccac caagggccca tcggtcttcc ccctggca 48 44 36 DNA
Unknown Genomic 44 gtaaagcttg agctcttacc cggagacagg gagagg 36 45 40
DNA Unknown Genomic 45 gtcccccggg tacctctaga atggattttc aagtgcagat
40 46 40 DNA Unknown Genomic 46 tttcagcttc ctgctaatca gtgcctcagt
catactctcg 40 47 40 DNA Unknown Genomic 47 ctctgggtca gctcgatgtc
cgagagtatg actgaggcac 40 48 40 DNA Unknown Genomic 48 tgattagcag
gaagctgaaa atctgcactt gaaaatccat 40 49 23 DNA Unknown Genomic 49
gtcccccggg tacctctaga atg 23 50 21 DNA Unknown Genomic 50
ctctgggtca gctcgatgtc c 21 51 27 DNA Unknown Genomic 51 gacatcgagc
tgacccagag cccggcg 27 52 22 DNA Unknown Genomic 52 cgcgctcgag
tttggtaccc tg 22 53 38 DNA Unknown Genomic 53 gcgcctcgag atcaaacgga
ctgtggctgc accatctg 38 54 32 DNA Unknown Genomic 54 gccggaattc
ctagcactct cccctgttga ag 32 55 40 DNA Unknown Genomic 55 ctctgcacca
gctgcacctg cgagagtatg actgaggcac 40 56 21 DNA Unknown Genomic 56
ctctgcacca gctgcacctg c 21 57 26 DNA Unknown Genomic 57 caggtgcagc
tggtgcagag cggtag 26 58 45 DNA Unknown Genomic 58 cgccggctcg
agacggtgac cgtggtccct tggccccagt aatcc 45 59 21 DNA Unknown Genomic
59 cgccggctcg agacggtgac c 21 60 26 DNA Unknown Genomic 60
gccgctcgag cgcatccacc aagggc 26 61 39 DNA Unknown Genomic 61
gccggaattc gctaaagctt acccggagac agggagagg 39 62 26 DNA Unknown
Genomic 62 gccctcccag cctccatcga gaaaac 26 63 26 DNA Unknown
Genomic 63 gttttctcga tggaggctgg gagggc 26 64 15 DNA Unknown
Genomic 64 taatacgact cacta 15 65 18 DNA Unknown Genomic 65
atttaggtga cactatag 18 66 1404 DNA Unknown Genomic 66 atggattttc
aagtgcagat tttcagcttc ctgctaatca gtgcctcagt catactctcg 60
caggtgcagc tggtgcagag cggtagcgaa ctgaaaaaac cgggtgcgag cgttaagatc
120 agctgcaaag cgagcggtta taccttcacc gattacggta tgaactgggt
taaacaggcg 180 ccgggtcaag gtctgaaatg gatgggttgg atcaacacct
acaccggtga aagcacctac 240 gttgacgatt tcaaaggtcg tttcgttttc
agcctggata ccagcgttag cgcggcctac 300 ctgcagatca gctctctgaa
agcggaagac accgcgacct acttctgcgc gcgtcgcggt 360 ttctacgcga
tggattactg gggccaaggg accacggtca ccgtctcgag cgcatccacc 420
aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg
480 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc
gtggaactca 540 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc
tacagtcctc aggactctac 600 tccctcagca gcgtggtgac cgtgccctcc
agcagcttgg gcacccagac ctacatctgc 660 aacgtgaatc acaagcccag
caacaccaag gtggacaaga gagttgagcc caaatcttgt 720 gacaaaactc
acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca
840 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac 900 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg
agcagtacaa cagcacgtac 960 cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag 1020 tgcaaggtct ccaacaaagc
cctcccagcc tccatcgaga aaaccatctc caaagccaaa 1080 gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1140
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag
1200 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt
gctggactcc 1260 gacggctcct tcttcctcta tagcaagctc accgtggaca
agagcaggtg gcagcagggg 1320 aacgtcttct catgctccgt gatgcatgag
gctctgcaca accactacac gcagaagagc 1380 ctctccctgt ctccgggtaa gctt
1404 67 468 PRT Artificial Sequence Synthetic 67 Met Asp Phe Gln
Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile
Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys 20 25 30
Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr 35
40 45 Phe Thr Asp Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln
Gly 50 55 60 Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu
Ser Thr Tyr 65 70 75 80 Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser
Leu Asp Thr Ser Val 85 90 95 Ser Ala Ala Tyr Leu Gln Ile Ser Ser
Leu Lys Ala Glu Asp Thr Ala 100 105 110 Thr Tyr Phe Cys Ala Arg Arg
Gly Phe Tyr Ala Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165
170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290
295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Ser Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410
415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 450 455 460 Pro Gly Lys Leu 465 68 699 DNA Unknown
Genomic 68 atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt
catactctcg 60 gacatcgagc tgacccagag cccggcgacc atgagcgcga
gcccgggtga acgtgttacc 120 ctgacctgca gcgcgagctc tagcatcagc
tatatgttct ggtatcatca gcgtccgggt 180 cagagcccgc gtctgttgat
ctatgatacc agcaacctgg cgagcggtgt tccggcgcgt 240 ttcagcggta
gcggtagcgg taccagctat agcctgacca tcagccgtat ggaaccggaa 300
gatttcgcga cctatttctg ccatcagagc tctagctatc cgttcacctt cggtcagggt
360 accaaactcg agatcaaacg gactgtggct gcaccatctg tcttcatctt
cccgccatct 420 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc
tgctgaataa cttctatccc 480 agagaggcca aagtacagtg gaaggtggat
aacgccctcc aatcgggtaa ctcccaggag 540 agtgtcacag agcaggacag
caaggacagc acctacagcc tcagcagcac cctgacgctg 600 agcaaagcag
actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660
agctcgcccg tcacaaagag cttcaacagg ggagagtgc 699 69 233 PRT
Artificial Sequence Synthetic 69 Met Asp Phe Gln Val Gln Ile Phe
Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Leu Ser Asp Ile
Glu Leu Thr Gln Ser Pro Ala Thr Met Ser 20 25 30 Ala Ser Pro Gly
Glu Arg Val Thr Leu Thr Cys Ser Ala Ser Ser Ser 35 40 45 Ile Ser
Tyr Met Phe Trp Tyr His Gln Arg Pro Gly Gln Ser Pro Arg 50 55 60
Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg 65
70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
Ser Arg 85 90 95 Met Glu Pro Glu Asp Phe Ala Thr Tyr Phe Cys His
Gln Ser Ser Ser 100 105 110 Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185
190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 70
41 DNA Unknown Genomic 70 cgcgctcgag atcaaacgga ccccgctggg
tgataccact c 41 71 40 DNA Unknown Genomic 71 cagttcacct ccggaggtat
gagtggtatc acccagcggg 40 72 40 DNA Unknown Genomic 72 atacctccgg
aggtgaactg gaagagctgt tgaaacatct 40 73 40 DNA Unknown Genomic 73
gacctttcag cagttctttc agatgtttca acagctcttc 40 74 40 DNA Unknown
Genomic 74 gaaagaactg ctgaaaggtc cgcggaaagg tgaactggag 40 75 40 DNA
Unknown Genomic 75 ttcaggtgct tcagcaattc ctccagttca cctttccgcg 40
76 40 DNA Unknown Genomic 76
gaattgctga agcacctgaa agagctgttg aaaggtaccc 40 77 40 DNA Unknown
Genomic 77 atgggtagta tcacctaggg gggtaccttt caacagctct 40 78 40 DNA
Unknown Genomic 78 ccctaggtga tactacccat accagcggtc aggtgcaact 40
79 42 DNA Unknown Genomic 79 cgcggaattc gcgttcgcga ctagttgcac
ctgaccgctg gt 42 80 21 DNA Unknown Genomic 80 cgcggtatac tgacccagag
c 21 81 22 DNA Unknown Genomic 81 cgcgctcgag tttggtaccc tg 22 82 29
DNA Unknown Genomic 82 cgcgactagt gcagagcggt agcgaactg 29 83 26 DNA
Unknown Genomic 83 gccagtgaat tctattagtg gtgatg 26 84 1626 DNA
Unknown Genomic 84 caggtgcagc tggtgcagag cggtagcgaa ctgaaaaaac
cgggtgcgag cgttaagatc 60 agctgcaaag cgagcggtta taccttcacc
gattacggta tgaactgggt taaacaggcg 120 ccgggtcaag gtctgaaatg
gatgggttgg atcaacacct acaccggtga aagcacctac 180 gttgacgatt
tcaaaggtcg tttcgttttc agcctggata ccagcgttag cgcggcctac 240
ctgcagatca gctctctgaa agcggaagac accgcgacct acttctgcgc gcgtcgcggt
300 ttctacgcga tggattactg gggccaaggg accacggtca ccgtctcctc
aggtggaggc 360 ggttcaggcg gaggtggctc tggcggtggc ggatcggaca
tcgtactgac ccagagcccg 420 gcgaccatga gcgcgagccc gggtgaacgt
gttaccctga cctgcagcgc gagctctagc 480 atcagctata tgttctggta
tcatcagcgt ccgggtcaga gcccgcgtct gttgatctat 540 gataccagca
acctggcgag cggtgttccg gcgcgtttca gcggtagcgg tagcggtacc 600
agctatagcc tgaccatcag ccgtatggaa ccggaagatt tcgcgaccta tttctgccat
660 cagagctcta gctatccgtt caccttcggt cagggtacca aactcgagat
caaacggacc 720 ccgctgggtg ataccactca tacctccgga ggtgaactgg
aagagctgtt gaaacatctg 780 aaagaactgc tgaaaggtcc gcggaaaggt
gaactggagg aattgctgaa gcacctgaaa 840 gagctgttga aaggtacccc
cctgggtgat actacccata ccagcggtca ggtgcaacta 900 gtgcagagcg
gtagcgaact gaaaaaaccg ggtgcgagcg ttaagatcag ctgcaaagcg 960
agcggttata ccttcaccga ttacggtatg aactgggtta aacaggcgcc gggtcaaggt
1020 ctgaaatgga tgggttggat caacacctac accggtgaaa gcacctacgt
tgacgatttc 1080 aaaggtcgtt tcgttttcag cctggatacc agcgttagcg
cggcctacct gcagatcagc 1140 tctctgaaag cggaagacac cgcgacctac
ttctgcgcgc gtcgcggttt ctacgcgatg 1200 gattactggg gccaagggac
cacggtcacc gtctcctcag gtggaggcgg ttcaggcgga 1260 ggtggctctg
gcggtggcgg atcggacatc gtactgaccc agagcccggc gaccatgagc 1320
gcgagcccgg gtgaacgtgt taccctgacc tgcagcgcga gctctagcat cagctatatg
1380 ttctggtatc atcagcgtcc gggtcagagc ccgcgtctgt tgatctatga
taccagcaac 1440 ctggcgagcg gtgttccggc gcgtttcagc ggtagcggta
gcggtaccag ctatagcctg 1500 accatcagcc gtatggaacc ggaagatttc
gcgacctatt tctgccatca gagctctagc 1560 tatccgttca ccttcggtca
gggtaccaaa ctcgagatca aacggcacca tcaccatcac 1620 cactaa 1626 85 541
PRT Artificial Sequence Synthetic 85 Gln Val Gln Leu Val Gln Ser
Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Gly Met Asn
Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly
Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Val Asp Asp Phe 50 55
60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Ala Ala Tyr
65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Thr Tyr
Phe Cys 85 90 95 Ala Arg Arg Gly Phe Tyr Ala Met Asp Tyr Trp Gly
Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Asp Ile Val Leu
Thr Gln Ser Pro Ala Thr Met Ser 130 135 140 Ala Ser Pro Gly Glu Arg
Val Thr Leu Thr Cys Ser Ala Ser Ser Ser 145 150 155 160 Ile Ser Tyr
Met Phe Trp Tyr His Gln Arg Pro Gly Gln Ser Pro Arg 165 170 175 Leu
Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg 180 185
190 Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg
195 200 205 Met Glu Pro Glu Asp Phe Ala Thr Tyr Phe Cys His Gln Ser
Ser Ser 210 215 220 Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys Arg Thr 225 230 235 240 Pro Leu Gly Asp Thr Thr His Thr Ser
Gly Gly Glu Leu Glu Glu Leu 245 250 255 Leu Lys His Leu Lys Glu Leu
Leu Lys Gly Pro Arg Lys Gly Glu Leu 260 265 270 Glu Glu Leu Leu Lys
His Leu Lys Glu Leu Leu Lys Gly Thr Pro Leu 275 280 285 Gly Asp Thr
Thr His Thr Ser Gly Gln Val Gln Leu Val Gln Ser Gly 290 295 300 Ser
Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala 305 310
315 320 Ser Gly Tyr Thr Phe Thr Asp Tyr Gly Met Asn Trp Val Lys Gln
Ala 325 330 335 Pro Gly Gln Gly Leu Lys Trp Met Gly Trp Ile Asn Thr
Tyr Thr Gly 340 345 350 Glu Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg
Phe Val Phe Ser Leu 355 360 365 Asp Thr Ser Val Ser Ala Ala Tyr Leu
Gln Ile Ser Ser Leu Lys Ala 370 375 380 Glu Asp Thr Ala Thr Tyr Phe
Cys Ala Arg Arg Gly Phe Tyr Ala Met 385 390 395 400 Asp Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly 405 410 415 Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu 420 425 430
Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly Glu Arg Val Thr 435
440 445 Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met Phe Trp Tyr
His 450 455 460 Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Asp
Thr Ser Asn 465 470 475 480 Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly Thr 485 490 495 Ser Tyr Ser Leu Thr Ile Ser Arg
Met Glu Pro Glu Asp Phe Ala Thr 500 505 510 Tyr Phe Cys His Gln Ser
Ser Ser Tyr Pro Phe Thr Phe Gly Gln Gly 515 520 525 Thr Lys Leu Glu
Ile Lys Arg His His His His His His 530 535 540 86 3423 DNA Unknown
Genomic 86 ttccggggat ctctcaccta ccaaacaatg cccccctgca aaaaataaat
tcatataaaa 60 aacatacaga taaccatctg cggtgataaa ttatctctgg
cggtgttgac ataaatacca 120 ctggcggtga tactgagcac atcagcagga
cgcactgacc accatgaagg tgacgctctt 180 aaaaattaag ccctgaagaa
gggcaggggt accaggaggt ttaaatcatg gtaagatcaa 240 gtagtcaaaa
ttcgagtgac aagcctgtag cccacgtcgt agcaaaccac caagtggagg 300
agcagtaacc atggttactg gagaaggggg accaactcag cgctgaggtc aatctgccca
360 agtctagagt cgacctgcag cccaagcttg gctgttttgg cggatgagag
aagattttca 420 gcctgataca gattaaatca gaacgcagaa gcggtctgat
aaaacagaat ttgcctggcg 480 gcagtagcgc ggtggtccca cctgacccca
tgccgaactc agaagtgaaa cgccgtagcg 540 ccgatggtag tgtggggtct
ccccatgcga gagtagggaa ctgccaggca tcaaataaaa 600 cgaaaggctc
agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct 660
ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
720 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca
gaaggccatc 780 ctgacggatg gcctttttgc gtttctacaa actcttttgt
ttatttttct aaatacattc 840 aaatatgtat ccgctcatga gacaataacc
ctgataaatg cttcaataat aaaaggatct 900 aggtgaagat cctttttgat
aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 960 actgagcgtc
agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 1020
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
1080 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
cagataccaa 1140 atactgtcct tctagtgtag ccgtagttag gccaccactt
caagaactct gtagcaccgc 1200 ctacatacct cgctctgcta atcctgttac
cagtggctgc tgccagtggc gataagtcgt 1260 gtcttaccgg gttggactca
agacgatagt taccggataa ggcgcagcgg tcgggctgaa 1320 cggggggttc
gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 1380
tacagcgtga gcattgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
1440 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
ggaaacgcct 1500 ggtatcttta tagtcctgtc gggtttcgcc acctctgact
tgagcgtcga tttttgtgat 1560 gctcgtcagg ggggcggagc ctatggaaaa
acgccagcaa cgcggccttt ttacggttcc 1620 tggccttttg ctggcctttt
gctcacatgt tctttcctgc gttatcccct gattctgtgg 1680 ataaccgtat
taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 1740
gcagcgagtc agtgagcgag gaagcggaag agcgctgact tccgcgtttc cagactttac
1800 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
tttgcagcag 1860 cagtcgcttc acgttcgctc gcgtatcggt gattcattct
gctaaccagt aaggcaaccc 1920 cgccagccta gccgggtcct caacgacagg
agcacgatca tgcgcacccg tggccaggac 1980 ccaacgctgc ccgagatgcg
ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg 2040 ttctgccaag
ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt 2100
cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
2160 ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
gcgcctacaa 2220 tccatgccaa cccgttccat gtgctcgccg aggcggcata
aatcgccgtg acgatcagcg 2280 gtccagtgat cgaagttagg ctggtaagag
ccgcgagcga tccttgaagc tgtccctgat 2340 ggtcgtcatc tacctgcctg
gacagcatgg cctgcaacgc gggcatcccg atgccgccgg 2400 aagcgagaag
aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga 2460
cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
2520 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
aataccgcaa 2580 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
ctcgccgaaa atgacccaga 2640 gcgctgccgg cacctgtcct acgagttgca
tgataaagaa gacagtcata agtgcggcga 2700 cgatagtcat gccccgcgcc
caccggaagg agctgactgg gttgaaggct ctcaagggca 2760 tcggtcggcg
ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga 2820
ggccgttgag caccgccgcc gcaaggaatg gtgcatgtaa ggagatggcg cccaacagtc
2880 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
agcccgaagt 2940 ggcgagcccg atcttcccca tcggtgatgt cggcgatata
ggcgccagca accgcacctg 3000 tggcgccggt gatgccggcc acgatgcgtc
cggcgtagag aatccacagg acgggtgtgg 3060 tcgccatgat cgcgtagtcg
atagtggctc caagtagcga agcgagcagg actgggcggc 3120 ggccaaagcg
gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat 3180
atagcgctag cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca
3240 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg
gtgacggtgc 3300 cgaggatgac gatgagcgca ttgttagatt tcatacacgg
tgcctgactg cgttagcaat 3360 ttaactgtga taaactaccg cattaaagct
aatcgatgat aagctgtcaa acatgagaat 3420 taa 3423 87 41 DNA Unknown
Genomic 87 cccaagcttg gcggaggctc acaggtgcag ctggtgcaga g 41 88 30
DNA Unknown Genomic 88 cggaattcta ccgtttgatc tcgagtttgg 30 89 2133
DNA Unknown Genomic 89 atggattttc aagtgcagat tttcagcttc ctgctaatca
gtgcctcagt catactctcg 60 caggtgcagc tggtgcagag cggtagcgaa
ctgaaaaaac cgggtgcgag cgttaagatc 120 agctgcaaag cgagcggtta
taccttcacc gattacggta tgaactgggt taaacaggcg 180 ccgggtcaag
gtctgaaatg gatgggttgg atcaacacct acaccggtga aagcacctac 240
gttgacgatt tcaaaggtcg tttcgttttc agcctggata ccagcgttag cgcggcctac
300 ctgcagatca gctctctgaa agcggaagac accgcgacct acttctgcgc
gcgtcgcggt 360 ttctacgcga tggattactg gggccaaggg accacggtca
ccgtctcgag cgcatccacc 420 aagggcccat cggtcttccc cctggcaccc
tcctccaaga gcacctctgg gggcacagcg 480 gccctgggct gcctggtcaa
ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgccctga
ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
660 aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc
caaatcttgt 720 gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 780 ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 840 tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 900 ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
1020 tgcaaggtct ccaacaaagc cctcccagcc tccatcgaga aaaccatctc
caaagccaaa 1080 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1140 aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1200 tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260 gacggctcct
tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1320
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
1380 ctctccctgt ctccgggtaa gcttggcgga ggctcacagg tgcagctggt
gcagagcggt 1440 agcgaactga aaaaaccggg tgcgagcgtt aagatcagct
gcaaagcgag cggttatacc 1500 ttcaccgatt acggtatgaa ctgggttaaa
caggcgccgg gtcaaggtct gaaatggatg 1560 ggttggatca acacctacac
cggtgaaagc acctacgttg acgatttcaa aggtcgtttc 1620 gttttcagcc
tggataccag cgttagcgcg gcctacctgc agatcagctc tctgaaagcg 1680
gaagacaccg cgacctactt ctgcgcgcgt cgcggtttct acgcgatgga ttactggggc
1740 caagggacca cggtcaccgt ctcctcaggt ggaggcggtt caggcggagg
tggctctggc 1800 ggtggcggat cggacatcgt actgacccag agcccggcga
ccatgagcgc gagcccgggt 1860 gaacgtgtta ccctgacctg cagcgcgagc
tctagcatca gctatatgtt ctggtatcat 1920 cagcgtccgg gtcagagccc
gcgtctgttg atctatgata ccagcaacct ggcgagcggt 1980 gttccggcgc
gtttcagcgg tagcggtagc ggtaccagct atagcctgac catcagccgt 2040
atggaaccgg aagatttcgc gacctatttc tgccatcaga gctctagcta tccgttcacc
2100 ttcggtcagg gtaccaaact cgagatcaaa cgg 2133 90 711 PRT
Artificial Sequence Synthetic 90 Met Asp Phe Gln Val Gln Ile Phe
Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Leu Ser Gln Val
Gln Leu Val Gln Ser Gly Ser Glu Leu Lys 20 25 30 Lys Pro Gly Ala
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr 35 40 45 Phe Thr
Asp Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly 50 55 60
Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr 65
70 75 80 Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr
Ser Val 85 90 95 Ser Ala Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala
Glu Asp Thr Ala 100 105 110 Thr Tyr Phe Cys Ala Arg Arg Gly Phe Tyr
Ala Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185
190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310
315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Ser Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435
440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 450 455 460 Pro Gly Lys Leu Gly Gly Gly Ser Gln Val Gln Leu Val
Gln Ser Gly 465 470 475 480 Ser Glu Leu Lys Lys Pro Gly Ala Ser Val
Lys Ile Ser Cys Lys Ala 485 490 495 Ser Gly Tyr Thr Phe Thr Asp Tyr
Gly Met Asn Trp Val Lys Gln Ala 500 505 510 Pro Gly Gln Gly Leu Lys
Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly 515 520 525 Glu Ser Thr Tyr
Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu 530 535 540 Asp Thr
Ser Val Ser Ala Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala 545 550
555
560 Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Arg Gly Phe Tyr Ala Met
565 570 575 Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly
Gly Gly 580 585 590 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile Val Leu 595 600 605 Thr Gln Ser Pro Ala Thr Met Ser Ala Ser
Pro Gly Glu Arg Val Thr 610 615 620 Leu Thr Cys Ser Ala Ser Ser Ser
Ile Ser Tyr Met Phe Trp Tyr His 625 630 635 640 Gln Arg Pro Gly Gln
Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn 645 650 655 Leu Ala Ser
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr 660 665 670 Ser
Tyr Ser Leu Thr Ile Ser Arg Met Glu Pro Glu Asp Phe Ala Thr 675 680
685 Tyr Phe Cys His Gln Ser Ser Ser Tyr Pro Phe Thr Phe Gly Gln Gly
690 695 700 Thr Lys Leu Glu Ile Lys Arg 705 710 91 240 PRT
Artificial Sequence Synthetic 91 Gln Val Gln Leu Val Gln Ser Gly
Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Gly Met Asn Trp
Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp
Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Val Asp Asp Phe 50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Ala Ala Tyr 65
70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Thr Tyr
Phe Cys 85 90 95 Ala Arg Arg Gly Phe Tyr Ala Met Asp Tyr Trp Gly
Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Asp 115 120 125 Ile Val Leu Thr Gln Ser Pro Ala
Thr Met Ser Ala Ser Pro Gly Glu 130 135 140 Arg Val Thr Leu Thr Cys
Ser Ala Ser Ser Ser Ile Ser Tyr Met Phe 145 150 155 160 Trp Tyr His
Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Asp 165 170 175 Thr
Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 180 185
190 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Pro Glu Asp
195 200 205 Phe Ala Thr Tyr Phe Cys His Gln Ser Ser Ser Tyr Pro Phe
Thr Phe 210 215 220 Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg His His
His His His His 225 230 235 240 92 702 DNA Unknown Genomic 92
caggtgcagc tggtgcagag cggtagcgaa ctgaaaaaac cgggtgcgag cgttaagatc
60 agctgcaaag cgagcggtta taccttcacc gattacggta tgaactgggt
taaacaggcg 120 ccgggtcaag gtctgaaatg gatgggttgg atcaacacct
acaccggtga aagcacctac 180 gttgacgatt tcaaaggtcg tttcgttttc
agcctggata ccagcgttag cgcggcctac 240 ctgcagatca gctctctgaa
agcggaagac accgcgacct acttctgcgc gcgtcgcggt 300 ttctacgcga
tggattactg gggccaaggg accacggtca ccgtctcctc aggcggaggt 360
ggctctggcg gtggcggatc ggacatcgta ctgacccaga gcccggcgac catgagcgcg
420 agcccgggtg aacgtgttac cctgacctgc agcgcgagct ctagcatcag
ctatatgttc 480 tggtatcatc agcgtccggg tcagagcccg cgtctgttga
tctatgatac cagcaacctg 540 gcgagcggtg ttccggcgcg tttcagcggt
agcggtagcg gtaccagcta tagcctgacc 600 atcagccgta tggaaccgga
agatttcgcg acctatttct gccatcagag ctctagctat 660 ccgttcacct
tcggtcaggg taccaaactc gagatcaaac gg 702 93 235 PRT Artificial
Sequence Synthetic 93 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln
Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr
Tyr Thr Gly Glu Ser Thr Tyr Val Asp Asp Phe 50 55 60 Lys Gly Arg
Phe Val Phe Ser Leu Asp Thr Ser Val Ser Ala Ala Tyr 65 70 75 80 Leu
Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Thr Tyr Phe Cys 85 90
95 Ala Arg Arg Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Val Leu
Thr Gln 115 120 125 Ser Pro Ala Thr Met Ser Ala Ser Pro Gly Glu Arg
Val Thr Leu Thr 130 135 140 Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met
Phe Trp Tyr His Gln Arg 145 150 155 160 Pro Gly Gln Ser Pro Arg Leu
Leu Ile Tyr Asp Thr Ser Asn Leu Ala 165 170 175 Ser Gly Val Pro Ala
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr 180 185 190 Ser Leu Thr
Ile Ser Arg Met Glu Pro Glu Asp Phe Ala Thr Tyr Phe 195 200 205 Cys
His Gln Ser Ser Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys 210 215
220 Leu Glu Ile Lys Arg His His His His His His 225 230 235 94 687
DNA Unknown Genomic 94 caggtgcagc tggtgcagag cggtagcgaa ctgaaaaaac
cgggtgcgag cgttaagatc 60 agctgcaaag cgagcggtta taccttcacc
gattacggta tgaactgggt taaacaggcg 120 ccgggtcaag gtctgaaatg
gatgggttgg atcaacacct acaccggtga aagcacctac 180 gttgacgatt
tcaaaggtcg tttcgttttc agcctggata ccagcgttag cgcggcctac 240
ctgcagatca gctctctgaa agcggaagac accgcgacct acttctgcgc gcgtcgcggt
300 ttctacgcga tggattactg gggccaaggg accacggtca ccgtctcctc
aggcggtggc 360 ggatcggaca tcgtactgac ccagagcccg gcgaccatga
gcgcgagccc gggtgaacgt 420 gttaccctga cctgcagcgc gagctctagc
atcagctata tgttctggta tcatcagcgt 480 ccgggtcaga gcccgcgtct
gttgatctat gataccagca acctggcgag cggtgttccg 540 gcgcgtttca
gcggtagcgg tagcggtacc agctatagcc tgaccatcag ccgtatggaa 600
ccggaagatt tcgcgaccta tttctgccat cagagctcta gctatccgtt caccttcggt
660 cagggtacca aactcgagat caaacgg 687 95 34 DNA Unknown Genomic 95
ggccgctctt cgaaatacct attgcctacg gcag 34 96 70 DNA Unknown Genomic
96 ctgggtcagt acgatgtcag agccacctcc gcctgaaccg cctccacctg
aggagacggt 60 gaccgtggtc 70 97 67 DNA Unknown Genomic 97 gtcaccgtct
cctcaggtgg aggcggttca ggcggaggtg gctctgacat cgtactgacc 60 cagagcc
67 98 26 DNA Unknown Genomic 98 gccagtgaat tctattagtg gtgatg 26 99
55 DNA Unknown Genomic 99 ctgggtcagt acgatgtctg aaccgcctcc
acctgaggag acggtgaccg tggtc 55 100 52 DNA Unknown Genomic 100
gtcaccgtct cctcaggtgg aggcggttca gacatcgtac tgacccagag cc 52 101
672 DNA Unknown Genomic 101 caggtgcagc tggtgcagag cggtagcgaa
ctgaaaaaac cgggtgcgag cgttaagatc 60 agctgcaaag cgagcggtta
taccttcacc gattacggta tgaactgggt taaacaggcg 120 ccgggtcaag
gtctgaaatg gatgggttgg atcaacacct acaccggtga aagcacctac 180
gttgacgatt tcaaaggtcg tttcgttttc agcctggata ccagcgttag cgcggcctac
240 ctgcagatca gctctctgaa agcggaagac accgcgacct acttctgcgc
gcgtcgcggt 300 ttctacgcga tggattactg gggccaaggg accacggtca
ccgtctcctc agacatcgta 360 ctgacccaga gcccggcgac catgagcgcg
agcccgggtg aacgtgttac cctgacctgc 420 agcgcgagct ctagcatcag
ctatatgttc tggtatcatc agcgtccggg tcagagcccg 480 cgtctgttga
tctatgatac cagcaacctg gcgagcggtg ttccggcgcg tttcagcggt 540
agcggtagcg gtaccagcta tagcctgacc atcagccgta tggaaccgga agatttcgcg
600 acctatttct gccatcagag ctctagctat ccgttcacct tcggtcaggg
taccaaactc 660 gagatcaaac gg 672 102 230 PRT Artificial Sequence
Synthetic 102 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro
Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr
Gly Glu Ser Thr Tyr Val Asp Asp Phe 50 55 60 Lys Gly Arg Phe Val
Phe Ser Leu Asp Thr Ser Val Ser Ala Ala Tyr 65 70 75 80 Leu Gln Ile
Ser Ser Leu Lys Ala Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala
Arg Arg Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr 100 105
110 Val Thr Val Ser Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Met
115 120 125 Ser Ala Ser Pro Gly Glu Arg Val Thr Leu Thr Cys Ser Ala
Ser Ser 130 135 140 Ser Ile Ser Tyr Met Phe Trp Tyr His Gln Arg Pro
Gly Gln Ser Pro 145 150 155 160 Arg Leu Leu Ile Tyr Asp Thr Ser Asn
Leu Ala Ser Gly Val Pro Ala 165 170 175 Arg Phe Ser Gly Ser Gly Ser
Gly Thr Ser Tyr Ser Leu Thr Ile Ser 180 185 190 Arg Met Glu Pro Glu
Asp Phe Ala Thr Tyr Phe Cys His Gln Ser Ser 195 200 205 Ser Tyr Pro
Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 210 215 220 His
His His His His His 225 230 103 40 DNA Unknown Genomic 103
ctgggtcagt acgatgtctg aggagacggt gaccgtggtc 40 104 37 DNA Unknown
Genomic 104 gtcaccgtct cctcagacat cgtactgacc cagagcc 37
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