U.S. patent application number 13/130656 was filed with the patent office on 2011-10-20 for polypeptides, antibody variable domains & antagonists.
Invention is credited to Ian Richard Catchpole, Fiona Cook, Gerald Gough, Laurent Jespers, Michael Steward.
Application Number | 20110256122 13/130656 |
Document ID | / |
Family ID | 39791116 |
Filed Date | 2011-10-20 |
United States Patent
Application |
20110256122 |
Kind Code |
A1 |
Catchpole; Ian Richard ; et
al. |
October 20, 2011 |
POLYPEPTIDES, ANTIBODY VARIABLE DOMAINS & ANTAGONISTS
Abstract
The present disclosure relates to immunoglobulin single variable
domains (dAbs) e.g., dAbs which are protease resistant, and also to
formulations, and compositions comprising such dAbs for ocular
delivery and to their uses to treat ocular diseases and
conditions.
Inventors: |
Catchpole; Ian Richard;
(Stevenage, GB) ; Cook; Fiona; (Stevenage, GB)
; Gough; Gerald; (Stevenage, GB) ; Jespers;
Laurent; (Cambridge, GB) ; Steward; Michael;
(Stevenage, GB) |
Family ID: |
39791116 |
Appl. No.: |
13/130656 |
Filed: |
November 4, 2009 |
PCT Filed: |
November 4, 2009 |
PCT NO: |
PCT/EP09/64654 |
371 Date: |
May 23, 2011 |
Current U.S.
Class: |
424/130.1 ;
530/387.1; 530/387.3; 530/389.1; 530/389.2; 530/389.6 |
Current CPC
Class: |
A61K 9/0078 20130101;
A61P 11/06 20180101; A61P 37/08 20180101; A61P 25/00 20180101; A61P
31/06 20180101; A61K 2039/544 20130101; A61P 11/00 20180101; A61P
35/00 20180101; C07K 16/22 20130101; C07K 2317/73 20130101; A61P
39/00 20180101; A61P 1/04 20180101; A61P 1/06 20180101; A61P 37/02
20180101; A61P 1/00 20180101; A61P 27/02 20180101; C07K 16/2878
20130101; C07K 2317/76 20130101; C07K 2317/90 20130101; A61P 31/16
20180101; C07K 2317/565 20130101; A61P 9/12 20180101; C07K 16/005
20130101; C07K 16/2866 20130101; C07K 2317/569 20130101; A61P 11/02
20180101; A61P 27/04 20180101; C07K 2317/94 20130101; A61P 37/00
20180101; A61P 19/02 20180101; C07K 2317/567 20130101; C07K 2317/92
20130101; A61P 29/00 20180101; A61P 31/04 20180101; A61P 37/06
20180101; A61P 7/02 20180101; A61P 27/06 20180101; A61P 43/00
20180101; A61P 9/00 20180101 |
Class at
Publication: |
424/130.1 ;
530/387.1; 530/389.2; 530/389.1; 530/389.6; 530/387.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/22 20060101 C07K016/22; C07K 16/18 20060101
C07K016/18; A61P 27/04 20060101 A61P027/04; C07K 16/24 20060101
C07K016/24; C07K 16/46 20060101 C07K016/46; A61P 27/02 20060101
A61P027/02; A61P 27/06 20060101 A61P027/06; C07K 16/00 20060101
C07K016/00; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 26, 2008 |
US |
12/323632 |
Claims
1. A composition comprising an immunoglobulin single variable
domain that binds to a target molecule, for ocular delivery.
2. The composition according to claim 1, wherein said
immunoglobulin single variable domain is resistant to a protease,
wherein the protease is selected from the group consisting of:
ocular proteases, caspases, calpains, matrix metalloproteases,
disintegrins, metalloproteinases, ADAMs, ADAM with motifs,
proteosomes, tissue plasminogen activator, secretases, cathepsin B,
cathepsin D, cystatin C, serine protease PRSS1, and ubiquitin
proteosome pathway.
3. A composition according to claim 1, which comprises an
immunoglobulin single variable domain which binds to a target
molecule selected from the group consisting of: VEGF, TNFalpha,
TNFalphaR, IL-1, IL-1r, TNFalphaR1, TGFbeta, IL-6, IL-8, IL-17,
IL-21, IL-23, CD20, Nogo-a, myelin associated glycoprotein and beta
amyloid.
4. The composition according to claim 3, wherein said
immunoglobulin single variable domain which binds to VEGF comprises
an amino acid sequence that is at least 97% identical to an amino
acid sequence selected from the group consisting of: (a) the amino
acid sequence of DOM15-26-593 shown in SEQ ID NO 1 and (b) the
amino acid sequence of DOM15-26-593-Fc shown in SEQ ID NO 2.
5. The composition according to claim 4, wherein the immunoglobulin
single variable domain comprises valine at position 6, wherein
numbering is according to Kabat.
6. The composition according to claim 4, wherein the immunoglobulin
single variable domain comprises leucine at position 99, wherein
numbering is according to Kabat.
7. The composition according to claim 4, wherein the immunoglobulin
single variable domain comprises lysine at position 30, wherein
numbering is according to Kabat.
8. The composition according to claim 4, wherein the immunoglobulin
single variable domain comprises an amino acid sequence that is
identical to an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of DOM15-26-593 shown in
SEQ ID NO 1 and (b) the amino acid sequence of DOM15-26-593 Fc
shown in SEQ ID NO 2.
9. The composition according to claim 3, wherein said
immunoglobulin single variable domain which binds to IL-1 comprises
an amino acid sequence that is at least 97% identical to an amino
acid sequence selected from the group consisting of: (a) the amino
acid sequence of DOM 4-130-54 shown in SEQ ID NO 5 and (b) the
amino acid sequence of DOM 0400 PEG shown in SEQ ID NO 4.
10. The composition according to claim 9, wherein said
immunoglobulin single variable domain which binds to IL-1 comprises
an amino acid sequence that is identical to an amino acid sequence
selected from the group consisting of: (a) the amino acid sequence
of DOM 4-130-54 shown in SEQ ID NO 5 and (b) the amino acid
sequence of DOM 0400 PEG shown in SEQ ID NO 4.
11. The composition according to claim 3, wherein said
immunoglobulin single variable domain which binds to TNFalphaR1 and
comprises an amino acid sequence that is at least 97% identical to
the amino acid sequence of Dom 1h-131-206 shown in SEQ ID NO 6.
12. The composition according to claim 3, wherein said
immunoglobulin single variable domain which binds to TNFalphaR1 and
comprises an amino acid sequence that is identical the amino acid
sequence of Dom 1 h-131-206 shown in SEQ ID NO 6.
13. A composition according to claim 1 which further comprises a
domain of an antibody constant region, wherein the antibody
constant region is an antibody Fc region.
14. The composition according to claim 9, wherein said antibody Fc
region has the amino acid. Fc sequence shown in SEQ ID NO 3.
15. The composition according to claim 9, wherein the
immunoglobulin single variable domain is present as a fusion with
an Fc and has an amino acid sequence identical to the amino acid
sequence of the DOM15-26-593-Fc fusion shown in SEQ ID NO 2.
16. A composition comprising a naked immunoglobulin single variable
domain which binds to a target molecule for delivery to one or more
of the ocular regions selected from the group consisting of: the
vitreous humour, the aqueous humour, the retina and the
choroid.
17. The composition according to claim 16 wherein the target
molecule is selected from the group consisting of: VEGF, a VEGF
antagonist, TNFalpha receptor, TNFalpha receptor, IL-1, TNFalphaR1,
IL-6, IL-8, IL-17, IL-21, IL-23, Nogo-a, myelin associated
glycoprotein and beta amyloid.
18. A composition according, to claim 16, wherein said
immunoglobulin single variable domain is resistant to a protease,
wherein the protease is selected from the group consisting of:
ocular proteases, caspases, calpains, matrix metalloproteases,
disintegrins, metalloproteinases, ADAMs, ADAM with thrombospondin
motifs, proteosomes, tissue plasminogen activator, secretases,
cathepsin B, cathepsin D, cystatin C, serine protease PRSS1, and
ubiquitin proteosome pathway.
19. A composition according to claim 16, wherein said
immunoglobulin single variable domain is selected from the group
consisting of: (a) an immunoglobulin single variable domain which
binds to VEGF and which comprises an amino acid sequence that is at
least 97% identical to an amino acid selected from the group
consisting of: (i) the amino acid sequence of DOM15-26-593 or shown
in SEQ ID NO 1 and (ii) the amino acid sequence of DOM15-26-593-Fc
shown in SEQ ID NO 2; (b) an immunoglobulin single variable domain
which binds to IL-1 and comprises an amino acid sequence that is at
least 97% identical to an amino acid sequence selected from the
group consisting of: (i) the amino acid sequence of DOM 4-130-54
shown in SEQ ID NO 5 and (ii) the amino acid sequence of DOM 0400
PEG shown in SEQ ID NO 4; and (c) are immunoglobulin single
variable domain which binds to TNFalphaR1 and comprises an amino
acid sequence that is at least 97% identical to the amino acid
sequence of Dom 1h-131-206 shown in SEQ ID NO 6.
20. A composition comprising a formatted immunoglobulin single
variable domain which binds to a target molecule for delivery to
one or more of the ocular regions selected, from the group
consisting of: the retina, the choroid and the lachrymal fluid.
21. The composition according to claim 20, wherein the target
molecule is selected from the group consisting of: VEGF, a VEGF
antagonist, TNFalpha, TNFalpha receptor, IL-1, TNFalphaR1, IL-17,
IL-21, IL-23, Nogo-a, myelin associated glycoprotein and beta
amyloid.
22. The composition according to claim 20, wherein said
immunoglobulin single variable domain is resistant to a protease,
wherein the protease is selected from the group consisting of:
ocular proteases, caspases, calpains, matrix metalloproteases,
disintegrins, metalloproteinases, ADAMs, ADAM with thrombospondin
motifs, proteosomes, tissue plasminogen activator, secretases,
cathepsin B, cathepsin D, cystatin C, serine protease PRSS1, and
ubiquitin proteosome pathway.
23. The composition according to claim 16, wherein said
immunoglobulin single variable domain is selected from the group
consisting of: (a) an immunoglobulin single variable domain which
binds to VEGF and which comprises an amino acid sequence that is at
least 97% identical to an amino acid sequence selected from the
group consisting of: (i) the amino acid sequence of DOM 5-26-593
shown in SEQ ID NO 1 and (ii) the amino acid sequence of DOM
15-26-593 Fc shown in SEQ ID NO 2; (b) an immunoglobulin single,
variable domain which binds to IL-1 and comprises an amino acid
sequence that is at least 97% identical to an amino acid sequence
selected from the group consisting of: (i) the amino acid sequence
of DOM 4-130-54 shown in SEQ ID NO 5 and (ii) the amino acid
sequence of DOM 0400 PEG shown in SEQ ID NO 4, and (c) an
immunoglobulin single variable domain which binds to TNFalphaR1
comprises an amino acid sequence that is at least 97% identical to
the amino acid sequence of Dom 1h-131-206 shown in SEQ ID NO 6.
24. A composition according to claim 20, wherein the immunoglobulin
single variable domain has a molecular weight of around 50 KDa.
25. A composition according to claim 20, wherein the immunoglobulin
single variable domain is formatted by pegylation or fusion to an
antibody Fc.
26. A composition according to claim 1, which further comprises one
or more enhancers selected from the group consisting of: an ocular
penetration enhancer and a viscosity enhancer.
27. A composition according to claim 1, which further comprises a
component selected from the group consisting of: a pharmaceutically
acceptable carrier, a physiologically acceptable carrier, a
diluent, and an excipient.
28. A method of delivering a composition according to claim 1,
directly to the eye, which comprises administering said composition
to the eye by a method for topical delivery to selected from the
group consisting of: eye drops, intra-ocular injection, peri-ocular
administration, and a slow release formulation.
29. A method for treating an eye condition which comprises
administering a composition according to claim 1 directly to the
eye by a method selected from the group consisting of: intra-ocular
injection, intra-vitreal injection, eye drops, peri-ocular
administration, and a slow release formulation.
30. A method according to claim 28 or 29, wherein the composition
is administered to a one or more regions of the eye selected from
the group consisting of: a surface of the eye, tear ducts,
lachrymal glands, an infra-ocular region, anterior chamber,
posterior chamber and the vitreous humour.
31. A method of delivering a composition according to claim 26, to
at least one region of the eye selected from the group consisting
of: the vitreous humour, the aqueous humour, the retina, and the
choroid; which comprises administering said composition to the eye
by topical delivery.
32. A method of delivering a composition according to claim 20 to
at least one region of the eye selected from the group consisting
of: the retina, the choroid, and the lachrymal fluid; which
comprises administering said composition to the eye by topical
delivery.
33. A process for producing a pharmaceutical composition
comprising: (a) mixing a composition according to claim 1 with (b)
a pharmaceutically acceptable carrier, diluent or excipient.
34. A process according to claim 33, wherein said pharmaceutical
composition is for treating an eye condition or disease.
35. A process according to claim 34, wherein said eye condition or
disease is selected from the group consisting of: age-related
macular degeneration, uveitis, glaucoma, dry eye, diabetic
retinopathy, and diabetic macular oedema.
Description
[0001] This application is filed under 35 USC .sctn.371 from
International Application No. PCT/EP2009/064654 filed Nov. 4, 2009
which claims the benefit of U.S. application Ser. No. 12/323,632
filed Nov. 26, 2008 which is incorporated herein in its
entirety.
[0002] The present disclosure relates to immunoglobulin single
variable domains (dAbs) e.g, dAbs which are protease resistant, and
also to formulations, and compositions comprising such dAbs for
ocular delivery and to their uses to treat ocular diseases and
conditions.
BACKGROUND OF THE DISCLOSURE
[0003] A difficulty of treating ocular diseases and conditions has
been the inefficiency of delivering therapeutic agents to the eye.
When a drug is delivered to the eye it very often clears extremely
rapidly from the ocular tissues. Additionally, when therapeutics
are delivered topically to the eye a problem has been that they may
not reach the posterior segments of the eye (the retina, vitreous
and choroid). Hence, many posterior segment ocular conditions have
been treated by administering drugs intravenously or by
intravitreal administration. Many of these diseases, e.g, AMD,
glaucoma, diabetic retinopathies cannot be treated optimally.
Therefore a need exists to provide further agents which can be
suitable for ocular delivery and which can treat or prevent ocular
diseases and conditions.
[0004] Polypeptides and peptides have become increasingly important
agents for use as medical, therapeutic and diagnostic agents.
However in certain in vivo environments e.g., the eye and in
certain physiological states, such as Cancer and inflammatory
states, the amount of proteases present in a tissue, organ or
animal can increase. This increase in proteases can result in
accelerated degradation and inactivation of endogenous proteins and
of therapeutic peptides, polypeptides and proteins that are
administered to treat disease. Accordingly, some agents that have
potential for in vivo use (e.g., use in treating, diagnosing or
preventing disease) have only limited efficacy because they are
rapidly degraded and inactivated by proteases.
[0005] Protease resistant polypeptides provide several advantages.
For example, protease resistant polypeptides remaining active in
vivo longer than protease sensitive agents and, accordingly,
remaining functional for a period of time that is sufficient to
produce biological effects.
[0006] VEGF is a secreted, heparin-binding, homodimeric
glycoprotein existing in several alternate forms due to alternative
splicing of its primary transcript (Leung et al., 1989, Science
246: 1306). VEGF is also known as vascular permeability factor
(VPF) due to its ability to induce vascular leakage, a process
important in inflammation.
[0007] In the eye VEGF and VEGF-receptors are known to stimulate
both choroidal and retinal vessel angiogenesis and regulate the
vascular permeability of such vessels. Both these features
contribute to retinal damage and consequential visual acuity
deterioration which results from a number of retinal inflammatory
conditions, vasculopathies and maculopathies. Attempts to regulate
VEGF activity or VEGF-receptor activity has previously been shown
to effectively manage the vascular permeability in both animal
models and human disease (Gragoudas et al., 2004: N. Engl. J. Med
351: 2805)
[0008] Targeting VEGF with currently available therapeutics is not
effective in all patients. Thus, a need exists for improved agents
for treating pathological conditions mediated by VEGF e.g, vascular
proliferative diseases (e.g, age related macular degeneration
(AMD)).
[0009] TNF-.alpha. (Tumour Necrosis Factor-.alpha.) is a
pro-inflammatory cytokine which has been implicated in a number of
ophthalmic inflammatory conditions such as uveitis and AMD and in
the generation of retinal vasculopathies in which there is an
inflammatory component. The generation of choroidal neovascular
lesions associated with age-related macular disease has been
demonstrated to have an associated inflammatory component.
Effective management of this associated inflammatory component has
been demonstrated to directly effect the development of the
choroidal neo-angiogenic lesion and the vascular permeability both
of which can impact human disease. Recent evidence in human AMD
patients have suggested that the use of anti-TNF.alpha.
therapeutics can impact disease in patients which are unresponsive
to anti-VEGF therapies (Theodossiadis et al., 2009: Am. J.
Ophthalmol. 147: 825-830).
[0010] Interleukin 1 (IL-1) is an important mediator of the immune
response that has biological effects on several types of cells.
Interleukin 1 binds to two receptors Interleukin 1 Receptor type 1
(IL-1R1, CD121a, p80), which transduces signal into cells upon
binding IL-1, and Interleukin 1 Receptor type 2 (IL-1R1, CDw121b),
which does not transduce signals upon binding IL-1 and acts as an
endogenous regulator of IL-1. Another endogenous protein that
regulates the interaction of IL-1 with IL-1R1 is Interleukin 1
receptor antagonist (IL-1ra). IL-1ra binds IL-1R1, but does not
activate IL-1R1 to transduce signals.
[0011] Signals transduced through IL-1R1 upon binding IL-1 (e.g.,
IL-1.alpha. or IL-1.beta.) induce a wide spectrum of biological
activities that can be pathogenic. For example, signals transduced
through IL-1R1 upon binding of IL-1 can lead to local or systemic
inflammation, and the elaboration of additional inflammatory
mediators (e.g., IL-6, IL-8, TNF). Accordingly, the interaction of
IL-1 with IL-1R1 has been implicated in the pathogenesis of ocular
diseases.
[0012] Certain agents that bind Interleukin 1 Receptor Type 1
(IL-1R1) and neutralize its activity (e.g., IL-1ra) have proven to
be effective therapeutic agents for certain inflammatory
conditions.
SUMMARY OF THE DISCLOSURE
[0013] In a first aspect the disclosure provides a composition
which comprises or consists of an immunoglobulin single variable
domain (or dAb) which can bind to a desired target molecule (e.g,
VEGF, IL-1, or TNF-.alpha.), e.g, at the site of delivery, for
administration to the eye.
[0014] The disclosure also provides compositions which comprise or
consist of an immunoglobulin single variable domain (or dAb) which
can bind to a desired target molecule (e.g, VEGF, IL-1, or
TNF-.alpha., TNFR1, TNFR2, IL-1r), for use to treat, prevent or
diagnose ocular diseases or conditions, such as age related macular
degeneration (AMD), uveitis, glaucoma, dry eye, diabetic
retinopathy, and diabetic macular oedema.
[0015] In an embodiment the immunoglobulin single variable domain
can be protease resistant, e.g, resistant to one or more of the
following: serine protease, cysteine protease, aspartate proteases,
thiol proteases, matrix metalloprotease, carboxypeptidase (e.g.,
carboxypeptidase A, carboxypeptidase B), trypsin, chymotrypsin,
pepsin, papain, elastase, leukozyme, pancreatin, thrombin, plasmin,
cathepsins (e.g., cathepsin G), proteinase (e.g., proteinase 1,
proteinase 2, proteinase 3), thermolysin, chymosin,
enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4,
caspase 5, caspase 9, caspase 12, caspase 13), calpain, ficain,
clostripain, actinidain, bromelain, and separase. In particular
embodiments, the protease is trypsin, elastase or leucozyme. Such
protease resistant polypeptides are especially suitable for
delivery to protease rich environments in vivo such as the eye. The
protease can also be provided by a biological extract, biological
homogenate or biological preparation. In one embodiment, the
protease is one found in the eye and/or tears. Examples of such
proteases found in the eye include caspases, calpains, matrix
metalloproteases, disintegrin, metalloproteinases, ADAMs, ADAM with
thrombospondin motifs, the proteosomes, tissue plasminogen
activator, secretases, cathepsin B and D, cystatin C, serine
protease PRSS1, ubiquitin proteosome pathway (UPP). In one
embodiment, the protease is a non-bacterial protease. In an
embodiment, the protease is an animal, e.g., mammalian, e.g.,
human, protease.
[0016] The composition can be delivered to different regions of the
eye, to the surface of the eye, the cornea, or tear ducts or
lachrymal glands or there can be intraocular delivery (e.g., to the
anterior or posterior chambers of the eye such as the vitreous
humour) and to ocular structures such as the iris, ciliary body,
lachrymal gland, and the composition can bind to target molecules
VEGF, IL-1, or TNF-.alpha.) in these parts of the eye. The
composition can also be delivered to the peri-ocular region of the
eye.
[0017] The target molecule may for example be VEGF, IL-1, or
TNF-.alpha. or it can be any other desired target e.g., a target
molecule present in the eye, for example on the surface of the eye,
within the eye or in tear ducts or lacrimal glands, e.g., the
target can be IL-1, IL-17 or TNF receptor such as TNFR1, TGFbeta,
IL-6, IL-8, IL-21, IL-23, CD20, Nogo-a, myelin associated
glycoprotein (MAG) or Beta amyloid.
[0018] In one embodiment the disclosure provides a protease
resistant immunoglobulin single variable domain (or dAb) for
administration to the eye, e.g., in the form of eye drops or as a
gel or e.g., in an implant. The dAb can for example bind to a
target molecule present in the eye e.g., VEGF, IL-1, or
TNF-.alpha..
[0019] Administration to the eye can be for example by topical
administration, e.g., in the form of eye drops; or alternatively it
can be by injection into the eye.
[0020] It can be useful to target the delivery of the
immunoglobulin single variable domain into particular regions of
the eye such as the surface of the eye, or the tear ducts or
lachrymal glands or there can be intra-ocular delivery (e.g., to
the anterior or posterior chambers of the eye such as the vitreous
humour). Hence the disclosure further provides a method of
delivering a composition directly to the eye which comprises
administering said composition to the eye by a method selected
from: infra-ocular injection, topical delivery, eye drops,
peri-ocular administration and use of a slow release formulations
(such as a polymeric nano or microparticle or gel) or by using
delivery devices making use of iontophoresis.
[0021] It can also be useful if the immunoglobulin single variable
domain is delivered to the eye e.g., by topical delivery e.g., as
eye drops, along with an ocular penetration enhancer e.g., sodium
caprate, or with a viscosity enhancer e.g.,
hydroxypropylmethylcellulose (HPMC). Accordingly the disclosure
further provides compositions comprising (a) an immunoglobulin
single variable domain that bind to a target molecule e.g., in the
eye (e.g., to VEGF, IL-1, or TNF-.alpha.), and also (b) an ocular
penetration enhancer and/or (c) a viscosity enhancer e.g., for
topical delivery to the eye.
[0022] In one aspect, the immunoglobulin single variable domain to
be delivered to the eye can be any one of the VEGF dAbs, disclosed
in WO 2008/149146, WO 2008149147, or WO 2008149150 which bind to
VEGF, For example it can be a polypeptide encoded by an amino acid
sequence that is at least 80% identical to the amino acid sequence
of DOM 15-26-593 (shown in FIG. 1a: SEQ ID NO 1). In one
embodiment, the percent identity is at least 70, 80, 85, 90, 91,
92, 93, 94, 95, 96, 97, 98 or 99% or 100%. In one embodiment the
protease resistant polypeptide is Obtainable by the method
described herein for isolating protease resistant polypeptides. The
DOM 15-26-593 for delivery to the eye may also further comprises a
domain of an antibody constant region. For example it may have an
amino acid sequence identical to the amino acid sequence of DOM
15-26-593-Fc fusion (shown in FIG. 1b: SEQ ID NO 2) or the percent
identity may be at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96,
97, 98 or 99% to that shown in FIG. 1b: SEQ ID NO 2.
[0023] In one aspect the VEGF dAb which is encoded by an amino acid
sequence that is at least 80% identical to the amino acid sequence
of DOM15-26-593 (e.g., by one which is 97% identical or more) can
comprise valine at position 6 and/or leucine at position 99, and/or
lysine at position 30 (Kabat numbering) as described in WO
2008149150 and WO 2008149147 (the contents of which are
incorporated herein by reference).
[0024] In a further aspect, the immunoglobulin single variable
domain to be delivered to the eye can be any one of the anti TNFR1
dAbs disclosed in WO 2008/149144, or WO 2008/149148.
[0025] In one embodiment the immunoglobulin single variable domain
which binds to .alpha.-TNF-.alpha.R1 can comprise an amino acid
sequence that is at least 97% (e.g., 98%, 99% or 100% identical)
identical to the amino acid sequence of Dom 1h-131-206 (shown in
FIG. 4; SEQ ID NO 6). Preparation and selection of Dom 1h-131-206
is described in WO2008149148.
[0026] In yet a further aspect, the immunoglobulin single variable
domain to be delivered to the eye can be any one of the anti-IL-1R1
dAbs disclosed in WO 2008/149149.
[0027] In one embodiment the immunoglobulin single variable domain
which binds to IL-1 can comprise an amino acid sequence that is at
least 97% (e.g., 98%, 99% or 100% identical) identical to: (a) the
amino acid sequence of DOM 4-130-54 (shown in FIG. 3; SEQ ID NO 5);
or to (b) the amino acid sequence of DOM 0400 PEG (shown in FIG. 2;
SEQ ID NO 4).
[0028] Preparation and selection of DOM 4-130-54 is described in WO
2007063311 and also WO2008149149. To prepare Dom 0400, the DOM
4-130-54 dAb sequence is taken and is mutated such that a cysteine
at position 80 replaces the proline present in DOM 4-130-54, this
dAb is then attached to a 40 KDa linear PEG molecule (obtained from
NOF Corporation, Europe) by standard maleimide coupling to the free
cysteine at position 80 of the dAb.
[0029] The disclosure also provides for use of any of the
compositions comprising or consisting of an immunoglobulin single
variable domains in the manufacture of a medicament for the
treatment, prevention or diagnosis of an eye condition or disease
e.g., wherein said eye disease is age related macular degeneration
(AMD), uveitis, glaucoma, dry eye, diabetic retinopathy, or
diabetic macular oedema.
[0030] The disclosure also provides a composition comprising or
consisting of an immunoglobulin single variable domain e.g., a
VEGF, IL-1, or TNF-.alpha. dAb, for use in the treatment,
prevention or diagnosis of an eye condition or disease e.g., AMD,
uveitis, glaucoma, dry eye, diabetic retinopathy, or diabetic
macular oedema.
[0031] The disclosure also provides for use of any of the
compositions comprising or consisting of an immunoglobulin single
variable domain in the manufacture of a medicament for the
treatment, prevention or diagnosis of an eye condition or disease
e.g., wherein said eye disease is age related macular degeneration
(AMD), uveitis, glaucoma, dry eye, diabetic retinopathy, or
diabetic macular oedema.
[0032] In one alternative embodiment the immunoglobulin single
variable domain for delivery to the eye can be one which is not the
amino acid sequence of DOM15-26-593 (shown in FIG. 1a; SEQ ID NO 1)
or which is not the amino acid sequence of DOM15-26-593-Fc fusion
(shown in FIG. 1b; SEQ ID NO 2).
[0033] In another alternative embodiment the immunoglobulin single
variable domain for delivery to the eye can be one which is not a
molecule which comprises or consists of any of the molecules
disclosed in the following applications: PCT/GB2008/050399,
PCT/GB2008/050400, PCT/GB2008/050406, PCT/GB2008/050405,
PCT/GB2008/050403, PCT/GB2008/050404, PCT/GB2008/050407.
[0034] In another alternative embodiment the immunoglobulin single
variable domain for delivery to the eye can be one which is not the
amino acid sequence of Dom1h-131-511, Dom1h-131-201, Dom1h-131-202,
Dom1h-131-203, Dom1h-131-204, Dom1h-131-205 as disclosed in
PCT/GB2008/050400.
[0035] In another alternative embodiment the immunoglobulin single
variable domain for delivery to the eye can be one which is not the
amino acid sequence of Dom-4-130-202 as disclosed in
PCT/GB2008/050406.
[0036] In another alternative embodiment the immunoglobulin single
variable domain for delivery to the eye can be one which is not the
amino acid sequence of Dom 1h-131-206 as disclosed in
PCT/GB2008/050405.
[0037] It can also be useful to deliver other agents to the eye in
combination or association with the immunoglobulin single variable
domains, for example it can be useful to deliver penetration
enhancers such as sodium caprate or a viscosity agent such as
Hydroxypropylmethylcellulose (HPMC).
[0038] The single immunoglobulin variable domains (dAbs) for ocular
delivery (e.g., that bind to VEGF, IL-1, or TNF-.alpha.), can be
formatted to have a larger hydrodynamic size, for example, by
attachment of a PEG group, serum albumin, transferrin, transferrin
receptor or at least the transferrin-binding portion thereof, an
antibody Fc region, or by conjugation to an antibody domain. For
example, the dAb monomer (e.g., VEGF dAb), can be formatted as a
larger antigen-binding fragment of an antibody (e.g., formatted as
a Fab, Fab', F(ab).sub.2, F(ab').sub.2, IgG, scFv). The
hydrodynamic size of the dAb and its serum half-life can also be
increased by conjugating or linking it to a binding domain (e.g.,
an antibody or antibody fragment) that binds an antigen or epitope
that increases half-live in vivo, as described herein (see, Annex 1
of WO2006038027 incorporated herein by reference in its entirety).
For example, the VEGF dAb can be conjugated or linked to an
anti-serum albumin or anti-neonatal Fc receptor antibody or
antibody fragment, e.g., an anti-SA or anti-neonatal Fc receptor
dAb, Fab, Fab' or scFv, or to an anti-SA affibody or anti-neonatal
Fc receptor affibody.
[0039] Examples of suitable albumin, albumin fragments or albumin
variants for use in compositions described herein e.g., linked with
VEGF-binding dAbs, are described in WO 2005/077042A2 and
WO2006038027, which are incorporated herein by reference in their
entirety.
[0040] Formatted dAbs (e.g., dAbs formatted by PEGylation) can have
a molecular weight which is e.g., between 30 KDa and 100 KDa e.g.,
around 50-60 KDa and can be useful for delivery to the retina
and/or the choroids and/or the lachrymal fluid.
[0041] Naked (unformatted) dAbs which have a molecular weight
around 15 KDa can be useful for delivery to the vitreous and/or
aqueous humour and/or retina and/or choroids.
[0042] In other embodiments of the disclosure described throughout
this disclosure, instead of the use of a single immunoglobulin
variable domain or "dAb" in an antagonist or ligand of the
disclosure, it is contemplated that the skilled addressee can use a
domain that comprises the CDRs of a dAb that binds e.g., VEGF,
IL-1, or TNF-.alpha. (e.g., CDRs grafted onto a suitable protein
scaffold or skeleton, e.g., an affibody, an SpA scaffold, an LDL
receptor class A domain or an EGF domain) or can be a protein
domain comprising a binding site for VEGF, IL-1, or TNF-.alpha.
e.g., wherein the domain is selected from an affibody, an SpA
domain, an LDL receptor class A domain or an EGF domain. The
disclosure as a whole is to be construed accordingly to provide
disclosure of antagonists, ligands and methods using such domains
in place of a dAb.
[0043] Protease resistant dAbs described herein can be selected
using the methods and teachings described in WO 2008149143, the
contents of which are incorporated herein by reference.
[0044] In one aspect, the disclosure provides a protease resistant
immunoglobulin single variable domain comprising e.g., a VEGF,
IL-1, or TNF-.alpha. binding site, wherein the variable domain is
resistant to protease when incubated with
(i) a concentration (c) of at least 10 micrograms/ml protease at
37.degree. C. for time (t) of at least one hour; or (ii) a
concentration (c') of at least 40 micrograms/ml protease at
30.degree. C. for time (t) of at least one hour. In one embodiment,
the ratio (on a mole/mole basis) of protease, e.g., trypsin, to
variable domain is 8,000 to 80,000 protease:variable domain, e.g.,
when C is 10 micrograms/ml, the ratio is 800 to 80,000
protease:variable domain; or when C or C' is 100 micrograms/ml, the
ratio is 8,000 to 80,000 protease:variable domain. In one
embodiment the ratio (on a weight/weight, e.g., microgram/microgram
basis) of protease (e.g., trypsin) to variable domain is 16,000 to
160,000 protease:variable domain e.g., when C is 10 micrograms/ml,
the ratio is 1,600 to 160,000 protease:variable domain; or when C
or C' is 100 micrograms/ml, the ratio is 16,000 to 160,000
protease:variable domain. In one embodiment, the concentration (c
or c') is at least 100 or 1000 micrograms/ml protease. In one
embodiment, the concentration (c or c') is at least 100 or 1000
micrograms/ml protease. Reference is made to the description herein
of the conditions suitable for proteolytic activity of the protease
for use when working with repertoires or libraries of peptides or
polypeptides (e.g., w/w parameters). These conditions can be used
for conditions to determine the protease resistance of a particular
immunoglobulin single variable domain. In one embodiment, time (t)
is or is about one, three or 24 hours or overnight (e.g., about
12-16 hours). In one embodiment, the variable domain is resistant
under conditions (i) and the concentration (c) is or is about 10 or
100 micrograms/ml protease and time (t) is 1 hour. In one
embodiment, the variable domain is resistant under conditions (ii)
and the concentration (c') is or is about 40 micrograms/ml protease
and time (t) is or is about 3 hours. In one embodiment, the
protease is selected from trypsin, elastase, leucozyme and
pancreatin. In one embodiment, the protease is trypsin. In one
embodiment, the protease is a protease found in sputum, mucus
(e.g., gastric mucus, nasal mucus, bronchial mucus),
bronchoalveolar lavage, lung homogenate, lung extract, pancreatic
extract, gastric fluid, saliva or tears or the eye. In one
embodiment, the protease is one found in the eye and/or tears. In
one embodiment, the protease is a non-bacterial protease. In an
embodiment, the protease is an animal, e.g., mammalian, e.g.,
human, protease.
[0045] In one embodiment, the variable domain is resistant to
trypsin and/or at least one other protease selected from elastase,
leucozyme and pancreatin. For example, resistance is to trypsin and
elastase; trypsin and leucozyme; trypsin and pacreatin; trypsin,
elastase and leucozyme; trypsin, elastase and pancreatin; trypsin,
elastase, pancreatin and leucozyme; or trypsin, pancreatin and
leucozyme.
[0046] In one embodiment, the variable domain is displayed on
bacteriophage when incubated under condition (i) or (ii) for
example at a phage library size of 10.sup.6 to 10.sup.13, e.g.,
10.sup.8 to 10.sup.12 replicative units (infective virions).
[0047] In one embodiment, the variable domain specifically binds
VEGF, IL-1, or TNF-.alpha. following incubation under condition (i)
or (ii), e.g., assessed using BIACORE.TM. or ELISA, e.g., phage
ELISA or monoclonal phage ELISA.
[0048] In one embodiment, the variable domains specifically bind
protein A or protein L. In one embodiment, specific binding to
protein A or L is present following incubation under condition (i)
or (ii).
[0049] In one embodiment, the variable domains may have an
OD.sub.450 reading in ELISA, e.g., phage ELISA or monoclonal phage
ELISA) of at least 0.404, e.g., following incubation under
condition (i) or (ii).
[0050] In one embodiment, the variable domains display
(substantially) a single band in gel electrophoresis, e.g.,
following incubation under condition (i) or (ii).
[0051] In another embodiment, an agent (dAb) can be locally
administered to the eye via an implantable delivery device. Thus,
in one embodiment, the disclosure provides a implantable delivery
device containing e.g., the VEGF, IL-1, or TNF-.alpha. dAb, for
ocular delivery.
[0052] In a further aspect, the disclosure provides a
pharmaceutical composition comprising an immunoglobulin single
variable domain (e.g., VEGF, IL-1, or TNF-.alpha. dAb), and a
pharmaceutically or physiologically acceptable carrier, excipient
or diluent for ocular delivery.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1a: Depicts the amino acid sequence of
DOM15-26-593.
[0054] FIG. 1b: Depicts the amino acid sequence of DOM15-26-593-FC
fusion.
[0055] FIG. 1c: Depicts the amino acid sequence of an antibody
Fc.
[0056] FIG. 2: Depicts the amino acid sequence of DOM 0400 PEG (a
pegylated anti-IL1 dAb, molecular weight about 52 KDa).
[0057] FIG. 3: Depicts the amino acid sequence of DOM4-130-54 (An
anti-IL1 dAb).
[0058] FIG. 4: Depicts the amino acid sequence of Dom 1h-131-206
(An anti TNF alpha R1 dAb).
DETAILED DESCRIPTION OF THE DISCLOSURE
[0059] Within this specification it is intended and should be
appreciated that embodiments may be variously combined or separated
without parting from the disclosure.
[0060] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art (e.g., in cell culture, molecular
genetics, nucleic acid chemistry, hybridization techniques and
biochemistry). Standard techniques are used for molecular, genetic
and biochemical methods (see generally, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,
Short Protocols in Molecular Biology (1999) 4.sup.th Ed, John Wiley
& Sons, Inc. which are incorporated herein by reference) and
chemical methods.
[0061] As used herein, the term "antagonist of vascular endothelial
growth factor (VEGF)" or "anti-VEGF antagonist" or the like refers
to an agent (e.g., a molecule, a compound) which binds VEGF and can
inhibit a (i.e., one or more) function of VEGF.
[0062] As used herein, "peptide" refers to about two to about 50
amino acids that are joined together via peptide bonds.
[0063] As used herein, "polypeptide" refers to at least about 50
amino acids that are joined together by peptide bonds. Polypeptides
generally comprise tertiary structure and fold into functional
domains.
[0064] As used herein, a peptide or polypeptide (e.g., a domain
antibody (dAb)) that is "resistant to protease degradation" is not
substantially degraded by a protease when incubated with the
protease under conditions suitable for protease activity. A
polypeptide (e.g., a dAb) is not substantially degraded when no
more than about 25%, no more than about 20%, no more than about
15%, no more than about 14%, no more than about 13%, no more than
about 12%, no more than about 11%, no more than about 10%, no more
than about 9%, no more than about 8%, no more than about 7%, no
more than about 6%, no more than about 5%, no more than about 4%,
no more than about 3%, no more that about 2%, no more than about
1%, or substantially none of the protein is degraded by protease
after incubation with the protease for about one hour at a
temperature suitable for protease activity. For example at 37 or 50
degrees C. Protein degradation can be assessed using any suitable
method, for example, by SDS-PAGE or by functional assay (e.g.,
ligand binding) as described herein.
[0065] As used herein, "target ligand" refers to a ligand which is
specifically or selectively bound by a polypeptide or peptide. For
example, when a polypeptide is an antibody or antigen-binding
fragment thereof, the target ligand can be any desired antigen or
epitope. Binding to the target antigen is dependent upon the
polypeptide or peptide being functional.
[0066] As used herein an antibody refers to IgG, IgM, IgA, IgD or
IgE or a fragment (such as a Fab, F(ab').sub.2, Fv, disulphide
linked Fv, scFv, closed conformation multispecific antibody,
disulphide-linked scFv, diabody) whether derived from any species
naturally producing an antibody, or created by recombinant DNA
technology; whether isolated from serum, B-cells, hybridomas,
transfectomas, yeast or bacteria.
[0067] As used herein, "antibody format" refers to any suitable
polypeptide structure in which one or more antibody variable
domains can be incorporated so as to confer binding specificity for
antigen on the structure. A variety of suitable antibody formats
are known in the art, such as, chimeric antibodies, humanized
antibodies, human antibodies, single chain antibodies, bispecific
antibodies, antibody heavy chains, antibody light chains,
homodimers and heterodimers of antibody heavy chains and/or light
chains, antigen-binding fragments of any of the foregoing (e.g., a
Fv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv),
a Fab fragment, a Fab' fragment, a F(ab').sub.2 fragment), a single
antibody variable domain (e.g., a dAb, V.sub.H, V.sub.HH, V.sub.L),
and modified versions of any of the foregoing (e.g., modified by
the covalent attachment of polyethylene glycol or other suitable
polymer or a humanized V.sub.HH).
[0068] The phrase "immunoglobulin single variable domain" refers to
an antibody variable domain (V.sub.H, V.sub.HH, V.sub.L) that
specifically binds an antigen or epitope independently of other V
regions or domains. An immunoglobulin single variable domain can be
present in a format (e.g., homo- or hetero-multimer) with other
variable regions or variable domains where the other regions or
domains are not required for antigen binding by the single
immunoglobulin variable domain (i.e., where the immunoglobulin
single variable domain binds antigen independently of the
additional variable domains). A "domain antibody" or "dAb" is the
same as an "immunoglobulin single variable domain" as the term is
used herein. A "single immunoglobulin variable domain" is the same
as an "immunoglobulin single variable domain" as the term is used
herein. A "single antibody variable domain" is the same as an
"immunoglobulin single variable domain" as the term is used herein.
An immunoglobulin single variable domain is in one embodiment a
human antibody variable domain, but also includes single antibody
variable domains from other species such as rodent (for example, as
disclosed in WO 00/29004, the contents of which are incorporated
herein by reference in their entirety), nurse shark and Camelid
V.sub.HH dAbs. Camelid V.sub.HH are immunoglobulin single variable
domain polypeptides that are derived from species including camel,
llama, alpaca, dromedary, and guanaco, which produce heavy chain
antibodies naturally devoid of light chains. The V.sub.HH may be
humanized.
[0069] A "domain" is a folded protein structure which has tertiary
structure independent of the rest of the protein. Generally,
domains are responsible for discrete functional properties of
proteins, and in many cases may be added, removed or transferred to
other proteins without loss of function of the remainder of the
protein and/or of the domain. A "single antibody variable domain"
is a folded polypeptide domain comprising sequences characteristic
of antibody variable domains. It therefore includes complete
antibody variable domains and modified variable domains, for
example, in which one or more loops have been replaced by sequences
which are not characteristic of antibody variable domains, or
antibody variable domains which have been truncated or comprise N-
or C-terminal extensions, as well as folded fragments of variable
domains which retain at least the binding activity and specificity
of the full-length domain.
[0070] As used herein, the term "dose" refers to the quantity of
ligand administered to a subject all at one time (unit dose), or in
two or more administrations over a defined time interval. For
example, dose can refer to the quantity of ligand (e.g., ligand
comprising an immunoglobulin single variable domain that binds
target antigen) administered to a subject over the course of one
day (24 hours) (daily dose), two days, one week, two weeks, three
weeks or one or more months (e.g., by a single administration, or
by two or more administrations). The interval between doses can be
any desired amount of time.
[0071] The phrase, "half-life," refers to the time taken for the
serum concentration of the ligand (e.g., dAb, polypeptide or
antagonist) to reduce by 50%, in vivo, for example due to
degradation of the ligand and/or clearance or sequestration of the
ligand by natural mechanisms. The ligands of the disclosure are
stabilized in vivo and their half-life increased by binding to
molecules which resist degradation and/or clearance or
sequestration. Typically, such molecules are naturally occurring
proteins which themselves have a long half-life in vivo. The
half-life of a ligand is increased if its functional activity
persists, in vivo, for a longer period than a similar ligand which
is not specific for the half-life increasing molecule. For example,
a ligand specific for human serum albumin (HAS) and a target
molecule is compared with the same ligand wherein the specificity
to HSA is not present, that is does not bind HSA but binds another
molecule. For example, it may bind a third target on the cell.
Typically, the half-life is increased by 10%, 20%, 30%, 40%, 50% or
more. Increases in the range of 2.times., 3.times., 4.times.,
5.times., 10.times., 20.times., 30.times., 40.times., 50.times. or
more of the half-life are possible. Alternatively, or in addition,
increases in the range of up to 30.times., 40.times., 50.times.,
60.times., 70.times., 80.times., 90.times., 100.times., 150.times.
of the half-life are possible.
[0072] As used herein, "hydrodynamic size" refers to the apparent
size of a molecule (e.g, a protein molecule, ligand) based on the
diffusion of the molecule through an aqueous solution. The
diffusion, or motion of a protein through solution can be processed
to derive an apparent size of the protein, where the size is given
by the "Stokes radius" or "hydrodynamic radius" of the protein
particle. The "hydrodynamic size" of a protein depends on both mass
and shape (conformation), such that two proteins having the same
molecular mass may have differing hydrodynamic sizes based on the
overall conformation of the protein.
[0073] As referred to herein, the term "competes" means that the
binding of a first target to its cognate target binding domain is
inhibited in the presence of a second binding domain that is
specific for said cognate target. For example, binding may be
inhibited sterically, for example by physical blocking of a binding
domain or by alteration of the structure or environment of a
binding domain such that its affinity or avidity for a target is
reduced. See WO2006038027 for details of how to perform competition
ELISA and competition BIACORE.TM. experiments to determine
competition between first and second binding domains.
[0074] Calculations of "homology" or "identity" or "similarity"
between two sequences (the terms are used interchangeably herein)
are performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid or nucleic acid sequence for
optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). In an embodiment, the length of a
reference sequence aligned for comparison purposes is at least 30%,
or at least 40%, or at least 50%, or at least 60%, or at least 70%,
80%, 90%, 100% of the length of the reference sequence. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "homology" is equivalent to amino acid
or nucleic acid "identity"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences. Amino acid and nucleotide sequence
alignments and homology, similarity or identity, as defined herein
may be prepared and determined using the algorithm BLAST 2
Sequences, using default parameters (Tatusova, T. A. et al., FEMS
Microbiol Lett, 174:187-188 (1999).
Protease Resistance:
[0075] The disclosure in one embodiment relates to dAbs, e.g.,
anti-VEGF dAbs, TNFR1 dAbs, IL-1 dAbs, for delivery to the eye,
which have been selected by a method of selection for protease
resistant dAbs that have a desired biological activity e.g.,
binding to VEGF, TNFR1 or IL-1. Two selective pressures are used in
the method to produce an efficient process for selecting
polypeptides that are highly stable and resistant to protease
degradation, and that have desired biological activity. As
described herein, protease resistant peptides and polypeptides
generally retain biological activity. In contrast, protease
sensitive peptides and polypeptides are cleaved or digested by
protease in the methods described herein, and therefore, lose their
biological activity. Accordingly, protease resistant peptides or
polypeptides are generally selected based on their biological
activity, such as binding activity.
[0076] The ocular environment is one which is rich in proteases and
hence use of protease resistant dAbs for ocular delivery as
described herein provides several advantages. For example, variable
domains that are selected for resistance to proteolytic degradation
by one protease (e.g., trypsin), are also resistant to degradation
by other proteases (e.g., elastase, leucozyme). Protease resistance
can correlate with a higher melting temperature (Tm) of the peptide
or polypeptide. Higher melting temperatures are indicative of more
stable variable domains, antagonists, peptides and polypeptides.
Resistance to protease degradation can also correlate with high
affinity binding to target ligands. Thus, the methods described and
referenced herein (in WO 2008149143) provide an efficient way to
select, isolate and/or recover dAbs that have a desired biological
activity and that are well suited for in vivo therapeutic and/or
diagnostic ocular uses because they are protease resistant and
stable. In one embodiment protease resistance can correlate with an
improved PK, for example improved over a variable domain,
antagonist, peptide or polypeptide that is not protease resistant.
Improved PK may be an improved AUC (area under the curve) and/or an
improved half-life. Protease resistance can also correlate with an
improved stability of the variable domain, antagonist, peptide or
polypeptide to shear and/or thermal stress and/or a reduced
propensity to aggregate during nebulisation, for example improved
over an variable domain, antagonist, peptide or polypeptide that is
not protease resistant. In one embodiment protease resistance
correlates with an improved storage stability, for example improved
over an variable domain, antagonist, peptide or polypeptide that is
not protease resistant. In one aspect, one, two, three, four or all
of the advantages are provided, the advantages being resistance to
protease degradation, higher Tm and high affinity binding to target
ligand.
[0077] The methods described and referenced herein (in WO
2008/149143) can be used as part of a program to isolate protease
resistant peptides or polypeptides, e.g., dAbs, that can comprise,
if desired, other suitable selection methods. In these situations,
the methods described herein can be employed at any desired point
in the program, such as before or after other selection methods are
used.
[0078] In certain embodiments, the dAb for ocular delivery is
selected for resistance to degradation by trypsin, elastase or
leucozyme and specifically binds VEGF. In these embodiments, a
library or repertoire comprising dAbs is provided and combined with
trypsin, elastase or leucozyme (or a biological preparation,
extract or homogenate comprising trypsin) under conditions suitable
for proteolytic digestion. Trypsin, elastase or leucozyme resistant
dAbs are selected that bind VEGF. For example, the protease
resistant dAb is not substantially degraded when incubated at
37.degree. C. in a 0.04% (w/w) solution of protease for a period of
at least about 2 hours. In another example, the protease resistant
dAb is not substantially degraded when incubated at 37.degree. C.
in a 0.04% (w/w) solution of protease for a period of at least
about 3 hours. In another example, the protease resistant dAb is
not substantially degraded when incubated at 37.degree. C. in a
0.04% (w/w) solution of protease for a period of at least about 4
hours, at least about 5 hours, at least about 6 hours, at least
about 7 hours, at least about 8 hours, at least about 9 hours, at
least about 10 hours, at least about 11 hours, or at least about 12
hours.
[0079] In another aspect, there is provided a method of producing a
repertoire of protease resistant peptides or polypeptides (eg,
dAbs). The method comprises providing a repertoire of peptides or
polypeptides; combining the repertoire of peptides or polypeptides
and a protease under suitable conditions for protease activity; and
recovering a plurality of peptides or polypeptides that
specifically bind VEGF, whereby a repertoire of protease resistant
peptides or polypeptides is produced. Proteases, display systems,
conditions for protease activity, and methods for selecting
peptides or polypeptides that are suitable for use in the method
are described herein with respect to the other methods.
[0080] In some embodiments, a display system (e.g., a display
system that links coding function of a nucleic acid and functional
characteristics of the peptide or polypeptide encoded by the
nucleic acid) that comprises a repertoire of peptides or
polypeptides is used, and the method further comprises amplifying
or increasing the copy number of the nucleic acids that encode the
plurality of selected peptides or polypeptides. Nucleic acids can
be amplified using any suitable method, such as by phage
amplification, cell growth or polymerase chain reaction.
[0081] In particular embodiments, there is provided a method of
producing a repertoire of protease resistant polypeptides that
comprise anti-VEGF dAbs. The method comprises providing a
repertoire of polypeptides that comprise anti-VEGF dAbs; combining
the repertoire of peptides or polypeptides and a protease (e.g.,
trypsin, elastase, leucozyme) under suitable conditions for
protease activity; and recovering a plurality of polypeptides that
comprise dAbs that have binding specificity for VEGF. The method
can be used to produce a naive repertoire, or a repertoire that is
biased toward a desired binding specificity, such as an affinity
maturation repertoire based on a parental dAb that has binding
specificity for VEGF.
Selection/Isolation/Recovery
[0082] A protease resistant peptide or polypeptide (e.g., a
population of protease resistant polypeptides) can be selected,
isolated and/or recovered from a repertoire or library (e.g., in a
display system) using any suitable method. In one embodiment, a
protease resistant polypeptide is selected or isolated based on a
selectable characteristic (e.g., physical characteristic, chemical
characteristic, functional characteristic). Suitable selectable
functional characteristics include biological activities of the
peptides or polypeptides in the repertoire, for example, binding to
a generic ligand (e.g., a superantigen), binding to a target ligand
(e.g., an antigen, an epitope, a substrate), binding to an antibody
(e.g., through an epitope expressed on a peptide or polypeptide),
and catalytic activity. (see e.g., Tomlinson et al., WO 99/20749;
WO 01/57065; WO 99/58655). In one embodiment, the selection is
based on specific binding to VEGF. In another embodiment, selection
is on the basis of the selected functional characteristic to
produce a second repertoire in which members are protease
resistant, followed by selection of a member from the second
repertoire that specifically binds VEGF.
[0083] In some embodiments, the protease resistant peptide or
polypeptide is selected and/or isolated from a library or
repertoire of peptides or polypeptides in which substantially all
protease resistant peptides or polypeptides share a common
selectable feature. For example, the protease resistant peptide or
polypeptide can be selected from a library or repertoire in which
substantially all protease resistant peptides or polypeptides bind
a common generic ligand, bind a common target ligand, bind (or are
bound by) a common antibody, or possess a common catalytic
activity. This type of selection is particularly useful for
preparing a repertoire of protease resistant peptides or
polypeptides that are based on a parental peptide or polypeptide
that has a desired biological activity, for example, when
performing affinity maturation of an immunoglobulin single variable
domain.
[0084] Selection based on binding to a common generic ligand can
yield a collection or population of peptides or polypeptides that
contain all or substantially all of the protease resistant peptides
or polypeptides that were components of the original library or
repertoire. For example, peptides or polypeptides that bind a
target ligand or a generic ligand, such as protein A, protein L or
an antibody, can be selected, isolated and/or recovered by panning
or using a suitable affinity matrix. Panning can be accomplished by
adding a solution of ligand (e.g., generic ligand, target ligand)
to a suitable vessel (e.g., tube, petri dish) and allowing the
ligand to become deposited or coated onto the walls of the vessel.
Excess ligand can be washed away and peptides or polypeptides
(e.g., a repertoire that has been incubated with protease) can be
added to the vessel and the vessel maintained under conditions
suitable for peptides or polypeptides to bind the immobilized
ligand. Unbound peptides or polypeptides can be washed away and
bound peptides or polypeptides can be recovered using any suitable
method, such as scraping or lowering the pH, for example.
[0085] Suitable ligand affinity matrices generally contain a solid
support or bead (e.g., agarose) to which a ligand is covalently or
noncovalently attached. The affinity matrix can be combined with
peptides or polypeptides (e.g., a repertoire that has been
incubated with protease) using a batch process, a column process or
any other suitable process under conditions suitable for binding of
peptides or polypeptides to the ligand on the matrix. Peptides or
polypeptides that do not bind the affinity matrix can be washed
away and bound peptides or polypeptides can be eluted and recovered
using any suitable method, such as elution with a lower pH buffer,
with a mild denaturing agent (e.g., urea), or with a peptide that
competes for binding to the ligand. In one example, a biotinylated
target ligand is combined with a repertoire under conditions
suitable for peptides or polypeptides in the repertoire to bind the
target ligand (VEGF). Bound peptides or polypeptides are recovered
using immobilized avidin or streptavidin (e.g., on a bead).
[0086] In some embodiments, the generic ligand is an antibody or
antigen binding fragment thereof. Antibodies or antigen binding
fragments that bind structural features of peptides or polypeptides
that are substantially conserved in the peptides or polypeptides of
a library or repertoire are particularly useful as generic ligands.
Antibodies and antigen binding fragments suitable for use as
ligands for isolating, selecting and/or recovering protease
resistant peptides or polypeptides can be monoclonal or polyclonal
and can be prepared using any suitable method.
Nucleic Acids, Host Cells and Methods for Producing Protease
Resistant Polypeptides:
[0087] The protease resistant peptide or polypeptide selected by
the method described herein can also be produced in a suitable in
vitro expression system e.g., E. coli or Pichia species e.g., P.
pastoris, by chemical synthesis or by any other suitable
method.
[0088] Polypeptides, dAbs & Antagonists:
[0089] As described herein, protease resistant dAbs generally bind
their target ligand with high affinity.
[0090] For example, the VEGF dAb can bind VEGF with an affinity
(KD; KD=K.sub.off (kd)/K.sub.on (ka) as determined by surface
plasmon resonance) of 300 nM to 1 pM (i.e., 3.times.10.sup.-7 to
5.times.10.sup.-12M), e.g., 50 nM to 1 pM, 5 nM to 1 pM and e.g., 1
nM to 1 pM; for example K.sub.D of 1.times.10.sup.-7 M or less,
e.g., 1.times.10.sup.-8 M or less, e.g., 1.times.10.sup.-9 M or
less, e.g., 1.times.10.sup.-10 M or less and e.g.,
1.times.10.sup.-11 M or less; and/or a K.sub.off rate constant of
5.times.10.sup.-1 s.sup.-1 to 1.times.10.sup.-7 s.sup.-1, e.g.,
1.times.10.sup.-2 s.sup.-1 to 1.times.10.sup.-6 s.sup.-1, e.g.,
5.times.10.sup.-3 s.sup.-1 to 1.times.10.sup.-5 s.sup.-1, for
example 5.times.10.sup.-1 s.sup.-1 or less, e.g., 1.times.10.sup.-2
s.sup.-1 or less, e.g., 1.times.10.sup.-3 s.sup.-1 or less, e.g.,
1.times.10.sup.-4 s.sup.-1 or less, e.g., 1.times.10.sup.-5
s.sup.-1 or less, and e.g., 1.times.10.sup.-6 s.sup.-1 or less as
determined by surface plasmon resonance.
[0091] Although we are not bound by any particular theory, peptides
and polypeptides that are resistant to proteases are believed to
have a lower entropy and/or a higher stabilization energy. Thus,
the correlation between protease resistance and high affinity
binding may be related to the compactness and stability of the
surfaces of the peptides and polypeptides and dAbs selected by the
method described herein.
[0092] In one embodiment, A VEGF dAb inhibits binding of VEGF at a
concentration 50 (IC50) of IC50 of about 1 .mu.M or less, about 500
nM or less, about 100 nM or less, about 75 nM or less, about 50 nM
or less, about 10 nM or less or about 1 nM or less.
[0093] In certain embodiments, the VEGF dAb specifically binds
VEGF, e.g., human VEGF, and dissociates from human VEGF with a
dissociation constant (K.sub.D) of 300 nM to 1 pM or 300 nM to 5 pM
or 50 nM to 1 pM or 50 nM to 5 pM or 50 nM to 20 pM or about 10 pM
or about 15 pM or about 20 pM as determined by surface plasmon
resonance. In certain embodiments, the polypeptide, dAb or
antagonist specifically binds VEGF, e.g., human VEGF, and
dissociates from human VEGF with a K.sub.off rate constant of
5.times.10.sup.-1 s.sup.-1 to 1.times.10.sup.-7 s.sup.-1, e.g.
1.times.10.sup.-2 s.sup.-1 to 1.times.10.sup.-6 s.sup.-1, e.g.,
5.times.10.sup.-3 s.sup.-1 to 1.times.10.sup.-5 s.sup.-1, for
example 5.times.10.sup.-1 s.sup.-1 or less, e.g., 1.times.10.sup.-2
s.sup.-1 or less, e.g., 1.times.10.sup.-3 s.sup.-1 or less, e.g.,
1.times.10.sup.-4 s.sup.-1 or less, e.g., 1.times.10.sup.-5
s.sup.-1 or less, and e.g., 1.times.10.sup.-6 s.sup.-1 or less as
determined by surface plasmon resonance.
[0094] In certain embodiments, VEGF dAb specifically binds VEGF,
e.g., human VEGF, with a K.sub.on of 1.times.10.sup.-3 M.sup.-1
s.sup.-1 to 1.times.10.sup.-7 M.sup.-1 s.sup.-1 or
1.times.10.sup.-3 M.sup.-1 s.sup.-1 to 1.times.10.sup.-6 M.sup.-1
s.sup.-1 or about 1.times.10.sup.-4 M.sup.-1 s.sup.-1 or about
1.times.10.sup.-5 M.sup.-1 s.sup.-1. In one embodiment, the
polypeptide, dAb or antagonist specifically binds VEGF, e.g., human
VEGF, and dissociates from human VEGF with a dissociation constant
(K.sub.D) and a K.sub.off as defined in this paragraph. In one
embodiment, the polypeptide, dAb or antagonist specifically binds
VEGF, e.g., human VEGF, and dissociates from human VEGF with a
dissociation constant (K.sub.D) and a K.sub.on as defined in this
paragraph. In some embodiments, the polypeptide or dAb specifically
binds VEGF (e.g., human VEGF) with a K.sub.D and/or K.sub.off
and/or K.sub.on as recited in this paragraph and comprises an amino
acid sequence that is at least or at least about 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the
amino acid sequence of a dAb with the amino acid sequence of
DOM15-26-593.
[0095] The dAb can be expressed in E. coli or in Pichia species
(e.g., P. pastoris). In one embodiment, the ligand or dAb monomer
is secreted in a quantity of at least about 0.5 mg/L when expressed
in E. coli or in Pichia species (e.g., P. pastoris). Although, the
ligands and dAb monomers described herein can be secretable when
expressed in E. coli or in Pichia species P. pastoris), they can be
produced using any suitable method, such as synthetic chemical
methods or biological production methods that do not employ E. coli
or Pichia species.
[0096] In some embodiments, the polypeptide, dAb or antagonist does
not comprise a Camelid immunoglobulin variable domain, or one or
more framework amino acids that are unique to immunoglobulin
variable domains encoded by Camelid germline antibody gene
segments, eg at position 108, 37, 44, 45 and/or 47.
[0097] Antagonists of VEGF can be monovalent or multivalent. In
some embodiments, the antagonist is monovalent and contains one
binding site that interacts with VEGF, the binding site provided by
a polypeptide or dAb of the disclosure. Monovalent antagonists bind
one VEGF and may not induce cross-linking or clustering of VEGF on
the surface of cells which can lead to activation of the receptor
and signal transduction.
[0098] Alternatively the antagonist of VEGF is multivalent.
Multivalent antagonists of VEGF can contain two or more copies of a
particular binding site for VEGF or contain two or more different
binding sites that bind VEGF, at least one of the binding sites
being provided by a dAb of the disclosure. For example, as
described herein the antagonist of VEGF can be a dimer, trimer or
multimer comprising two or more copies of a dAb that binds VEGF, or
two or more different dAbs that bind VEGF.
[0099] In other embodiments, the, dAb specifically binds VEGF with
a K.sub.D described herein and inhibits tumour growth in a standard
murine xenograft model (e.g., inhibits tumour growth by at least
about 10%, as compared with a suitable control). In one embodiment,
the polypeptide, dAb or antagonist inhibits tumour growth by at
least about 10% or by at least about 25%, or by at least about 50%,
as compared to a suitable control in a standard murine xenograft
model when administered at about 1 mg/kg or more, for example about
5 or 10 mg/kg.
[0100] In other embodiments, the polypeptide, dAb or antagonist
binds VEGF and antagonizes the activity of the VEGF in a standard
cell assay with an ND.sub.50 of .ltoreq.100 nM.
[0101] In certain embodiments, the dAbs are efficacious in animal
models of ocular disease when an effective amount is administered.
Generally an effective amount is about 1 mg/kg to about 10 mg/kg
(e.g., about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg,
about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9
mg/kg, or about 10 mg/kg). The dAb can be administered at a dosing
frequency of e.g., once or twice daily, once or twice weekly, once
or twice monthly.
[0102] Generally, the dAbs will be utilized in purified form
together with pharmacologically appropriate carriers for ocular
delivery. Typically, these carriers can include aqueous or
alcoholic/aqueous solutions, emulsions or suspensions, any
including saline and/or buffered media. Suitable
physiologically-acceptable adjuvants, if necessary to keep a
polypeptide complex in suspension, may be chosen from thickeners
such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and
alginates. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may
also be present (Mack (1982) Remington's Pharmaceutical Sciences,
16th Edition), A variety of suitable formulations can be used,
including extended release formulations. These might comprise,
implants, gels, nanoparticles, and microparticles. Drug loaded PLA
nano- and microparticles have been used to deliver drug to the
posterior segment of the eye after sub-conjunctival delivery of the
formulation (Kompella et al. IOVS 2003 44(3) 1192-1201). In
particular, microspheres are retained at the site of delivery and
appear to be more appropriate for retinal drug delivery compared to
nanoparticles which may be cleared more readily (Amrite et al ARVO
abstract #5067/B391 2003).
[0103] The ligands (e.g., antagonists) of the present disclosure
may be used as separately administered compositions or in
conjunction with other agents. These can include various drugs for
oclar delivery to the eye and/or ocular penetration enhancers
and/or viscosity enhancers.
[0104] Pharmaceutical compositions can include "cocktails" of
various other agents in conjunction with the ligands of the present
disclosure, or even combinations of ligands according to the
present disclosure having different specificities, such as ligands
selected using different target antigens or epitopes, whether or
not they are pooled prior to administration.
[0105] The precise dosage and frequency of administration of the
dAbs to the eye will depend on the age, sex and condition of the
patient, concurrent administration of other drugs,
counterindications and other parameters to be taken into account by
the clinician.
[0106] The dAbs of this disclosure can be lyophilised for storage
and reconstituted in a suitable carrier prior to use. This
technique has been shown to be effective with conventional
immunoglobulins and art-known lyophilisation and reconstitution
techniques can be employed. It will be appreciated by those skilled
in the art that lyophilisation and reconstitution can lead to
varying degrees of antibody activity loss (e.g., with conventional
immunoglobulins, IgM antibodies tend to have greater activity loss
than IgG antibodies) and that use levels may have to be adjusted
upward to compensate.
[0107] The compositions containing the present dAbs or a cocktail
thereof can be administered for prophylactic and/or therapeutic
treatments. In certain therapeutic applications, an adequate amount
to accomplish at least partial inhibition, suppression, modulation,
killing, or some other measurable parameter, of a population of
selected cells can be defined as a "therapeutically-effective
dose". Amounts needed to achieve this dosage will depend upon the
severity of the disease and the general state of the patient's own
immune system. The skilled clinician will be able to determine the
appropriate dosing interval to treat, suppress or prevent
disease.
[0108] Treatment or therapy performed using the compositions
described herein is considered "effective" if one or more symptoms
are reduced (e.g., by at least 10% or at least one point on a
clinical assessment scale), relative to such symptoms present
before treatment, or relative to such symptoms in an individual
(human or model animal) not treated with such composition or other
suitable control. Symptoms will obviously vary depending upon the
disease or disorder targeted, but can be measured by an ordinarily
skilled clinician or technician. Such symptoms can be measured, for
example, by monitoring the level of one or more biochemical
indicators of the disease or disorder (e.g., levels of an enzyme or
metabolite correlated with the disease, affected cell numbers,
etc.), by monitoring physical manifestations or by an accepted
clinical assessment scale.
[0109] Similarly, prophylaxis performed using a composition as
described herein is "effective" if the onset or severity of one or
more symptoms is delayed, reduced or abolished relative to such
symptoms in a similar individual (human or animal model) not
treated with the composition.
[0110] In one embodiment, the disclosure is a method for treating,
suppressing or preventing ocular disease or condition, selected
from for example cancer (e.g., a solid tumour), inflammatory
disease, autoimmune disease, vascular proliferative disease (e.g.,
AMD (age related macular degeneration)) comprising administering to
a mammal in need thereof a therapeutically-effective dose or amount
of a polypeptide, dAb which binds to VEGF or antagonist of VEGF
according to the disclosure or to IL-1, or TNF-.alpha.. Examples of
such ocular diseases or conditions include AMD, uveitis, dry eye,
diabetic retinopathy and diabetic macular oedema.
Formats:
[0111] Increased half-life is useful in in vivo applications of
immunoglobulins, especially antibodies and most especially antibody
fragments of small size. Such fragments (Fvs, disulphide bonded
Fvs, Fabs, scFvs, dAbs) can suffer from rapid clearance from the
body; thus, whilst they are able to reach most parts of the body
rapidly, and are quick to produce and easier to handle, their in
vivo applications have been limited by their only brief persistence
in vivo. Hence the dAbs described herein can be modified to provide
increased half-life in vivo and consequently longer persistence
times in the body.
[0112] Methods for pharmacokinetic analysis and determination of
ligand half-life will be familiar to those skilled in the art.
Details may be found in Kenneth, A et al: Chemical Stability of
Pharmaceuticals: A Handbook for Pharmacists and in Peters et al,
Pharmacokinetic analysis: A Practical Approach (1996). Reference is
also made to "Pharmacokinetics", M Gibaldi & D Perron,
published by Marcel Dekker, 2.sup.nd Rev. ex edition (1982), which
describes pharmacokinetic parameters such as t alpha and t beta
half lives and area under the curve (AUC).
[0113] Half lives (t1/2 alpha and t1/2 beta) and AUC can be
determined from a curve of serum concentration of ligand against
time. The WinNonlin analysis package (available from Pharsight
Corp., Mountain View, Calif. 94040, USA) can be used, for example,
to model the curve. In a first phase (the alpha phase) the ligand
is undergoing mainly distribution in the patient, with some
elimination. A second phase (beta phase) is the terminal phase when
the ligand has been distributed and the serum concentration is
decreasing as the ligand is cleared from the patient. The t alpha
half life is the half life of the first phase and the t beta half
life is the half life of the second phase. Thus, in one embodiment,
the present disclosure provides a ligand or a composition
comprising a ligand according to the disclosure having a t.alpha.
half-life in the range of 15 minutes or more. In one embodiment,
the lower end of the range is 30 minutes, 45 minutes, 1 hour, 2
hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11
hours or 12 hours. In addition, or alternatively, a ligand or
composition according to the disclosure will have a t.alpha. half
life in the range of up to and including 12 hours. In one
embodiment, the upper end of the range is 11, 10, 9, 8, 7, 6 or 5
hours. An example of a suitable range is 1 to 6 hours, 2 to 5 hours
or 3 to 4 hours.
[0114] In one embodiment, the dAb or a composition comprising a dAb
according to the disclosure has a t.beta. half-life in the range of
30 minutes or more. In one embodiment, the lower end of the range
is 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours,
7 hours, 10 hours, 11 hours, or 12 hours. In addition, or
alternatively, a ligand or composition according to the disclosure
has a t.beta. half-life in the range of up to and including 21
days. In one embodiment, the upper end of the range is 12 hours, 24
hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20 days. In one
embodiment a ligand or composition according to the disclosure will
have a t.beta. half life in the range 12 to 60 hours. In a further
embodiment, it will be in the range 12 to 48 hours. In a further
embodiment still, it will be in the range 12 to 26 hours.
[0115] In addition, or alternatively to the above criteria, the
present disclosure provides a dAb or a composition comprising a
ligand according to the disclosure having an AUC value (area under
the curve) in the range of 1 mgmin/ml or more. In one embodiment,
the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300
mgmin/ml. In addition, or alternatively, a ligand or composition
according to the disclosure has an AUC in the range of up to 600
mgmin/ml. In one embodiment, the upper end of the range is 500,
400, 300, 200, 150, 100, 75 or 50 mgmin/ml. In one embodiment a
ligand according to the disclosure will have a AUC in the range
selected from the group consisting of the following: 15 to 150
mgmin/ml, 15 to 100 mgmin/ml, 15 to 75 mgmin/ml, and 15 to 50
mgmin/ml.
[0116] dAbs of the disclosure can be formatted to have a larger
hydrodynamic size, for example, by attachment of a PEG group, serum
albumin, transferrin, transferrin receptor or at least the
transferrin-binding portion thereof, an antibody Fc region, or by
conjugation to an antibody domain. For example, dAbs can be
formatted as a larger antigen-binding fragment of an antibody, or
as an antibody (e.g., formatted as a Fab, Fab', F(ab).sub.2,
F(ab').sub.2, IgG, scFv). In another embodiment dAbs according to
the disclosure on can be formatted as a fusion or conjugate with
another polypeptide or peptide.
[0117] Hydrodynamic size of the ligands (e.g., dAb monomers and
multimers) of the disclosure may be determined using methods which
are well known in the art. For example, gel filtration
chromatography may be used to determine the hydrodynamic size of a
ligand. Suitable gel filtration matrices for determining the
hydrodynamic sizes of ligands, such as cross-linked agarose
matrices, are well known and readily available.
[0118] The size of a ligand i.e., dAb format (e.g., the size of a
PEG moiety attached to a dAb monomer), can be varied depending on
the desired application e.g., if it is desired to have the dAb
remain in the systemic circulation for a longer period of time the
size of can be increased, for example by formatting as an Ig like
protein.
Half-Life Extension by Targeting an Antigen or Epitope that
Increases Half-Live In Vivo
[0119] The hydrodynamic size of a ligand and its serum half-life
can also be increased by conjugating or associating a dAb to a
binding domain (e.g., antibody or antibody fragment) that binds an
antigen or epitope that increases half-live in vivo, as described
herein. For example, the VEGF dAb can be conjugated or linked to an
anti-serum albumin or anti-neonatal Fc receptor antibody or
antibody fragment, e.g., an anti-SA or anti-neonatal Fc receptor
dAb, Fab, Fab' or scFv, or to an anti-SA affibody or anti-neonatal
Fc receptor Affibody or an anti-SA avimer, or an anti-SA binding
domain which comprises a scaffold selected from, but preferably not
limited to, the group consisting of CTLA-4, lipocallin, SpA, an
affibody, an avimer, GroEl and fibronectin (see PCT/GB2008/000453
filed 8 Feb. 2008 for disclosure of these binding domain, which
domains and their sequences are incorporated herein by reference
and form part of the disclosure of the present text). Conjugating
refers to a composition comprising polypeptide, dAb or antagonist
of the disclosure that is bonded (covalently or noncovalently) to a
binding domain that binds serum albumin.
[0120] Suitable polypeptides that enhance serum half-life in vivo
include, for example, transferrin receptor specific
ligand-neuropharmaceutical agent fusion proteins (see U.S. Pat. No.
5,977,307, the teachings of which are incorporated herein by
reference), brain capillary endothelial cell receptor, transferrin,
transferrin receptor (e.g., soluble transferrin receptor), insulin,
insulin-like growth factor 1 (IGF 1) receptor, insulin-like growth
factor 2 (IGF 2) receptor, insulin receptor, blood coagulation
factor X, .alpha.1-antitrypsin and HNF 1.alpha.. Suitable
polypeptides that enhance serum half-life also include alpha-1
glycoprotein (orosomucoid; AAG), alpha-1 antichymotrypsin (ACT),
alpha-1 microglobulin (protein HC; AIM), antithrombin III (AT III),
apolipoprotein A-1 (Apo A-1), apolipoprotein B (Apo B),
ceruloplasmin (Cp), complement component C3 (C3), complement
component C4 (C4), C1 esterase inhibitor (C1 INH), C-reactive
protein (CRP), ferritin (FER), hemopexin (HPX), lipoprotein(a)
(Lp(a)), mannose-binding protein (MBP), myoglobin (Myo), prealbumin
(transthyretin; PAL), retinol-binding protein (RBP), and rheumatoid
factor (RF).
[0121] Suitable proteins from the extracellular matrix include, for
example, collagens, laminins, integrins and fibronectin. Collagens
are the major proteins of the extracellular matrix. About 15 types
of collagen molecules are currently known, found in different parts
of the body, e.g., type I collagen (accounting for 90% of body
collagen) found in bone, skin, tendon, ligaments, cornea, internal
organs or type II collagen found in cartilage, vertebral disc,
notochord, and vitreous humor of the eye.
[0122] Suitable proteins from the blood include, for example,
plasma proteins (e.g., fibrin, .alpha.-2 macroglobulin, serum
albumin, fibrinogen (e.g., fibrinogen A, fibrinogen B), serum
amyloid protein A, haptoglobin, profilin, ubiquitin, uteroglobulin
and .beta.-2-microglobulin), enzymes and enzyme inhibitors (e.g.,
plasminogen, lysozyme, cystatin C, alpha-1-antitrypsin and
pancreatic trypsin inhibitor), proteins of the immune system, such
as immunoglobulin proteins (e.g., IgA, IgD, IgE, IgG, IgM,
immunoglobulin light chains (kappa/lambda)), transport proteins
(e.g., retinol binding protein, .alpha.-1 microglobulin), defensins
(e.g., beta-defensin 1, neutrophil defensin 1, neutrophil defensin
2 and neutrophil defensin 3) and the like.
[0123] Suitable proteins found at the blood brain barrier or in
neural tissue include, for example, melanocortin receptor, myelin,
ascorbate transporter and the like.
[0124] Suitable polypeptides that enhance serum half-life in vivo
also include proteins localized to the kidney (e.g., polycystin,
type IV collagen, organic anion transporter K1, Heymann's antigen),
proteins localized to the liver (e.g., alcohol dehydrogenase,
G250), proteins localized to the lung (e.g., secretory component,
which binds IgA), proteins localized to the heart (e.g., HSP 27,
which is associated with dilated cardiomyopathy), proteins
localized to the skin (e.g., keratin), bone specific proteins such
as morphogenic proteins (BMPs), which are a subset of the
transforming growth factor .beta. superfamily of proteins that
demonstrate osteogenic activity (e.g., BMP-2, BMP-4, BMP-5, BMP-6,
BMP-7, BMP-8), tumor specific proteins (e.g., trophoblast antigen,
herceptin receptor, oestrogen receptor, cathepsins (e.g., cathepsin
B, which can be found in liver and spleen)).
[0125] Suitable disease-specific proteins include, for example,
antigens expressed only on activated T-cells, including LAG-3
(lymphocyte activation gene), osteoprotegerin ligand (OPGL; see
Nature 402, 304-309 (1999)), OX40 (a member of the TNF receptor
family, expressed on activated T cells and specifically
up-regulated in human T cell leukemia virus type-I
(HTLV-I)-producing cells; see Immunol. 165 (1):263-70 (2000)).
Suitable disease-specific proteins also include, for example,
metalloproteases (associated with arthritis/cancers) including
CG6512 Drosophila, human paraplegin, human FtsH, human AFG3L2,
murine ftsH; and angiogenic growth factors, including acidic
fibroblast growth factor (FGF-1), basic fibroblast growth factor
(FGF-2), vascular endothelial growth factor/vascular permeability
factor (VEGF/VPF), transforming growth factor-.alpha. (TGF
.alpha.), tumor necrosis factor-alpha (TNF-.alpha.), angiogenin,
interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived
endothelial growth factor (PD-ECGF), placental growth factor
(P1GF), midkine platelet-derived growth factor-BB (PDGF), and
fractalkine.
[0126] Suitable polypeptides that enhance serum half-life in vivo
also include stress proteins such as heat shock proteins (HSPs).
HSPs are normally found intracellularly. When they are found
extracellularly, it is an indicator that a cell has died and
spilled out its contents. This unprogrammed cell death (necrosis)
occurs when as a result of trauma, disease or injury, extracellular
HSPs trigger a response from the immune system. Binding to
extracellular HSP can result in localizing the compositions of the
disclosure to a disease site.
[0127] Suitable proteins involved in Fc transport include, for
example, Brambell receptor (also known as FcRB). This Fc receptor
has two functions, both of which are potentially useful for
delivery. The functions are (1) transport of IgG from mother to
child across the placenta (2) protection of IgG from degradation
thereby prolonging its serum half-life. It is thought that the
receptor recycles IgG from endosomes. (See, Holliger et al, Nat
Biotechnol 15(7):632-6 (1997).)
dAbs that Bind Serum Albumin
[0128] The disclosure in one embodiment a first dAb that binds to
an ocular target molecule, e.g., VEGF, IL-1, or TNF-.alpha., and a
second dAb that binds serum albumin (SA), the second dAb binding SA
with a K.sub.D as determined by surface plasmon resonance of 1 nM
to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 100, 200, 300, 400 or
500 .mu.M (i.e., .times.10.sup.-9 to 5.times.10.sup.-4), or 100 nM
to 10 .mu.M, or 1 to 5 .mu.M or 3 to 70 nM or 10 nM to 1, 2, 3, 4
or 5 .mu.M. For example 30 to 70 nM as determined by surface
plasmon resonance. In one embodiment, the first dAb (or a dAb
monomer) binds SA (e.g., HSA) with a K.sub.D as determined by
surface plasmon resonance of approximately 1, 50, 70, 100, 150,
200, 300 nM or 1, 2 or 3 .mu.M. In one embodiment, for a dual
specific ligand comprising a first anti-SA dAb and a second dAb to
VEGF, the affinity (e.g., K.sub.D and/or K.sub.off as measured by
surface plasmon resonance, e.g., using BIACORE.TM.) of the second
dAb for its target is from 1 to 100000 times (e.g., 100 to 100000,
or 1000 to 100000, or 10000 to 100000 times) the affinity of the
first dAb for SA. In one embodiment, the serum albumin is human
serum albumin (HSA). For example, the first dAb binds SA with an
affinity of approximately 10 .mu.M, while the second dAb binds its
target with an affinity of 100 pM. In one embodiment, the serum
albumin is human serum albumin (HSA). In one embodiment, the first
dAb binds SA (e.g., HSA) with a K.sub.D of approximately 50, for
example 70, 100, 150 or 200 nM. Details of dual specific ligands
are found in WO03002609, WO04003019 and WO04058821.
[0129] The dAbs of the disclosure can in one embodiment comprise a
dAb that binds serum albumin (SA) with a K.sub.D as determined by
surface plasmon resonance of 1 nM to 1, 2, 3, 4, 5, 10, 20, 30, 40,
50, 60, 70, 100, 200, 300, 400 or 500 .mu.M (i.e., .times.10-9 to
5.times.10-4), or 100 nM to 10 .mu.M, or 1 to 5 .mu.M or 3 to 70 nM
or 10 nM to 1, 2, 3, 4 or 5 .mu.M. For example 30 to 70 nM as
determined by surface plasmon resonance. In one embodiment, the
first dAb (or a dAb monomer) binds SA (e.g., HSA) with a K.sub.D as
determined by surface plasmon resonance of approximately 1, 50, 70,
100, 150, 200, 300 nM or 1, 2 or 3 .mu.M. In one embodiment, the
first and second dAbs are linked by a linker, for example a linker
of from 1 to 4 amino acids or from 1 to 3 amino acids, or greater
than 3 amino acids or greater than 4, 5, 6, 7, 8, 9, 10, 15 or 20
amino acids. In one embodiment, a longer linker (greater than 3
amino acids) is used to enhance potency (K.sub.D of one or both
dAbs in the antagonist).
[0130] In particular embodiments, the dAb binds human serum albumin
and competes for binding to albumin with a dAb selected from the
group consisting of
[0131] MSA-16, MSA-26 (See WO04003019 for disclosure of these
sequences, which sequences and their nucleic acid counterpart are
incorporated herein by reference and form part of the disclosure of
the present text),
[0132] DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474),
DOM7m-26 (SEQ ID NO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ
ID NO: 477), DOM7r-4 (SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479),
DOM7r-7 (SEQ ID NO: 480), DOM7r-8 (SEQ ID NO: 481), DOM7h-2 (SEQ ID
NO: 482), DOM7h-3 (SEQ ID NO: 483), DOM7h-4 (SEQ ID NO: 484),
DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQ ID NO: 486), DOM7h-7 (SEQ ID
NO: 487), DOM7h-22 (SEQ ID NO: 489), DOM7h-23 (SEQ ID NO: 490),
DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ ID NO: 492), DOM7h-26 (SEQ
ID NO: 493), DOM7h-21 (SEQ ID NO: 494), DOM7h-27 (SEQ ID NO: 495),
DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497), DOM7r-14 (SEQ
ID NO: 498), DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ ID NO: 500),
DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502), DOM7r-19 (SEQ
ID NO: 503), DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505),
DOM7r-22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507), DOM7r-24 (SEQ
ID NO: 508), DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510),
DOM7r-27 (SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512), DOM7r-29 (SEQ
ID NO: 513), DOM7r-30 (SEQ ID NO: 514), DOM7r-31 (SEQ ID NO: 515),
DOM7r-32 (SEQ ID NO: 516), DOM7r-33 (SEQ ID NO: 517) (See
WO2007080392 for disclosure of these sequences, which sequences and
their nucleic acid counterpart are incorporated herein by reference
and form part of the disclosure of the present text; the SEQ ID
No's in this paragraph are those that appear in WO2007080392),
[0133] dAb8 (dAb10), dAb 10, dAb36, dAb7r20 (DOM7r20), dAb7r21
(DOM7r21), dAb7r22 (DOM7r22), dAb7r23 (DOM7r23), dAb7r24 (DOM7r24),
dAb7r25 (DOM7r25), dAb7r26 (DOM7r26), dAb7r27 (DOM7r27), dAb7r28
(DOM7r28), dAb7r29 (DOM7r29), dAb7r29 (DOM7r29), dAb7r31 (DOM7r31),
dAb7r32 (DOM7r32), dAb7r33 (DOM7r33), dAb7r33 (DOM7r33), dAb7h22
(DOM7h22), dAb7h23 (DOM7h23), dAb7h24 (DOM7h24), dAb7h25 (DOM7h25),
dAb7h26 (DOM7h26), dAb7h27 (DOM7h27), dAb7h30 (DOM7h30), dAb7h31
(DOM7h31), dAb2 (dAbs 4,7,41), dAb4, dAb7, dAb11, dAb12 (dAb7 m12),
dAb13 (dAb 15), dAb15, dAb16 (dAb21, dAb7 m16), dAb17, dAb18,
dAb19, dAb21, dAb22, dAb23, dAb24, dAb25 (dAb26, dAb7 m26), dAb27,
dAb30 (dAb35), dAb31, dAb33, dAb34, dAb35, dAb38 (dAb54), dAb41,
dAb46 (dAbs 47, 52 and 56), dAb47, dAb52, dAb53, dAb54, dAb55,
dAb56, dAb7 m12, dAb7 m16, dAb7 m26, dAb7r1 (DOM 7r1), dAb7r3
(DOM7r3), dAb7r4 (DOM7r4), dAb7r5 (DOM7r5), dAb7r7 (DOM7r7), dAb7r8
(DOM7r8), dAb7r13 (DOM7r13), dAb7r14 (DOM7r14), dAb7r15 (DOM7r15),
dAb7r16 (DOM7r16), dAb7r17 (DOM7r17), dAb7r18 (DOM7r18), dAb7r19
(DOM7r19), dAb7h1 (DOM7h1), dAb7h2 (DOM7h2), dAb7h6 (DOM7h6),
dAb7h7 (DOM7h7), dAb7h8 (DOM7h8), dAb7h9 (DOM7h9), dAb7h10
(DOM7h10), dAb7h11 (DOM7h11), dAb7h12 (DOM7h12), dAb7h13 (DOM7h13),
dAb7h14 (DOM7h14), dAb7p1 (DOM7p1), and dAb7p2 (DOM7p2) (see
PCT/GB2008/000453 filed 8 Feb. 2008 and published as WO 2008/096158
for disclosure of these sequences, which sequences and their
nucleic acid counterpart are incorporated herein by reference and
form part of the disclosure of the present text). Alternative names
are shown in brackets after the dAb, dAb8 has an alternative name
which is dAb10 i.e., dAb8 (dAb10).
[0134] In certain embodiments, the dAb binds human serum albumin
and comprises an amino acid sequence that has at least about 80%,
or at least about 85%, or at least about 90%, or at least about
95%, or at least about 96%, or at least about 97%, or at least
about 98%, or at least about 99% amino acid sequence identity with
the amino acid sequence of a dAb selected from the group consisting
of
[0135] MSA-16, MSA-26,
[0136] DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474),
DOM7m-26 (SEQ ID NO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ
ID NO: 477), DOM7r-4 (SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479),
DOM7r-7 (SEQ ID NO: 480), DOM7r-8 (SEQ ID NO: 481), DOM7h-2 (SEQ ID
NO: 482), DOM7h-3 (SEQ ID NO: 483), DOM7h-4 (SEQ ID NO: 484),
DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQ ID NO: 486), DOM7h-7 (SEQ ID
NO: 487), DOM7h-22 (SEQ ID NO: 489), DOM7h-23 (SEQ ID NO: 490),
DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ ID NO: 492), DOM7h-26 (SEQ
ID NO: 493), DOM7h-21 (SEQ ID NO: 494), DOM7h-27 (SEQ ID NO: 495),
DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497), DOM7r-14 (SEQ
ID NO: 498), DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ ID NO: 500),
DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502), DOM7r-19 (SEQ
ID NO: 503), DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505),
DOM7r-22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507), DOM7r-24 (SEQ
ID NO: 508), DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510),
DOM7r-27 (SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512), DOM7r-29 (SEQ
ID NO: 513), DOM7r-30 (SEQ ID NO: 514), DOM7r-31 (SEQ ID NO: 515),
DOM7r-32 (SEQ ID NO: 516), DOM7r-33 (SEQ ID NO: 517) (the SEQ ID
No's in this paragraph are those that appear in WO2007080392),
[0137] dAb8, dAb 10, dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23,
dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30,
dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24,
Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11,
dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22,
dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34,
dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55,
dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,
dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17,
dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9,
dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and
dAb7p2.
[0138] For example, the dAb that binds human serum albumin can
comprise an amino acid sequence that has at least about 90%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99% amino acid sequence
identity with DOM7h-2 (SEQ ID NO:482), DOM7h-3 (SEQ ID NO:483),
DOM7h-4 (SEQ ID NO:484), DOM7h-6 (SEQ ID NO:485), DOM7h-1 (SEQ ID
NO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8 (SEQ ID NO:496), DOM7r-13
(SEQ ID NO:497), DOM7r-14 (SEQ ID NO:498), DOM7h-22 (SEQ ID
NO:489), DOM7h-23 (SEQ ID NO:490), DOM7h-24 (SEQ ID NO:491),
DOM7h-25 (SEQ ID NO:492), DOM7h-26 (SEQ ID NO:493), DOM7h-21 (SEQ
ID NO:494), DOM7h-27 (SEQ ID NO:495) (the SEQ ID No's in this
paragraph are those that appear in WO2007080392),
[0139] dAb8, dAb 10, dAb36, dAb7h21, dAb7h22, dAb7h23, Ab7h24,
Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11,
dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22,
dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34,
dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55,
dAb56, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10,
dAb7h11, dAb7h12, dAb7h13 and dAb7h14.
[0140] In certain embodiments, the dAb binds human serum albumin
and comprises an amino acid sequence that has at least about 80%,
or at least about 85%, or at least about 90%, or at least about
95%, or at least about 96%, or at least about 97%, or at least
about 98%, or at least about 99% amino acid sequence identity with
the amino acid sequence of a dAb selected from the group consisting
of DOM7h-2 (SEQ ID NO:482), DOM7h-6 (SEQ ID NO:485), DOM7h-1 (SEQ
ID NO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8 (SEQ ID NO:496),
DOM7h-22 (SEQ ID NO:489), DOM7h-23 (SEQ ID NO:490), DOM7h-24 (SEQ
ID NO:491), DOM7h-25 (SEQ ID NO:492), DOM7h-26 (SEQ ID NO:493),
DOM7h-21 (SEQ ID NO:494), DOM7h-27 (SEQ ID NO:495) (the SEQ ID No's
in this paragraph are those that appear in WO2007080392),
[0141] dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,
dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb38, dAb41, dAb7h1, dAb7h2,
dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13
and dAb7h14.
[0142] In more particular embodiments, the dAb is a V.sub..kappa.
dAb that binds human serum albumin and has an amino acid sequence
selected from the group consisting of
[0143] DOM7h-2 (SEQ ID NO:482), DOM7h-6 (SEQ ID NO:485), DOM7h-1
(SEQ ID NO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8 (SEQ ID NO:496)
(the SEQ ID No's in this paragraph are those that appear in
WO2007080392),
[0144] dAb2, dAb4, dAb7, dAb38, dAb41, dAb54, dAb7h1, dAb7h2,
dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13
and dAb7h14.
[0145] In more particular embodiments, the dAb is a V.sub.H dAb
that binds human serum albumin and has an amino acid sequence
selected from dAb7h30 and dAb7h31.
[0146] In more particular embodiments, the dAb is dAb7h11 or
dAb7h14.
[0147] In other embodiments, the dAb, ligand or antagonist binds
human serum albumin and comprises one, two or three of the CDRs of
any of the foregoing amino acid sequences, e.g., one, two or three
of the CDRs of dAb7h11 or dAb7h14.
[0148] Suitable Camelid V.sub.HH that bind serum albumin include
those disclosed in WO 2004/041862 (Ablynx N.V.) and in WO2007080392
(which V.sub.HH sequences and their nucleic acid counterpart are
incorporated herein by reference and form part of the disclosure of
the present text), such as Sequence A (SEQ ID NO:518), Sequence B
(SEQ ID NO:519), Sequence C (SEQ ID NO:520), Sequence D (SEQ ID
NO:521), Sequence E (SEQ ID NO:522), Sequence F (SEQ ID NO:523),
Sequence G (SEQ ID NO:524), Sequence H (SEQ ID NO:525), Sequence I
(SEQ ID NO:526), Sequence J (SEQ ID NO:527), Sequence K (SEQ ID
NO:528), Sequence L (SEQ ID NO:529), Sequence M (SEQ ID NO:530),
Sequence N (SEQ ID NO:531), Sequence 0 (SEQ ID NO:532), Sequence P
(SEQ ID NO:533), Sequence Q (SEQ ID NO:534), these sequence numbers
corresponding to those cited in WO2007080392 or WO 2004/041862
(Ablynx N.V.). In certain embodiments, the Camelid V.sub.HH binds
human serum albumin and comprises an amino acid sequence that has
at least about 80%, or at least about 85%, or at least about 90%,
or at least about 95%, or at least about 96%, or at least about
97%, or at least about 98%, or at least about 99% amino acid
sequence identity with ALB1 disclosed in WO2007080392 or with any
one of SEQ ID NOS:518-534, these sequence numbers corresponding to
those cited in WO2007080392 or WO 2004/041862.
[0149] In some embodiments, the dAb composition comprises an
anti-serum albumin dAb that competes with any anti-serum albumin
dAb disclosed herein for binding to serum albumin (e.g., human
serum albumin).
Conjugation to a Half-Life Extending Moiety (eg, Albumin)
[0150] In one embodiment, a (one or more) half-life extending
moiety (e.g., albumin, transferrin and fragments and analogues
thereof) is conjugated or associated with the VEGF-binding (or
IL-1, or TNF-.alpha. binding) dAb. Examples of suitable albumin,
albumin fragments or albumin variants for use in a VEGF (or IL-1,
or TNF-.alpha.)-binding format are described in WO 2005077042,
which disclosure is incorporated herein by reference and forms part
of the disclosure of the present text. In particular, the following
albumin, albumin fragments or albumin variants can be used in the
present disclosure: [0151] SEQ ID NO:1 (as disclosed in WO
2005077042, this sequence being explicitly incorporated into the
present disclosure by reference); [0152] Albumin fragment or
variant comprising or consisting of amino acids 1-387 of SEQ ID
NO:1 in WO 2005077042; [0153] Albumin, or fragment or variant
thereof, comprising an amino acid sequence selected from the group
consisting of: (a) amino acids 54 to 61 of SEQ ID NO:1 in WO
2005077042; (b) amino acids 76 to 89 of SEQ ID NO:1 in WO
2005077042; (c) amino acids 92 to 100 of SEQ ID NO:1 in WO
2005077042; (d) amino acids 170 to 176 of SEQ ID NO:1 in WO
2005077042; (e) amino acids 247 to 252 of SEQ ID NO:1 in WO
2005077042; (f) amino acids 266 to 277 of SEQ ID NO:1 in WO
2005077042; (g) amino acids 280 to 288 of SEQ ID NO:1 in WO
2005077042; (h) amino acids 362 to 368 of SEQ ID NO:1 in WO
2005077042; (i) amino acids 439 to 447 of SEQ ID NO:1 in WO
2005077042 (j) amino acids 462 to 475 of SEQ ID NO:1 in WO
2005077042; (k) amino acids 478 to 486 of SEQ ID NO:1 in WO
2005077042; and (l) amino acids 560 to 566 of SEQ ID NO:1 in WO
2005077042.
[0154] Further examples of suitable albumin, fragments and analogs
for use in a VEGF binding format are described in WO 03076567,
which disclosure is incorporated herein by reference and which
forms part of the disclosure of the present text. In particular,
the following albumin, fragments or variants can be used in the
present disclosure: [0155] Human serum albumin as described in WO
03076567, e.g., in FIG. 3 (this sequence information being
explicitly incorporated into the present disclosure by reference);
[0156] Human serum albumin (HA) consisting of a single
non-glycosylated polypeptide chain of 585 amino acids with a
formula molecular weight of 66,500 (See, Meloun, et al., FEBS
Letters 58:136 (1975); Behrens, et al., Fed. Proc. 34:591 (1975);
Lawn, et al., Nucleic Acids Research 9:6102-6114 (1981); Minghetti,
et al., J. Biol. Chem. 261:6747 (1986)); [0157] A polymorphic
variant or analog or fragment of albumin as described in Weitkamp,
et al., Ann. Hum. Genet. 37:219 (1973); [0158] An albumin fragment
or variant as described in EP 322094, e.g., HA(1-373, HA(1-388),
HA(1-389), HA(1-369), and HA(1-419) and fragments between 1-369 and
1-419; [0159] An albumin fragment or variant as described in EP
399666, e.g., HA(1-177) and HA(1-200) and fragments between
HA(1-X), where X is any number from 178 to 199.
[0160] Where a (one or more) half-life extending moiety (e.g.,
albumin, transferrin and fragments and analogues thereof) is used
to format the dAbs of the disclosure, it can be conjugated using
any suitable method, such as, by direct fusion, for example by
using a single nucleotide construct that encodes a fusion protein,
wherein the fusion protein is encoded as a single polypeptide chain
with the half-life extending moiety located N- or C-terminally to
the dAb. Alternatively, conjugation can be achieved by using a
peptide linker between moieties, e.g., a peptide linker as
described in WO 03076567 or WO 2004003019 (these linker disclosures
being incorporated by reference in the present disclosure to
provide examples for use in the present disclosure). Typically, a
polypeptide that enhances serum half-life in vivo is a polypeptide
which occurs naturally in vivo and which resists degradation or
removal by endogenous mechanisms which remove unwanted material
from the organism (e.g., human). For example, a polypeptide that
enhances serum half-life in vivo can be selected from proteins from
the extracellular matrix, proteins found in blood, proteins found
at the blood brain barrier or in neural tissue, proteins localized
to the kidney, liver, lung, heart, skin or bone, stress proteins,
disease-specific proteins, or proteins involved in Fc
transport.
[0161] The dAbs of the disclosure can be formatted as a fusion
protein that contains a first immunoglobulin single variable domain
that is fused directly to a second immunoglobulin single variable
domain. If desired such a format can further comprise a half-life
extending moiety. For example, the ligand can comprise a first
immunoglobulin single variable domain that is fused directly to a
second immunoglobulin single variable domain that is fused directly
to an immunoglobulin single variable domain that binds serum
albumin.
[0162] Generally the orientation of the polypeptide domains that
have a binding site with binding specificity for a target, and
whether the ligand comprises a linker, is a matter of design
choice. However, some orientations, with or without linkers, may
provide better binding characteristics than other orientations. All
orientations (e.g., dAb1-linker-dAb2; dAb2-linker-dAb1) are
encompassed by the disclosure are ligands that contain an
orientation that provides desired binding characteristics can be
easily identified by screening.
[0163] dAbs according to the disclosure, including dAb monomers,
dimers and trimers, can be linked to an antibody Fc region,
comprising one or both of C.sub.H2 and C.sub.H3 domains, and
optionally a hinge region. For example, vectors encoding ligands
linked as a single nucleotide sequence to an Fc region may be used
to prepare such polypeptides.
[0164] In embodiments of the disclosure the dAbs can be encoded by
codon optimized nucleotide sequences e.g., optimized for expression
by Pichia pastoris or E. coli e.g., as described in
WO2008149147.
EXEMPLIFICATION
Example 1
Topical Delivery of DOM15-26-593 (myc Tagged Anti-VEGF dAb) to Eyes
of Rabbits
[0165] DOM 15-26-593 can be selected and prepared as described in
WO2008149147 and has the amino sequence shown in FIG. 1a (SEQ ID NO
1).
[0166] Myc tagged DOM15-26-593 (--the Dom 15-26-593 dAb with amino
acid sequence shown in FIG. 1a (SEQ ID NO 1) was prepared and used
as a c-myc-tagged anti-VEGF dAb in this experiment) was prepared as
an endotoxin free preparation at a 2 mg/ml concentration formulated
in a 50 mM sodium acetate buffer (pH 7.0) supplemented with 104 mM
sodium chloride, 0.02% (w/v) Tween 80, 0.5% (w/v) Sodium caprate
and either 0.3% or 1.5% (w/v) Hydroxypropyl methylcellulose (HPMC).
Adult Chinchilla Bastard rabbits were obtained from Charles River,
Germany. The animals were allowed to acclimatise before use. The
left eyes of six female rabbits were dosed every 20 minutes over a
four hour period with 50 microlitres of 2 mg/ml solution of
anti-VEGF dAb. Each dose was placed in the subconjuntival sac.
Three rabbits received the anti-VEGF dAb formulated in 0.3% HPMC
and three with the drug formulated in 1.5% HPMC. Two hours after
the last dose the animals were culled. As close as possible to the
time that euthanasia had been confirmed both eyes from each animal
were enucleated. Each eye was washed in PBS to remove any excess
drug from the surface. Samples of aqueous and vitreous humour were
collected and stored frozen (-20.degree. C.) prior to analysis. The
samples of aqueous and vitreous humour were tested for
concentration of DOM15-26-593 (anti-VEGF-dAb) present using a
sandwich ELISA assay where the dAb was captured on recombinant
human VEGF protein coated plates and detected using an antibody
with specificity for a c-myc tag.
[0167] The VEGF dAb Elisa assay described above was performed as
follows:
[0168] The assay uses recombinant human VEGF (rVEGF, obtained from
R&D Systems) coated onto the surface of ELISA plates (obtained
from Nunc Immunosorb) to capture the VEGF dAb. The plates were
washed to remove any unbound dAb. Bound dAb was subsequently
detected using an antibody to the Myc tag of the VEGF dab (obtained
from 9E10, Sigma). Excess antibody was removed by washing and the
bound anti-myc antibody detected using an anti-mouse IgG peroxidase
conjugate (Sigma). The assay was developed using TMB solution and
stopped using acid. The signal from the assay is proportional to
the amount of dAb. The stages in the assay are summarized as
follows:
Coating the Plate:
[0169] 1. Prepare 20 mL of rVEGF at 0.25 .mu.g/mL (5 ml for each
ELISA plate) was prepared. This was done by for each plate, by
adding 25 .mu.L of stock VEGF to 5 mL of carbonate coating buffer
(0.2M sodium carbonate-bicarbonate coating buffer solution pH 9.4
(Pierce, Cat No: 28382)) and mixing by inversion. 2. 50 .mu.L of
rVEGF (0.25 .mu.g/mL) solution was added to each well of a Maxisorb
96-well ELISA plate using a multichannel pipette. 3. The plate was
covered with a plastic lid and stored at 4.degree. C. for
approximately 42 hours.
Washing and Blocking Plates:
[0170] 4. The plates were removed from 4.degree. C. storage 5. Each
plate was washed 6 times with PBS+0.1% Tween 20. 6. 100 .mu.L of
assay blocking buffer (1% BSA/PBS) was added to all wells of each
plate. 7. The plates were incubated at room temperature with
agitation for 1 hour.
Preparation of Samples and Standards:
[0171] 8. Standards and samples were diluted in assay diluent (0.1%
BSA/0.05% Tween20/PBS) while the plates were blocking. The standard
(reference material i.e Dom15-26-593) was serially (10-fold)
diluted to produce a log dilution curve.
Addition of Samples:
[0172] 9. Blocked plates were washed (as in 6 above). 10. 50 .mu.l
of diluted sample or standard was added to appropriate wells. 50
.mu.l/well assay diluent was added to wells to act as negative
controls. 11. Plates were incubated for 2 hours at room temperature
with agitation. 12. Plates were washed 6 times and blotted dry (as
in 6 above). 13. 50 .mu.L of anti-myc antibody was added (9E10
Sigma M5546) diluted 1:500 (in assay diluent: 0.1% BSA/0.05%
Tween20/PBS) to all wells (i.e., add 10 .mu.L of anti-myc antibody
(9E10) to 5 ml assay diluent for each plate). 14. Plates were
incubated on the rocker for at least 1 hour at room temperature.
15. Plates were washed 6 times and blotted dry (as in 6 above). 16.
50 .mu.L of anti-mouse Ig HRP at 1:10000 was added (Sigma A9309) to
all wells. (i.e., dilute stock antibody 1:10 by adding 54, of
anti-mouse Ig HRP antibody to 454 of assay diluent (0.1% BSA/0.05%
Tween20/PBS). For each plate add 5 .mu.L of the 1:10 diluted stock
to 5 ml assay diluent. 17. Plates were incubated on the rocker for
at least 1 hour at room temperature. 18. Plates were washed 6 times
and blotted dry (as in 6 above). 19. 50 .mu.L of TMB substrate was
added to all wells. As the development of this assay is quite fast,
it is advisable to add TMB to no more than 3 plates at a time. TMB
can be used directly from the fridge or at room temperature. 20.
The reaction was stopped (once sufficient colour has developed) by
adding 50 .mu.L of 1M HCl to every well. 21. Plates were read on a
96 well plate reader at 450 nm.
Results:
[0173] Results are shown in Table 1.
[0174] The dosing schedule was well tolerated with no signs of
redness, irritancy or abnormal animal behaviour observed. The
results of the ELISA assay carried out to investigate the level of
anti-VEGF dAb (DOM15-26-593) present in vitreous and aqueous humour
samples obtained from treated and contralateral (non-treated) eyes
showed that most of the dAb detected was present in the vitreous
humour of the treated eyes. The rabbit (animal 3) that had the
highest concentration in the vitreous humour also had detectable
levels of anti-VEGF dAb present in the aqueous humour of the
treated eye.
[0175] It was observed that the dAb formulated in 1.5% HPMC (which
was a more viscous solution) appeared to be retained in the eye
following each dose more effectively than the more fluid 0.3% HPMC
containing formulation. The rabbits dosed with the lower HPMC
concentration appeared to lose some of the later dosing material by
blinking it out.
TABLE-US-00001 TABLE 1 Concentrations of anti-VEGF dAb
(DOM15-26-593) present in vitreous and aqueous humour from
topically treated and contralateral eyes. Treatment consisted of 12
doses (each consisting of a 50 .mu.L volume of a 2 mg/ml solution)
administered in to the subconjunctival sac (every 20 minutes over a
4 hour period) Vitreous Rabbit Vitreous humour Aqueous Aqueous no.
humour (ng/ml) humour humour (ng/ml) (% (ng/ml) Contralateral
(ng/ml) Contralateral HPMC) Treated eye eye Treated eye eye 1 8
ng/ml ND* ND* ND* (1.5%) 2 5 ng/ml ND* ND* ND* (1.5%) 3 14 ng/ml
ND* 6 ng/ml ND* (1.5%) 4 5 ng/ml 4 ng/ml 5 ng/ml ND* (0.3%) 5 4
ng/ml 4 ng/ml ND* ND* (0.3%) 6 7 ng/ml ND* ND* ND* (0.3%) ND* = Not
Detected (</=2 ng/ml)
Conclusions:
[0176] The dose of anti-VEGF dAb was placed in the conjunctival
sac. It was expected that some of the dAb may penetrate through the
cornea and would subsequently be detected in the aqueous humour.
Surprisingly the majority of the anti-VEGF dAb detected was present
in the vitreous humour and this observation would be consistent
with the anti-VEGF dAb entering the eye by diffusion from the eye
socket across the sclera and choroidal membranes in order to enter
the posterior chamber.
[0177] Hydroxypropylcellulose (HPMC) had been included in the
formulation as a viscosity enhancer. The 1.5% formulation appeared
to be retained in the treated eye more effectively. The more fluid
0.3% formulation was less well retained and this may contribute to
movement of anti-VEGF dAb to the contralateral eyes observed in two
out of the three rabbits in this group.
Example 2
Pharmacokinetics of DOM15-26-593 Following Intravitreal
Administration to Eyes of Rabbits
[0178] An experiment was carried out to investigate the duration
that the anti-VEGF immunoglobulin single variable domain antibody
(anti-VEGF dAb) DOM15-26-593 was retained in the eye following
direct injection of the DOM15-26-593 into vitreous humour. Dom
15-26-593 dAb with amino acid sequence shown in FIG. 1a (SEQ ID NO
1) was prepared (2 mg/ml concentration formulated in a 50 mM sodium
acetate buffer (pH 7.0) supplemented with 104 mM sodium chloride,
0.02% (w/v) Tween 80), and used as a c-myc-tagged anti-VEGF dAb in
this experiment. Adult Chinchilla Bastard rabbits were obtained
from Charles River, Germany. The animals were allowed to
acclimatise before use. Each rabbit was anaesthetised and 10
microlitres of a 2 mg/ml solution (solution prepared as described
in Example 1) (total 20 .mu.g) of c-myc tagged anti-VEGF dAb
(DOM15-26-593) was injected directly into the vitreous humour of
the left eye. The rabbits were euthanased at various times (2, 24
and 30 hours) after the injection and both eyes were enucleated and
samples of aqueous and vitreous humour were collected. These
samples were stored frozen (-20.degree. C.) prior to analysis. The
samples of aqueous and vitreous humour were tested for
concentration of DOM15-26-593 present using a sandwich ELISA assay
where the dAb was captured on recombinant VEGF protein coated
plates and detected using an antibody with specificity for a c-myc
tag.
Results:
[0179] The concentrations of DOM15-26-593 (anti-VEGF dAb) are shown
in the Table 2 below:
TABLE-US-00002 TABLE 2 Concentrations of anti-VEGF dAb
(DOM15-26-593) present in vitreous and aqueous humour from
intravitreally dosed and contralateral eyes. Treatment consisted of
a single intravitreal injection (10 .mu.L of a 2 mg/ml solution) to
the left eye-rabbits were allowed to recover from anaesthesia and
were culled at 2, 24 and 30 hours after dosing. Vitreous Vitreous
Aqueous Aqueous humour humour humour humour Sample (ng/ml) (ng/ml)
(ng/ml) (ng/ml) Rabbit time Treated Contralateral Treated
Contralateral no. (hours) eye eye eye eye 1 2 121.48 ND* 0.75 ND* 2
2 115.43 ND* ND* ND* 3 24 88.61 0.10 1.58 ND* 4 24 157.79 ND* ND*
ND* 5 30 88.90 ND* ND* ND* 6 30 17.31 ND* 0.14 ND* ND* = not
detected (<0.1 ng/ml) Concentrations rounded to 2 decimal
places.
Conclusions:
[0180] The results of the experiment indicated that the
concentration of DOM15-26-593 was maintained at levels
approximating to the injected concentration at 24 hours after
dosing. The half-life of DOM15-26-593 in vitreous humour has not
yet been established but the demonstration that the domain antibody
is present in vitreous humour at 24 and 30 hours after dosing
suggests that a daily dosing regimen (for example using eye drops)
could be used to maintain therapeutic levels in the vitreous
humour.
[0181] Low concentrations of DOM15-26-593 (anti-VEGF dAb) were
detected in the aqueous humour of some of the treated eye. However,
there was minimal transfer of DOM15-26-593 to the contralateral
untreated eye.
Example 3
Rat Laser-Induced Choroidal Neovascularization (CNV) Model
[0182] Experimental choroidal neovascularization (CNV) was induced
unilaterally in groups of five 2-4 month old female Dark Agouti
(DA) rats. Laser light photocoagulation (PC) was used to rupture
Bruch's membrane of anaesthetised rats. Dye laser PC was performed
using a diode-pumped, 532 nm argon laser (Novus Omni, Coherent
Inc., Santa Clara, Calif.) attached to a slit lamp funduscope, and
a handheld planoconcave contact lens (Moorfields Eye hospital,
London, UK) applied to the cornea to neutralize ocular power. Five
lesions (532 nm, 150 mW, 0.2 second, 200 .mu.m diameter) were made
in a single eye of each experimental animal. Lesions were made in a
peripapillary distributed and standardized fashion centered on the
optic nerve at 500 .mu.m radius and avoiding major vessels. The
morphologic end point of the laser injury was identified as the
temporary appearance of a cavitation bubble, a sign associated with
the disruption of Bruch's membrane. Laser spots that did not result
in the formation of a bubble were excluded from the analysis.
Immediately after laser CNV induction, each animal was dosed
intravitreally with a 5 .mu.L volume (centered on the optic disc).
(This volume was selected as it was calculated that there would be
sufficient volume to cover the retinal area where the lesions had
been made). The dAb was formulated as a 2 mg/ml concentration in a
50 mM sodium acetate buffer (pH 7.0) supplemented with 104 mM
sodium chloride, 0.02% (w/v) Tween 80). The 5 .mu.L volume
contained 50 .mu.g of anti-VEGF dAb (DOM15-26-593; with the amino
acid sequence shown in FIG. 1a; SEQ ID NO 1), 50 .mu.g of anti-VEGF
DOM15-26-593-Fc fusion (with the amino acid sequence shown in FIG.
1b; SEQ ID NO 2) (or no compound (vehicle only, negative controls).
In vivo image data of CNV and associated leakage was generated
using confocal high-resolution SLO Fluorescein Angiography (0.2 ml
10% intra-abdominally injected Fluorescein Sodium, FS) and OCT
(Heidelberg Spectralis, Heidelberg, Germany) at 7 and 14 days after
lesion generation and injections. Baseline reflectance (at 488 nm
and 790 nm) and autofluorescence (ex. 488 nm, em. >498 nm)
images were made prior to injection of FS to help locate lesions in
fluorescein angiographic images. The artero-venous phase was
recorded immediately after FS injection. Fluorescein angiograms
were thereafter recorded one minute after injection and again four
minutes after injection. The effect of drug treatment was evaluated
by semi-quantitative assessment of late-phase fluorescein
angiography. Leakage was defined as the presence of a
hyperfluorescent lesion that increased in size with time in the
late-phase angiogram. The intensity and area of staining in
late-phase fluorescein angiography was graded by two examiners in a
masked fashion. When the two scores given for a particular lesion
did not coincide, the higher score was used for the analysis. Such
discrepant scoring was observed in <10% of lesions analyzed, and
the discrepancy was never by more than one grade. The study was
carried out in a masked manner and the substances were only
unmasked once all the data had been collected.
Results:
[0183] Results are shown below in Table 3.
[0184] At 7 and 14 days after induction of choroidal
neovascularization (CNV) using laser burns to rat retina,
fluoresecein angiography was used to observe each lesion. The
lesions were graded as follows: Grade 0=no leakage, Grade 1=Small
leakage, Grade 2=Medium leakage and Grade 3=Large leakage. The
results for groups of rats treated intravitreally with
anti-Vascular Endothelium Growth Factor domain antibody (anti-VEGF
dAb, DOM15-26-593), DOM15-26-593-FC fusion and for negative control
vehicle dosed groups are tabulated below. The results indicate that
treatment with anti-VEGF dAb (DOM15-26-593) or DOM15-26-593-FC
fusion reduced the extent of neovascularization and leakage
compared with control (sham-treated) rats.
TABLE-US-00003 TABLE 3 CNV lesion scores in rat eyes at 7 and 14
after induction by photocoagulation. Substance Lesions at Day 7
Lesions at Day 14 Anti-VEGF dAb-Fc** G0 = 19 G0 = 7
(DOM15-26-593-FC G1 = 1 G1 = 7 FUSION) G2 = 6 Anti-VEGF dAb** G0 =
14 G0 = 6 (DOM15-26-593) G1 = 6 G1 = 7 G2 = 5 G3 = 2 Negative
control G0 = 1 (Vehicle) G1 = 5 G1 = 5 G2 = 7 G2 = 11 G3 = 7 G3 = 4
**= data at both time points statistically significant from
controls (P < 0.05)
Conclusions:
[0185] The results indicated that DOM15-26-593-Fc fusion,
(anti-VEGF dAb-Fc) was efficacious in a rat model where
experimental choroidal neovascularization (CNV) induced by laser
photocoagulation of the RPE-choroid was characterized by
fluorescence angiography. Results for DOM15-26-593-Fc fusion were
significantly better than the control vehicle dosed group at both 7
and 14 days. This group appeared to retain slightly more activity
than the anti-VEGF dAb (DOM15-26-593) group. However, anti-VEGF dAb
(DOM15-26-593) was also efficacious (significantly better than the
control at both 7 and 14 days post-laser induced injury).
[0186] These results indicate that anti-VEGF dAb (DOM15-26-593) and
anti-VEGF dAb-Fc were efficacious in an experimental rat CNV model.
This demonstration of efficacy in an in vivo rodent model of
ophthalmic disease indicates that the domain antibodies may be
beneficial in the treatment of Choroidal Neovascularisation in
Age-related Macular Degeneration (AMD).
Example 4
Topical Delivery of an Anti-TNF-.alpha. Antibody, an Fc-Formatted
Anti-VEGF dAb, a Pegylated Anti-IL-1 dAb and an Anti-IL-1 dAb to
Rabbits
Method
[0187] Female, adult Chinchilla Bastard rabbits were obtained from
Charles River, Germany. The animals were allowed to acclimatise
before use. A blood sample was collected from the marginal ear vein
of every rabbit five days prior to commencement of dosing. The
blood was allowed to clot at room temperature and was centrifuged
(12000 rpm/2 minutes) to separate the serum. The serum was
transferred to fresh tubes and stored frozen (-20.degree. C.).
[0188] Preparation and selection of DOM 4-130-54 is described in WO
2007063311 and also WO2008149149. To prepare Dom 0400 the DOM
4430-54 dAb sequence is taken and is mutated such that a cysteine
at position 80 replaces the proline present in DOM 4-130-54, this
dAb is then attached to a 40 KDa linear PEG molecule (obtained
front NOF Corp., Europe) by standard maleimide coupling to the free
cysteine at position 80 of the dAb.
[0189] Domain antibodies (dAbs) with specificity for IL-1 in either
a naked format (DOM4-130-54; IL-1 naked dAb, 12.026 kDa; with amino
acid sequence shown in FIG. 3; SEQ ID NO 5) or a pegylated format
(DOM0400PEG; IL-1 pegylated dAb, 52.032 kDa; with amino acid
sequence shown in FIG. 2; SEQ ID NO 4) were formulated at 8.5 and
10.4 mg/ml respectively in 20 mM succinate, 5% sorbitol, pH 6.0. An
Fc-formatted .alpha.-VEGF dAb (VEGF 15-26-593 with amino acid
sequence sequence shown in FIG. 1b; SEQ ID NO 2) was formulated at
9.1 mg/ml in 50 mM phosphate, 1% L-arginine, 0.05 mM EDTA. 0.02%
polysorbate and 0.3% NaCl pH 7.0. Monoclonal antibody with
specificity for TNF-.alpha. (commercially available) was
reconstituted from a freeze-dried preparation at 10 mg/ml using
sterile distilled water. The left eyes of groups of four rabbits
were dosed five times daily (at 3 hour intervals) over a period of
4.2 days. Animals were allowed a rest period of 12 hours
(overnight) between each dosing day. Each dose consisted of 25
microlitres of a solution of the relevant compound placed under the
top eye lid. The animals were held still for at least 30 seconds
after dosing. At various times prior to and during the dosing
schedule samples of lachrymal (tear) fluid were collected by
placing a small absorbent strip of paper under the eyelid to absorb
some fluid. The area of paper impregnated with tear fluid was
placed into a tube containing 200 .mu.L of phosphate buffered
saline. The tube was centrifuged (12000 rpm/2 minutes), the paper
removed and the recovered sample stored frozen (-20.degree. C.)
prior to analysis.
[0190] One hour after the last dose a blood sample was collected
from the marginal ear vein of every rabbit. The blood was allowed
to clot so that serum could be separated and stored by the methods
described above. Immediately afterwards the animals were
euthanased. As close as possible to the time that euthanasia had
been confirmed both eyes from each animal were enucleated. Each eye
was washed in PBS to remove any excess drug from the surface.
Samples of aqueous and vitreous humour were collected and stored
frozen (-20.degree. C.) prior to analysis. Vitreous humour was
subjected to a single freeze/thaw cycle before being tested in an
assay. Eyes were dissected and the retina/choroid collected.
Retina/choroid samples were weighed and 100 microlitres of lysis
buffer (10 mM Tris pH 7.4; 0.1% SDS; with proteinase inhibitor
cocktail, (Roche)) was added to each 15 mg of retina/choroid
tissue. The samples were homogenised using ultrasonic disruption
(Covaris S2 SONOLAB.TM. Single) using a 2 minute cycle of repeated
high and low frequency bursts. Samples of retina/choroid were
centrifuged (12000 rpm/2 minutes) in a microfuge (Heraeus).
Supernatants were transferred to fresh tubes and stored frozen
(-20.degree. C.).
[0191] The drug content of each sample was tested and measured
using sandwich format ELISA assays. The .alpha.-TNF-.alpha.
antibody was captured using plates coated with recombinant human
TNF-.alpha. protein (Peprotech) and detected using an alkaline
phosphatase conjugated anti-human IgG (Fc specific) antibody
(Sigma). The IL-1 and pegylated IL-1 dAbs were captured using
plates coated with recombinant human IL-1 Receptor Type 1 Fc
(Axxora) and detected using Protein L-peroxidase (Sigma). VEGF-Fc
formatted dAb was captured using an in-house preparation of
recombinant VEGF protein and detected with an anti-human IgG (Fc
specific) Alkaline phosphatase conjungated antibody (Sigma).
Results
[0192] In all cases drug dosing was well tolerated with no signs of
redness, irritancy or abnormal animal behaviour.
[0193] The results of the various formats of domain antibodies and
for the .alpha.-TNF-.alpha. antibody in aqueous and vitreous humour
and in retina/choroid are shown in the following tables. Results
are shown as for the mean concentrations (from three independent
assays where each sample was tested in triplicate)+/-Standard
Deviation (shown in brackets)
TABLE-US-00004 TABLE 4 Concentration in Aqueous Humour after
topical dosing (ng/ml) 1 1 2 2 3 3 4 4 Left Right Left Right Left
Right Left Right .alpha.-TNF-.alpha. antibody ND ND 2.2 2.3 ND ND
ND ND (2.0) (0.7) VEGF-Fc dAb 7.6 ND 2.0 1.9 1.1 ND ND ND (7.5)
(1.3) (1.0) (0.4) IL-1 pegylated ND ND 1.7 1.7 ND ND ND ND
DOM0400PEG (2.9) (2.9) IL-1 131.2 3.0 2.3 2.3 1.7 1.9 1.6 1.3
DOM4-130-54 (53.0) (1.2) (0.3) (0.8) (0.3) (0.4) (0.2) (0.04) ND =
Not detected - below limit of quantitation (in at least 2 out of 3
repeat assays) Results in this table are rounded to one decimal
place.
TABLE-US-00005 TABLE 5 Concentration in Vitreous Humour after
topical dosing (ng/ml): 1 1 2 2 3 3 4 4 Left Right Left Right Left
Right Left Right .alpha.-TNF-.alpha. antibody ND ND ND ND ND ND ND
ND VEGF-Fc dAb ND ND ND ND ND ND ND ND IL-1 pegylated ND 6.7 15.0
1.7 ND ND 5.0 1.7 DOM0400PEG (11.5) (21.8) (2.9) (5.0) (2.9) IL-1
2.5 2.1 2.3 2.6 1.6 1.8 2.7 2.1 DOM4-130-54 (0.6) (0.5) (0.6) (0.9)
(0.1) (0.2) (2.7) (0.9) ND = Not detected - below limit of
quantitation (in at least 2 out of 3 repeat assays) Results in this
table are rounded to one decimal place.
TABLE-US-00006 TABLE 6 Concentration in Retina/Choroid after
topical dosing (ng/ml in samples where 100 .mu.L lysis buffer has
been added to 15 mg of tissue): 1 1 2 2 3 3 4 4 Left Right Left
Right Left Right Left Right .alpha.-TNF-.alpha. antibody ND ND 8.9
ND 7.3 ND ND ND (3.6) (2.6) VEGF-Fc dAb 7.5 29.5 1.1 2.0 7.6 2.2
0.6 ND (5.8) (14.2) (0.7) (0.4) (1.2) (0.001) (0.2) IL-1 pegylated
3.3 3.3 11.7 5.0 5.0 3.3 10 5.0 DOM0400PEG (2.9) (2.9) (7.6) (5.0)
(5.0) (2.9) (5.0) (5.0) IL-1 91.9 4.6 3.4 2.7 5.3 3.4 3.9 3.3
DOM4-130-54 (90.3) (1.4) (0.5) (1.3) (0.5) (0.3) (1.2) (0.7) ND =
Not detected - below limit of quantitation (in at least 2 out of 3
repeat assays) Results in this table are rounded to one decimal
place.
[0194] Lachrymal fluid (tear) samples were collected from rabbits
just prior to doses 20 and 21 results for concentrations of drug
present are shown in Tables 4 and 5 respectively. Material dosed
was detected in all of the left (dosed) eyes (although there was
quite a lot of variation in concentration detected between
individual rabbits) and some transfer to most of the contralateral
(right not dosed) eyes had also occurred. Dosing material was still
present in the eye at 12 hours after dose 20. DOM0400PEG (pegylated
IL-1 dAb) and VEGF-Fc (15-26-593) appeared to be retained in tears
at higher concentrations over the 12 hour period between doses 20
and 21 than the naked IL-1 dAb (DOM4-130-54).
[0195] The results for the concentrations of the various formats of
domain antibodies and for the antibody in lachrymal fluid (tears)
are shown in the following tables (only the Left eye was
dosed):
TABLE-US-00007 TABLE 7 Concentration in Lachrymal fluid samples
collected prior to dose 20 (3 hours after previous dose)
(.mu.g/ml). Standard deviation is in brackets: 1 1 2 2 3 3 4 4 Left
Right Left Right Left Right Left Right .alpha.-TNF-.alpha. antibody
13.0 18.6 211.9 ND 249.3 0.5 6.7 ND (1.0) (2.2) (46.2) (40.2) (0.3)
(0.7) VEGF-Fc dAb 28.6 ND 47.7 ND 19.4 0.3 32.5 0.2 (4.7) (7.0)
(1.2) (0.06) (4.6) (0.02) IL-1 pegylated 1.0 0.3 3.4 0.5 25.0 0.3
21.1 ND DOM0400PEG (0.2) (0) (0.6) (0.2) (11.1) (0) (6.4) IL-1 7.5
0.8 3.9 0.2 3.8 ND 5.7 0.3 DOM4-130-54 (4.3) (0.1) (0.7) (0.02)
(0.6) (1.8) (0.06) Results in this table are rounded to one decimal
place.
TABLE-US-00008 TABLE 8 Concentration in Lachrymal fluid samples
collected prior to dose 21 (12 hours after previous dose):
Concentration (.mu.g/ml). Standard deviation is in brackets: 1 1 2
2 3 3 4 4 Left Right Left Right Left Right Left Right
.alpha.-TNF-.alpha. antibody 17.2 5.3 81.7 0.3 2.9 ND 24.2 ND (2.4)
(3.0) (6.1) (0.2) (0.3) (1.2) VEGF-Fc dAb 21.2 1.3 12.5 ND 6.7 0.1
27.0 0.06 (1.3) (1.9) (0.7) (0.5) (0.03) (5.0) (0.02) IL-1
pegylated 0.4 ND 43.1 0.3 102.3 44.2 1.5 0.3 DOM0400PEG (0.2)
(15.8) (0) (3.2) (39.1) (2.6) (0) IL-1 5.6 0.9 3.6 ND 2.1 ND 3.0
0.1 DOM4-130-54 (3.4) (0.1) (0.9) (0.2) (0.7) (0.01) Results in
this table are rounded to one decimal place.
[0196] The results for the concentrations of the various formats of
domain antibodies and for the antibody in prebleeds and in serum
are shown in the following table:
TABLE-US-00009 TABLE 9 Concentration in prebleeds and serum
collected just prior to euthanasia Concentration (ng/ml) with
Standard Deviation in brackets: 1 1 2 2 3 3 4 4 Prebleed Serum
Prebleed Serum Prebleed Serum Prebleed Serum .alpha.-TNF-.alpha. ND
ND 62.6 ND 220.1* 102.7 ND 152.4 antibody (49.0) (115.0) (115.6)
(130.4) VEGF-Fc dAb ND 2.1 1.8 16.9 ND 16.9 ND 15.6 (1.8) (1.3)
(6.5) (15.6) (0.5) IL-1 pegylated ND 3.33 ND 3.33 ND 3.33 ND 3.33
DOM0400PEG (5.8) (5.8) (5.8) (5.8) IL-1 3.5 3.2 3.5 3.4 3.8 3.2 4.5
4.2 DOM4-130-54 (0.9) (0.6) (1.2) (1.2) (1.7) (0.9) (1.9) (1.9)
Results in this table are rounded to one decimal place. *Rabbit 3
in the .alpha.-TNF-.alpha. antibody treated group had a prebleed
that was red in colour owing to lysis and this may have contributed
to the apparently high result.
Example 5
Topical Delivery of .alpha.-TNF-.alpha.R1 dAb
Methods
[0197] Adult male Chinchilla Bastard rabbits were obtained from
Charles River, Germany. The animals were allowed to acclimatise
before use. A blood sample was collected from the marginal ear vein
of every rabbit seven days prior to commencement of dosing. The
blood was allowed to clot at room temperature before centrifugation
(12000 rpm/2 minutes) to separate the serum. The serum was
transferred to fresh tubes and stored frozen (-20.degree. C.).
[0198] A domain antibody (dAb) with specificity for TNF-.alpha.R1
(Dom 1h-131-206 with amino acid sequence shown in FIG. 4; SEQ ID NO
6) was formulated in phosphate buffered saline at 10 mg/ml. The
left eyes of a group of four rabbits were dosed 10 times on a
single day (at hourly intervals). Each dose consisted of 50
microlitres of 10 mg/ml .alpha.-TNF-.alpha.R1 dAb solution placed
under the top eye lid. The animals were held still for at least 30
seconds after dosing. At various times prior to and during the
dosing schedule samples of lachrymal (tear) fluid were collected by
placing a small absorbent strip of paper under the eyelid to absorb
some fluid. The area of paper impregnated with tear fluid was
placed into a tube containing 200 .mu.L of phosphate buffered
saline. The tube was centrifuged (12000 rpm/2 minutes), the paper
removed and the recovered sample stored frozen (-20.degree. C.)
prior to analysis.
[0199] One hour after the last dose a blood sample was collected
from the marginal ear vein of every rabbit. The blood was allowed
to clot so that serum could be separated by the methods described
above. Immediately afterwards the animals were euthanased. As close
as possible to the time that euthanasia had been confirmed both
eyes from each animal were enucleated. Each eye was washed in PBS
to remove any excess drug from the surface. Samples of aqueous and
vitreous humour were collected and stored frozen (-20.degree. C.)
prior to analysis. Vitreous humour was subjected to a single
freeze/thaw cycle before being tested in an assay. Eyes were
dissected and the retina/choroid was collected. The retina/choroid
samples were weighed and 900 microlitres of lysis buffer (10 mM
Tris pH 7.4; 0.1% SDS; with proteinase inhibitor cocktail, (Roche))
was added to each sample. The samples were homogenised using
ultrasonic disruption (Covaris S2 SONOLAB.TM. Single) using a 2
minute cycle of repeated high and low frequency bursts. Samples of
retina/choroid were centrifuged (12000 rpm/2 minutes) in a
microfuge (Heraeus). Supernatants were transferred to fresh tubes
and stored frozen (-20.degree. C.). The samples were tested for
concentration of .alpha.-TNF-.alpha.R1 dAb by a sandwich ELISA
assay where the dAb was captured using plates coated with
recombinant human TNF R1/TNFRSF1A/Fc chimera (R+D Systems) and
detected with specificity for human IgG (F(ab)2) fragments
(Thermo). This antibody was not conjugated, so an
anti-goat/sheep-HRP reagent (Sigma) was used to detect bound
antibody.
Results
[0200] Drug dosing was well tolerated with no signs of redness,
irritancy or abnormal animal behaviour observed.
[0201] Concentrations of .alpha.-TNF-.alpha.R1 dAb in ocular fluids
and serum are shown for samples tested in triplicate. The
.alpha.-TNF-.alpha.R1 dAb was detected in all of the ocular samples
tested.
TABLE-US-00010 TABLE 10 Concentrations of .alpha.-TNF-.alpha.R1 dAb
in ocular samples: Aqueous Humour Vitreous Humour Retina/Choroid
(ng/ml +/- s.e.) (ng/ml +/- s.e.) (ng/100 mg +/- s.e.) Rabbit 1 2.6
+/- 0.4 0.7 +/- 0.2 209.1 +/- 7.5 Left (dosed) eye Rabbit 1 0.7 +/-
0.3 0.1 +/- 0.1 3.5 +/- 0.7 Right (un- treated) Rabbit 2 10.8 +/-
5.4 0.4 +/- 0.2 229.4 +/- 42.0 Left (dosed) eye Rabbit 2 0.3 +/-
0.1 0.4 +/- 0.1 63.9 +/- 1.8 Right (un- treated) Rabbit 3 43.6 +/-
6.0 4.3 +/- 2.6 1086.6 +/- 20.7 Left (dosed) eye Rabbit 3 12.3 +/-
1.6 1.3 +/- 0.4 25.1 +/- 0.6 Right (un- treated) Rabbit 4 12.3 +/-
1.4 0.7 +/- 0.2 88.1 +/- 4.2 Left (dosed) eye Rabbit 4 0.5 +/- 0.1
0.2 +/- 0.03 74.3 +/- 4.3 Right (un- treated) Results in Table 10
are rounded to one decimal place. S.E. = Standard Error
[0202] Lachrymal fluid (tear) samples were collected from rabbits
just prior to doses 2, 6, 10 together with 1 hour following the
final dose and concentrations of .alpha.-TNF-.alpha.R1 dAb detected
in the samples are shown in Table 8. .alpha.-TNF-.alpha.R1 dAb was
detected in all of the left (dosed) eyes and some transfer to most
of the contralateral (right not dosed) eyes had also occurred.
TABLE-US-00011 TABLE 11 Concentrations of .alpha.-TNF-.alpha.R1 dAb
in lachrymal fluid (tear) samples: Left (dosed) eye Right
(contralateral) eye (Mean data for 4 rabbits) (Mean data for 4
rabbits) (.mu.g/ml +/- S.E.) (.mu.g/ml +/- S.E.) Prior to Second
dose 19.74 +/- 6.27 0.1 +/- 0.01 Prior to Sixth dose 20.75 +/- 5.15
1.77 +/- 0.47 Prior to Tenth dose 21.83 +/- 5.81 1.11 +/- 0.41 1 h
after Tenth dose 27.03 +/- 6.98 0.60 +/- 0.36 Results in Table 11
are rounded to two decimal places. S.E. = Standard Error
[0203] Blood was collected for serum prior to the first dose and at
the time of euthanasia. The resulting data is shown in Table 9. Low
concentrations of .alpha.-TNF-.alpha.R1 dAb were detected in serum
obtain from each of the four rabbits 1 hour following the final
dose.
TABLE-US-00012 TABLE 12 Concentrations of .alpha.-TNF-.alpha.Rl dAb
in serum samples: Prior to first dose 1 h following final dose
(ng/ml +/- s.e.) (ng/ml +/- s.e.) Rabbit 1 ND 0.36 +/- 0.21 Rabbit
2 ND 0.44 +/- 0.18 Rabbit 3 ND 0.51 +/- 0.08 Rabbit 4 ND 1.02 +/-
0.12 Results in Table 12 are rounded to two decimal places. ND =
Not detected S.E. = Standard Error
Sequence CWU 1
1
61116PRTArtificial Sequenceartficial sequence derived from homo
sapiens sequence 1Glu Val Gln Leu Leu Val Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Lys Ala Tyr 20 25 30Pro Met Met Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile Ser Pro Ser Gly Ser Tyr
Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp Pro Arg
Lys Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser
Ser 1152343PRTArtificial Sequenceartficial sequence derived from
homo sapiens sequence 2Glu Val Gln Leu Leu Val Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Lys Ala Tyr 20 25 30Pro Met Met Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile Ser Pro Ser Gly Ser
Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp Pro
Arg Lys Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val
Ser Ser Ala Ser Thr His Thr Cys Pro Pro Cys Pro Ala Pro 115 120
125Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
130 135 140Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val145 150 155 160Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp 165 170 175Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 180 185 190Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 195 200 205Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 210 215 220Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg225 230 235
240Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
245 250 255Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp 260 265 270Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys 275 280 285Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser 290 295 300Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser305 310 315 320Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 325 330 335Leu Ser Leu
Ser Pro Gly Lys 3403225PRThomo sapiens 3Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro1 5 10 15Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 20 25 30Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 35 40 45Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 50 55 60Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val65 70 75 80Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 85 90
95Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
100 105 110Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 115 120 125Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 130 135 140Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu145 150 155 160Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 165 170 175Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 180 185 190Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 195 200 205Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210 215
220Lys2254108PRTArtificial Sequenceartficial sequence derived from
homo sapiens sequence 4Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Ile Tyr Leu Asn 20 25 30Leu Asp Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Asn Phe Gly Ser Glu Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Tyr Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Cys65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Pro Ser Phe Tyr Phe Pro Tyr 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 1055108PRTArtificial
Sequenceartficial sequence derived from homo sapiens sequence 5Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Tyr Leu Asn
20 25 30Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Asn Phe Gly Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Pro Ser
Phe Tyr Phe Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 1056119PRTArtificial Sequenceartficial sequence derived
from homo sapiens sequence 6Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ala His Glu 20 25 30Thr Met Val Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser His Ile Pro Pro Asp Gly
Gln Asp Pro Phe Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95Ala Leu Leu
Pro Lys Arg Gly Pro Trp Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 115
* * * * *