WO2010060768A1 - Polypeptides, domaines variables d'anticorps et antagonistes - Google Patents
Polypeptides, domaines variables d'anticorps et antagonistes Download PDFInfo
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- WO2010060768A1 WO2010060768A1 PCT/EP2009/064654 EP2009064654W WO2010060768A1 WO 2010060768 A1 WO2010060768 A1 WO 2010060768A1 EP 2009064654 W EP2009064654 W EP 2009064654W WO 2010060768 A1 WO2010060768 A1 WO 2010060768A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/06—Antiglaucoma agents or miotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/241—Tumor Necrosis Factors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
- C07K16/245—IL-1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
Definitions
- the present invention 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.
- dAbs immunoglobulin single variable domains
- a difficulty of treating ocular diseases and conditions has been the inefficiency of delivering therapeutic agents to the eye.
- a drug is delivered to the eye it very often clears extremely rapidly from the ocular tissues.
- 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).
- 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.
- Polypeptides and peptides have become increasingly important agents for use as medical, therapeutic and diagnostic agents.
- 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.
- 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.
- 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.
- 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.
- VPF vascular permeability factor
- 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)
- VEGF vascular proliferative diseases
- AMD Age related macular degeneration
- TNF- ⁇ Tuour Necrosis Factor- ⁇
- TNF- ⁇ Tuour Necrosis Factor- ⁇
- ophthalmic inflammatory conditions such as uveitis and AMD
- 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.
- Interleukin 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-IRl, CD121a, p80), which transduces signal into cells upon binding IL-I, and Interleukin 1 Receptor type 2 (IL-IRl, CDwI 2 Ib), which does not transduce signals upon binding IL-I and acts as an endogenous regulator of IL-I.
- IL-IRl Interleukin 1 Receptor type 1
- IL-IRl Interleukin 1 Receptor type 2
- Another endogenous protein that regulates the interaction of IL-I with IL-IRl is Interleukin 1 receptor antagonist (IL- Ira).
- IL- Ira binds IL-IRl, but does not activate IL-IRl to transduce signals.
- Signals transduced through IL-IRl upon binding IL-I e.g., IL- l ⁇ or IL- l ⁇
- IL-I e.g., IL- l ⁇ or IL- l ⁇
- signals transduced through IL-IRl upon binding of IL-I can lead to local or systemic inflammation, and the elaboration of additional inflammatory mediators (e.g., IL-6, 11-8, TNF). Accordingly, the interaction of IL-I with IL-IRl has been implicated in the pathogenesis of ocular diseases.
- IL-IRl Interleukin 1 Receptor Type 1
- IL- Ira Interleukin 1 Receptor Type 1
- the invention 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-I, or TNF- ⁇ ), e.g. at the site of delivery, for administration to the eye.
- a desired target molecule e.g. VEGF, IL-I, or TNF- ⁇
- 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-I , or TNF- ⁇ , TNFRl , TNFR2, IL-Ir), 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.
- a desired target molecule e.g. VEGF, IL-I , or TNF- ⁇ , TNFRl , TNFR2, IL-Ir
- AMD Age related macular degeneration
- Uveitis glaucoma
- dry eye dry eye
- diabetic retinopathy diabetic macular oedema
- 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, f ⁇ cain
- 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.
- the protease is one found in the eye and/or tears.
- proteases found in the eye include caspases, calpains, matric metalloproteases, disintegrin, metalloproteinases (ADAMs) and ADAM with thrombospondin motifs, the proteosomes, tissue plasminogen activator, secretases, cathepsin B and D, cystatin C, serine protease PRSSl, ubiquitin proteosome pathway (UPP).
- the protease is a non-bacterial protease.
- the protease is an animal, e.g., mammalian, e.g., human, protease.
- the composition can be delivered to different regions of the eye, e.g. 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 (e.g. VEGF, IL-I, or TNF- ⁇ ) in these parts of the eye.
- target molecules e.g. VEGF, IL-I, or TNF- ⁇
- the composition can also be delivered to the peri-ocular region of the eye.
- the target molecule may for example be VEGF, IL-I, or TNF- ⁇ 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 lachrymal glands, e.g. the target can be IL- 1, IL-17 or TNF receptor such as TNFRl, TGFbeta, IL-6, IL-8, IL-21, IL-23, CD20, Nogo-a, Myelin associated glycoprotein (MAG) or Beta amyloid.
- a target molecule present in the eye, for example on the surface of the eye, within the eye or in tear ducts or lachrymal glands
- the target can be IL- 1, IL-17 or TNF receptor such as TNFRl, TGFbeta, IL-6, IL-8, IL-21, IL-23, CD20, Nogo-a, Myelin associated glycoprotein (MAG) or Beta amyloid.
- the invention 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-I, or TNF- ⁇ .
- 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.
- the invention further provides a method of delivering a composition directly to the eye which compises administering said composition to the eye by a method selected from: intra-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.
- a method of delivering a composition directly to the eye which compises administering said composition to the eye by a method selected from: intra-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.
- 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).
- an ocular penetration enhancer e.g. sodium caprate
- a viscosity enhancer e.g. Hydroxypropylmethylcellulose (HPMC).
- HPMC Hydroxypropylmethylcellulose
- the invention 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-I, or TNF- ⁇ ), and also (b) an ocular penetration enhancer and /or (c) a viscosity enhancer e.g. for topical delivery to the eye.
- 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.
- 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 figure Ia: SEQ ID NO 1).
- the percent identity is at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% or 100%.
- 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.
- it may have an amino acid sequence identical to the amino acid sequence of DOM 15-26-593-Fc fusion (shown in Figure Ib: 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 Figure Ib: SEQ ID NO 2.
- 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 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).
- the immunoglobulin single variable domain to be delivered to the eye can be any one of the anti TNFRl dAbs disclosed in WO 2008/149144, or WO 2008/149148.
- the immunoglobulin single variable domain which binds to ⁇ -TNF- ⁇ Rl 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 Ih- 131-206 (shown in f ⁇ gure 4; SEQ ID NO 6). Preparation and selection of Dom lh-131-206 is described in WO2008149148.
- the immunoglobulin single variable domain to be delivered to the eye can be any one of the anti-IL-lRl dAbs disclosed in WO 2008/149149.
- the immunoglobulin single variable domain which binds to IL-I 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 figure 3; SEQ ID NO 5); or to (b) the amino acid sequence of DOM 0400 PEG (shown in figure 2; SEQ ID NO 4).
- DOM 4-130-54 Preparation and selection of DOM 4-130-54 is described in WO 2007063311 and also WO2008149149.
- 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 40KDa linear PEG molecule (obtained from NOF Corporation, Europe) by standard maleimide coupling to the free cysteine at position 80 of the dAb.
- the invention 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.
- AMD Age related Macular Degeneration
- Uveitis glaucoma dry eye
- diabetic retinopathy diabetic macular oedema
- the invention also provides a composition comprising or consisting of an immunoglobulin single variable domain e.g. a VEGF, IL-I, or TNF- ⁇ 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.
- an eye condition or disease e.g. AMD, Uveitis glaucoma, dry eye, diabetic retinopathy, or diabetic macular oedema.
- the immunoglobulin single variable domain for delivery to the eye can be one which is not the amino acid sequence of DOM 15-26-593 (shown in Figure Ia; SEQ ID NO 1) or which is not the amino acid sequence of DOM15-26-593-Fc fusion (shown in Figure Ib; SEQ ID NO 2).
- 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.
- the immunoglobulin single variable domain for delivery to the eye can be one which is not the amino acid sequence of Domlh-131- 511, Domlh-131-201, Domlh-131-202, Domlh-131-203, Domlh-131-204, Domlh- 131-205 as disclosed in PCT/GB2008/050400.
- the immunoglobulin single variable domain for delivery to the eye can be one which is not the amino acid sequence of Dom4-130- 202 as disclosed in PCT/GB2008/050406.
- the immunoglobulin single variable domain for delivery to the eye can be one which is not the amino acid sequence of Domlh-131- 206 as disclosed in PCT/GB2008/050405.
- the single immunoglobulin variable domains (dAbs) for ocular delivery 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.
- 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) 2 , F(ab') 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).
- 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 aff ⁇ body.
- 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 aff ⁇ body.
- 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.
- Formatted dAbs can have a molecular weight which is e.g. between 30KDa 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.
- 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.
- a single immunoglobulin variable domain or "dAb" in an antagonist or ligand of the invention, it is contemplated that the skilled addressee can use a domain that comprises the CDRs of a dAb that binds e.g. VEGF, IL-I, or TNF- ⁇ (e.g., CDRs grafted onto a suitable protein scaffold or skeleton, e.g.
- an aff ⁇ body, 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-I, or TNF- ⁇ e.g., wherein the domain is selected from an aff ⁇ body, 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.
- 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.
- the invention provides a protease resistant immunoglobulin single variable domain comprising e.g. a VEGF, IL-I, or TNF- ⁇ 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°C for time (t) of at least one hour; or
- 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.
- 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 1,6000 to 160,000 protease: variable domain.
- 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.
- time (t) is or is about one, three or 24 hours or overnight (e.g., about 12-16 hours).
- 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.
- 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.
- the protease is selected from trypsin, elastase, leucozyme and pancreatin.
- the protease is trypsin.
- 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.
- 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.
- variable domain is resistant to trypsin and/or at least one other protease selected from elastase, leucozyme and pancreatin.
- 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.
- variable domain is displayed on bacteriophage when incubated under condition (i) or (ii) for example at a phage library size of 10 6 to 10 13 , e.g. 10 8 to 10 12 replicative units (infective virions).
- variable domain specifically binds VEGF, IL-I, or TNF- ⁇ following incubation under condition (i) or (ii), e.g. assessed using BiaCore TM or ELISA, e.g. phage ELISA or monoclonal phage ELISA.
- 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). In one embodiment, the variable domains may have an OD 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).
- ELISA e.g. phage ELISA or monoclonal phage ELISA
- variable domains display (substantially) a single band in gel electrophoresis, e.g. following incubation under condition (i) or (ii).
- an agent can be locally administered to the eye via an implantable delivery device.
- the invention provides an implantable delivery device containing e.g. the VEGF, IL-I, or TNF- ⁇ dAb, for ocular delivery
- the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an immunoglobulin single variable domain (e.g. VEGF, IL-I, or TNF- ⁇ dAb), and a pharmaceutically or physiologically acceptable carrier, excipient or diluent for ocular delivery.
- an immunoglobulin single variable domain e.g. VEGF, IL-I, or TNF- ⁇ dAb
- Fig. Ia Depicts the amino acid sequence of DOM 15-26-593
- Fig. Ib Depicts the amino acid sequence of DOM 15-26-593-Fc fusion
- Fig. Ic Depicts the amino acid sequence of an antibody Fc
- Fig. 2 Depicts the amino acid sequence of DOM 0400 PEG (a pegylated anti-
- Fig. 3 Depicts the amino acid sequence of DOM4-130-54 (An anti-ILl dAb)
- Fig. 4 Depicts the amino acid sequence of Dom Ih- 131-206 (An anti TNF alpha
- VEGF vascular endothelial growth factor
- anti-VEGF antagonist 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.
- peptide refers to about two to about 50 amino acids that are joined together via peptide bonds.
- 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.
- a peptide or polypeptide e.g. a domain antibody (dAb)
- dAb domain antibody
- 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.
- target ligand refers to a ligand which is specifically or selectively bound by a polypeptide or peptide.
- 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.
- an antibody refers to IgG, IgM, IgA, IgD or IgE or a fragment
- 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') 2 fragment), a single antibody variable domain (e.g., a dAb, V H , V HH , V 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 HH ).
- a Fv fragment e.g., single chain Fv (scFv), a disulfide bonded Fv
- Fab fragment e.g
- immunoglobulin single variable domain refers to an antibody variable domain (V H , V HH , V 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 HH dAbs.
- Camelid V 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 HH may be humanized.
- 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.
- 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.
- 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.
- 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.
- 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 invention can be 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.
- a ligand specific for human serum albumin (HSA) 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.
- the half- life is increased by 10%, 20%, 30%, 40%, 50% or more. Increases in the range of 2x, 3x, 4x, 5x, 10x, 2Ox, 30x, 4Ox, 5Ox or more of the half-life are possible.
- hydrodynamic size refers to the apparent size of a molecule
- 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.
- 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.
- 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 experiments to determine competition between first and second binding domains. Calculations of "homology” or "identity” or “similarity” between two sequences
- 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).
- 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.
- 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, 774:187-188 (1999).
- the invention in one embodiment relates to dAbs, e.g. anti-VEGF dAbs, TNFRl dAbs, IL-I 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, TNFRl or IL-I .
- 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.
- protease resistant peptides and polypeptides generally retain biological activity.
- 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.
- 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 e.g., trypsin
- 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.
- 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 a variable domain, antagonist, peptide or polypeptide that is not protease resistant.
- protease resistance correlates with an improved storage stability, for example improved over an variable domain, antagonist, peptide or polypeptide that is not protease resistant.
- 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.
- protease resistant peptides or polypeptides e.g. dAbs that can comprise, if desired, other suitable selection methods.
- the methods described herein can be employed at any desired point in the program, such as before or after other selection methods are used.
- the dAb for ocular delivery is selected for resistance to degradation by trypsin, elastase or leucozyme and specifically binds VEGF.
- 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.
- the protease resistant dAb is not substantially degraded when incubated at 37 0 C in a 0.04% (w/w) solution of protease for a period of at least about 2 hours.
- the protease resistant dAb is not substantially degraded when incubated at 37 0 C in a 0.04% (w/w) solution of protease for a period of at least about 3 hours.
- the protease resistant dAb is not substantially degraded when incubated at 37 0 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.
- a method of producing a repertoire of protease resistant peptides or polypeptides 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.
- protease resistant peptides or polypeptides e.g., dAbs.
- 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
- 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.
- a method of producing a repertoire of protease resistant polypeptides that comprise anti-VEGF dAbs 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 na ⁇ ve 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.
- a protease resistant peptide or polypeptide can be selected, isolated and/or recovered from a repertoire or library (e.g. , in a display system) using any suitable method.
- a protease resistant polypeptide is selected or isolated based on a selectable characteristic (e.g., physical characteristic, chemical characteristic, functional characteristic).
- 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
- catalytic activity See, e.g., Tomlinson et ⁇ l., WO 99/20749; WO 01/57065; WO 99/58655.
- the selection is based on specific binding to VEGF.
- 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.
- 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.
- 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.
- 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.
- 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.
- ligand e.g. , generic ligand, target ligand
- 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.
- peptides or polypeptides e.g., a repertoire that has been incubated with protease
- 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.
- 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).
- 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.
- 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.
- 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.
- protease resistant dAbs generally bind their target ligand with high affinity.
- KD K off (kd)/K on (ka) as determined by surface plasmon resonance
- 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.
- a VEGF dAb inhibits binding of VEGF at a concentration 50 (IC50) of IC50 of about 1 ⁇ 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.
- IC50 concentration 50
- the VEGF dAb specifically binds VEGF, eg, human VEGF, and dissociates from human VEGF with a dissociation constant (K D ) of 300 nM to IpM or 30OnM to 5pM or 5OnM to IpM or 5OnM to 5pM or 5OnM to 20 pM or about 10 pM or about 15pM or about 2OpM as determined by surface plasmon resonance.
- K D dissociation constant
- the polypeptide, dAb or antagonist specifically binds VEGF, eg, human VEGF, and dissociates from human VEGF with a K off rate constant of 5 x 10 "1 s " 1 to 1 x 10 "7 S “1 , e.g. 1 x 10 "2 s “1 to 1 x 10 "6 s “1 , e.g. 5 x 10 "3 s “1 to 1 x 10 "5 s "1 , for example 5 x 10 "1 s "1 or less, e.g. 1 x 10 "2 s “1 or less, e.g.l x 10 "3 s “1 or less, e.g. 1 x 10 "4 s "1 or less, e.g. 1 x 10 "5 s “1 or less, and e.g. 1 x 10 "6 s “1 or less as determined by surface plasmon resonance.
- VEGF dAb specifically binds VEGF, eg, human VEGF, with a K 0n of IxIO "3 M ' V 1 to IxIO "7 M ' V 1 or IxIO "3 M ' V 1 to IxIO "6 M ' V 1 or about IxIO "4 M 1 S 1 or about IxIO "5 M 1 S 1 .
- the polypeptide, dAb or antagonist specifically binds VEGF, eg, human VEGF, and dissociates from human VEGF with a dissociation constant (K D ) and a K o ff as defined in this paragraph.
- the polypeptide, dAb or antagonist specifically binds VEGF, eg, human VEGF, and dissociates from human VEGF with a dissociation constant (K D ) and a K 0n as defined in this paragraph.
- VEGF eg, human VEGF
- K D dissociation constant
- the polypeptide or dAb specifically binds VEGF (eg, human VEGF) with a K D and/or K o ff and/or K 0n 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 DOM 15-26-593.
- VEGF eg, human VEGF
- K D and/or K o ff and/or K 0n 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 DOM 15-26-593.
- the dAb can be expressed in E. coli or in Pichia species (e.g., P. pastoris).
- 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).
- the ligands and dAb monomers described herein can be secretable when expressed in E. coli or in Pichia species (e.g., 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.
- 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.
- Antagonists of VEGF can be monovalent or multivalent.
- the antagonist is monovalent and contains one binding site that interacts with VEGF, the binding site provided by a polypeptide or dAb of the invention.
- 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.
- 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 invention.
- 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.
- the, dAb specifically binds VEGF with a K 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).
- 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.
- the polypeptide, dAb or antagonist binds VEGF and antagonizes the activity of the VEGF in a standard cell assay with an ND 5O of ⁇ 100 nM.
- the dAbs are efficacious in animal models of ocular disease when an effective amount is administered.
- 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.
- the dAbs will be utilized in purified form together with pharmacologically appropriate carriers for ocular delivery.
- 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).
- the ligands (e.g., antagonists) of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include various drugs for ocular delivery to the eye and/or ocular penetration enhancers and/or viscosity enhancers.
- compositions can include "cocktails" of various other agents in conjunction with the ligands of the present invention, or even combinations of ligands according to the present invention having different specificities, such as ligands selected using different target antigens or epitopes, whether or not they are pooled prior to administration.
- 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.
- the dAbs of this invention can be lyophilized 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.
- compositions containing the present dAbs or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments.
- 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.
- 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.
- the invention 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 invention or to IL-I , or TNF- ⁇ or TNF- ⁇ R.
- ocular diseases or conditions include AMD, Uveitis, dry eye, diabetic retinopathy and diabetic macular oedema.
- Half lives (I 1 A alpha and I 1 A 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, CA94040, USA) can be used, for example, to model the curve.
- a first phase the alpha phase
- 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.
- the present invention provides a ligand or a composition comprising a ligand according to the invention having a t ⁇ half-life in the range of 15 minutes or more.
- 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.
- a ligand or composition according to the invention will have a t ⁇ half life in the range of up to and including 12 hours.
- 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.
- the dAb or a composition comprising a dAb according to the invention has a t ⁇ half-life in the range of 30 minutes or more.
- 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.
- a ligand or composition according to the invention has a t ⁇ half- life in the range of up to and including 21 days.
- the upper end of the range is 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20 days.
- a ligand or composition according to the invention will have a t ⁇ 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.
- the present invention provides a dAb or a composition comprising a ligand according to the invention having an AUC value (area under the curve) in the range of 1 mg.min/ml or more.
- the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300 mg.min/ml.
- a ligand or composition according to the invention has an AUC in the range of up to 600 mg.min/ml.
- the upper end of the range is 500, 400, 300, 200, 150, 100, 75 or 50 mg.min/ml.
- a ligand according to the invention will have a AUC in the range selected from the group consisting of the following: 15 to 150 mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50mg.min/ml.
- dAbs of the invention 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.
- 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) 2 , F(ab') 2 , IgG, scFv).
- dAbs according to the invention can be formatted as a fusion or conjugate with another polypeptide or peptide.
- Hydrodynamic size of the ligands (e.g., dAb monomers and multimers) of the invention 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.
- 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
- 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.
- a binding domain e.g., antibody or antibody fragment
- the VEGF dAb can be conjugated or linked to an anti-serum albumin or anti-neonatal Fc receptor antibody or antibody fragment, eg 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 th February 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).
- an anti-serum albumin or anti-neonatal Fc receptor antibody or antibody fragment eg an anti-SA or anti-neonatal Fc receptor dAb
- Conjugating refers to a composition comprising polypeptide, dAb or antagonist of the invention that is bonded (covalently or noncovalently) to a binding domain that binds serum albumin.
- Suitable polypeptides that enhance serum half-life in vivo include, for example, transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins (see U.S. Patent No. 5,977,307, the teachings of which are incorporated herein by reference), brain capillary endothelial cell receptor, transferrin, transferrin receptor (e.g.
- 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-I (Apo A-I), apolipoprotein B (Apo B), ceruloplasmin (Cp), complement component C3 (C3), complement component C4 (C4), Cl esterase inhibitor (Cl INH), C-reactive protein (CRP), ferritin (FER), hemopexin (HPX), lipoprotein(a) (Lp(a)), mannose- binding protein (MBP), myoglobin (Myo), prealbumin
- 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.
- Suitable proteins from the blood include, for example, plasma proteins (e.g., fibrin, ⁇ -2 macro globulin, serum albumin, fibrinogen (e.g., fibrinogen A, fibrinogen B), serum amyloid protein A, haptoglobin, profilin, ubiquitin, uteroglobulin and ⁇ -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, ⁇ -1 microglobulin), defensins (e.g., beta-defensin 1, neutrophil defensin 1, neutrophil def
- Suitable proteins found at the blood brain barrier or in neural tissue include, for example, melanocortin receptor, myelin, ascorbate transporter and the like.
- 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 Kl, Heymann's antigen
- proteins localized to the liver e.g., alcohol dehydrogenase, G250
- proteins localized to the lung e.g., secretary 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 ⁇ 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)).
- 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 (l):263-70 (2000)).
- LAG-3 lymphocyte activation gene
- osteoprotegerin ligand OPGL
- 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 (l):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-I), basic fibroblast growth factor (F GF -2), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), transforming growth factor- ⁇ (TGF ⁇ ), tumor necrosis factor-alpha (TNF- ⁇ ), angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelial growth factor (PD-ECGF), placental growth factor (PlGF), midkine platelet-derived growth factor-BB (PDGF), and fractalkine.
- metalloproteases associated with arthritis/cancers
- FGF-I acidic fibroblast growth factor
- F GF-2 basic fibroblast growth
- 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 invention to a disease site.
- Suitable proteins involved in Fc transport include, for example, Brambell receptor (also known as FcRB).
- FcRB Brambell receptor
- 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).)
- the invention in one embodiment a first dAb that binds to an ocular target molecule, e.g. VEGF, IL-I, or TNF- ⁇ , and a second dAb that binds serum albumin (SA), the second dAb binding SA with a K D as determined by surface plasmon resonance of InM to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 100, 200, 300, 400 or 500 ⁇ M (i.e., x 10 "9 to 5 x 10 "4 ), or 100 nM to 10 ⁇ M, or 1 to 5 ⁇ M or 3 to 70 nM or 1OnM to 1, 2, 3, 4 or 5 ⁇ M. For example 30 to 70 nM as determined by surface plasmon resonance.
- an ocular target molecule e.g. VEGF, IL-I, or TNF- ⁇
- SA serum albumin
- the first dAb (or a dAb monomer) binds SA ⁇ e.g., HSA) with a K D as determined by surface plasmon resonance of approximately 1, 50, 70, 100, 150, 200, 300 nM or 1, 2 or 3 ⁇ M.
- the affinity eg K D and/or K o ff as measured by surface plasmon resonance, eg using BiaCore
- the affinity 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.
- the serum albumin is human serum albumin (HSA).
- HSA human serum albumin
- the first dAb binds SA with an affinity of approximately 10 ⁇ M, while the second dAb binds its target with an affinity of 100 pM.
- the serum albumin is human serum albumin (HSA).
- the first dAb binds SA (eg, HSA) with a K D of approximately 50, for example 70, 100, 150 or 200 nM. Details of dual specific ligands are found in WO03002609, WO04003019 and WO04058821.
- the dAbs of the invention can in one embodiment comprise a dAb that binds serum albumin (SA) with a K D as determined by surface plasmon resonance of InM to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 100, 200, 300, 400 or 500 ⁇ M (i.e., x 10-9 to 5 x 10-4), or 100 nM to 10 ⁇ M, or 1 to 5 ⁇ M or 3 to 70 nM or 1OnM to 1, 2, 3, 4 or 5 ⁇ M. For example 30 to 70 nM as determined by surface plasmon resonance.
- the first dAb (or a dAb monomer) binds SA (e.g.
- 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.
- a longer linker greater than 3 amino acids is used to enhance potency (K D of one or both dAbs in the antagonist).
- the dAb binds human serum albumin and competes for binding to albumin with a dAb selected from the group consisting of
- 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),
- DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474), DOM7m-26 (SEQ ID NO: 475), DOM7r-l (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-l (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: 49
- 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 MSA- 16, MSA-26, D0M7m- 16 (SEQ ID NO : 473), D0M7m- 12 (SEQ ID NO : 474), DOM7m-26
- 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-l (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-23
- 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-l (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), dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb
- the dAb is a V ⁇ dAb that binds human serum albumin and has an amino acid sequence selected from the group consisting of DOM7h-2 (SEQ ID NO:482), DOM7h-6 (SEQ ID NO:485), DOM7h-l (SEQ ID NO:482), DOM7h-6 (SEQ ID NO:485), DOM7h-l (SEQ ID NO:482)
- the dAb is a VJJ dAb that binds human serum albumin and has an amino acid sequence selected from dAb7h30 and dAb7h31.
- the dAb is dAb7hl 1 or dAb7hl4.
- 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, eg one, two or three of the CDRs of dAb7hl 1 or dAb7hl4.
- Suitable Camelid Vfjfj that bind serum albumin include those disclosed in WO 2004/041862 (Ablynx N. V.) and in WO2007080392 (which VHH 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 O (SEQ ID NO:532)
- the Camelid VpfH 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 ALBldisclosed 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.
- 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).
- a (one or more) half- life extending moiety eg, albumin
- a (one or more) half- life extending moiety is conjugated or associated with the VEGF -binding (or IL-I, or TNF- ⁇ binding or TNF- ⁇ R binding) dAb.
- suitable albumin, albumin fragments or albumin variants for use in a VEGF (or IL-I, or TNF- ⁇ or TNF- ⁇ R) -binding format are described in WO 2005077042, which disclosure is incorporated herein by reference and forms part of the disclosure of the present text.
- the following albumin, albumin fragments or albumin variants can be used in the present invention:
- 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;
- 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.
- albumin, fragments or variants can be used in the present invention:
- HA Human serum albumin
- An albumin fragment or variant as described in EP 322094 eg, HA(l-373., HA(l-388), HA(l-389), HA(l-369), and HA(I -419) and fragments between 1- 369 and 1-419;
- a (one or more) half-life extending moiety eg, albumin, transferrin and fragments and analogues thereof
- 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.
- conjugation can be achieved by using a peptide linker between moieties, eg, 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 invention).
- 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).
- 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.
- the dAbs of the invention 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.
- 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.
- 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., dAbl-linker-dAb2; dAb2-linker-dAbl) are encompassed by the invention are ligands that contain an orientation that provides desired binding characteristics can be easily identified by screening.
- dAbs according to the invention can be linked to an antibody Fc region, comprising one or both of C R 2 and C R 3 domains, and optionally a hinge region.
- vectors encoding ligands linked as a single nucleotide sequence to an Fc region may be used to prepare such polypeptides.
- 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.
- DOM 15-26-593 can be selected and prepared as described in WO2008149147 and has the amino sequence shown in Figure Ia (SEQ ID NO 1).
- Myc tagged DOM 15-26-593 (- the Dom 15-26-593 dAb with amino acid sequence shown in figure Ia (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 acclimatize before use.
- the left eyes of six female rabbits were dosed every 20 minutes over a four hour period with 50 micro litres of 2 mg/ml solution of anti-VEGF dAb. Each dose was placed in the subconjunctival 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 0 C) prior to analysis.
- DOM 15-26-593 anti-VEGF-dAb
- the samples of aqueous and vitreous humour were tested for concentration of DOM 15-26-593 (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.
- the VEGF dAb ELISA assay described above was performed as follows: The assay uses recombinant human VEGF (rVEGF, obtained from R&D Systems) coated onto the surface of Immunsorb plates (obtained from Nunc) 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 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:
- rVEGF was prepared at 0.25 ⁇ g/mL to coat the plates (5 ml for each ELISA plate) was prepared. This was done by for each plate, by adding 25 ⁇ 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.
- carbonate coating buffer 0.2M sodium carbonate-bicarbonate coating buffer solution pH 9.4 (Pierce, Cat No: 28382)
- rVEGF 0.25 ⁇ g/mL
- 50 ⁇ L of rVEGF (0.25 ⁇ g/mL) solution was added to each well of a Immunsorb 96- well ELISA plate using a multichannel pipette.
- the plate was covered with a plastic lid and stored at 4 0 C for approximately 42 hours.
- 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 ⁇ L of IM HCl to every well.
- 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.
- Table 1 Concentrations of anti-VEGF dAb (DOM 15-26-593) present in vitreous and aqueous humour from topically treated and contralateral eyes. Treatment consisted of 12 doses (each consisting of a 50 ⁇ L volume of a 2 mg/ml solution) administered in to the subconjunctival sac (every 20 minutes over a 4 hour period)
- 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.
- HPMC Hydroxypropylcellulose
- Example 2 Pharmacokinetics of DOM 15-26-593 following intravitreal administration to eyes of rabbits:
- Dom 15-26-593 dAb with amino acid sequence shown in figure Ia was prepared (2 mg/ml concentration formulated in a 50 mM sodium acetate buffer (pH 7.0) supplemented with 104 rnM 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 acclimatize before use. Each rabbit was anaesthetized and 10 micro litres of a 2 mg/ml solution (solution prepared as described in Example 1) (total 20 ⁇ 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 euthanized 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 0 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.
- Table 2 Concentrations of anti-VEGF dAb (DOM 15-26-593) present in vitreous and aqueous humour from intravitreally dosed and contralateral eyes. Treatment consisted of a single intravitreal injection (10 ⁇ L of a 2 mg/ml solution) to the left eye - rabbits were allowed to recover from anesthesia and were culled at 2, 24 and 30 hours after dosing.
- ND* not detected ( ⁇ 0.1 ng/ml) Concentrations rounded to 2 decimal places.
- CNV Experimental choroidal neovascularization
- DA Dark Agouti
- PC Laser light photocoagulation
- Dye laser PC was performed using a diode-pumped, 532 nm argon laser (Novus Omni, Coherent Inc., Santa Clara, CA) attached to a slit lamp funduscope, and a handheld planoconcave contact lens (Moorf ⁇ elds Eye hospital, London, UK) applied to the cornea to neutralize ocular power.
- Five lesions (532 nm, 150 mW, 0.2 second, 200 ⁇ m diameter) were made in a single eye of each experimental animal.
- the dAb was formulated as a 2 mg/ml concentration in a 50 mM sodium acetate buffer (pH 5.5) supplemented with 104 mM sodium chloride, 0.02% (w/v) Tween 80).
- the 5 ⁇ L volume contained 50 ⁇ g of anti- VEGF dAb (DOM15-26-593; with the amino acid sequence shown in figure Ia; SEQ ID NO 1), 50 ⁇ g of anti-VEGF DOM 15 -26-593 -Fc fusion (with the amino acid sequence shown in figure Ib; SEQ ID NO 2) (or no compound (vehicle only, negative controls).
- 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 are shown below in Table 3.
- CNV choroidal neovascularization
- fluoresecein angiography was used to observe each lesion.
- 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 3 CNV lesion scores in rat eyes at 7 and 14 after induction by photocoagulation.
- DOM 4-130-54 Preparation and selection of DOM 4-130-54 is described in WO 2007063311 and also WO2008149149.
- 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 40KDa linear PEG molecule (obtained from NOF Corp., Europe) by standard maleimide coupling to the free cysteine at position 80 of the dAb.
- Domain antibodies with specificity for IL- 1 in either a naked format (D0M4- 130-54; IL-I naked dAb, 12.026 kDa; with amino acid sequence shown in figure 3; SEQ ID NO 5 ) or a pegylated format (DOM0400PEG; IL-I pegylated dAb, 52.032 kDa; with amino acid sequence sequence shown in figure 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 ⁇ -VEGF dAb (VEGF 15-26-593 with amino acid sequence shown in figure Ib; 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- ⁇ (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 micro litres 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 ⁇ L of phosphate buffered saline. The tube was centrifuged (12000 rpm/2 minutes), the paper removed and the recovered sample stored frozen (-20 C) prior to analysis.
- 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 euthanized. 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 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 micro litres 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 homogenized using ultrasonic disruption (Covaris S2 Sonolab Single) using a 2 minute cycle of repeated high and low frequency bursts.
- Samples of retina/choroid were centrifuged (12000 rpm/2minutes) in a micro fuge (Heraeus). Supernatants were transferred to fresh tubes and stored frozen (- 2O 0 C).
- the drug content of each sample was tested and measured using sandwich format ELISA assays.
- the ⁇ -TNF- ⁇ antibody was captured using plates coated with recombinant human TNF- ⁇ protein (Peprotech) and detected using an alkaline phosphatase conjugated anti-human IgG (Fc specific) antibody (Sigma).
- the IL-I and pegylated IL-I dAbs were captured using plates coated with recombinant human IL-I 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 conjugated antibody (Sigma).
- results of the various formats of domain antibodies and for the ⁇ -TNF- ⁇ 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)
- Results in this table are rounded to one decimal place.
- results in this table are rounded to one decimal place.
- 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-I dAb
- VEGF-Fc 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-I dAb (D0M4- 130-54).
- Results in this table are rounded to one decimal place.
- Results in this table are rounded to one decimal place.
- a domain antibody (dAb) with specificity for TNF- ⁇ Rl i.e. an anti-TNF ⁇ receptor type IdAb
- 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 ⁇ -TNF- ⁇ Rl dAb solution placed under the top eye lid. The animals were held still for at least 30 seconds after dosing.
- lachrymal (tear) fluid samples 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 ⁇ L of phosphate buffered saline. The tube was centrifuged (12000 rpm/2 minutes), the paper removed and the recovered sample stored frozen (-20 C) prior to analysis.
- 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 euthanized. 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 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 homogenized using ultrasonic disruption (Covaris S2 Sonolab 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 (-2O 0 C).
- the samples were tested for concentration of ⁇ -TNF- ⁇ Rl dAb by a sandwich ELISA assay where the dAb was captured using plates coated with recombinant human TNF
- Drug dosing was well tolerated with no signs of redness, irritancy or abnormal animal behaviour observed.
- Concentrations of ⁇ -TNF- ⁇ Rl dAb in ocular fluids and serum are shown for samples tested in triplicate. The ⁇ -TNF- ⁇ Rl dAb was detected in all of the ocular samples tested.
- Results in Table 10 are rounded to one decimal place.
- 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 ⁇ -TNF- ⁇ Rl dAb detected in the samples are shown in Table 8. ⁇ -TNF- ⁇ Rl 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.
- Results in Table 11 are rounded to two decimal places.
- Results in Table 12 are rounded to two decimal places.
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US13/130,656 US20110256122A1 (en) | 2007-06-06 | 2009-11-04 | Polypeptides, antibody variable domains & antagonists |
MX2011005540A MX2011005540A (es) | 2008-11-26 | 2009-11-04 | Polipeptidos dominios variables de anticuerpos, y antagonistas. |
CN2009801469998A CN102224169A (zh) | 2008-11-26 | 2009-11-04 | 多肽、抗体可变结构域和拮抗剂 |
AU2009319175A AU2009319175A1 (en) | 2008-11-26 | 2009-11-04 | Polypeptides, antibody variable domains & antagonists |
EA201100546A EA201100546A1 (ru) | 2007-06-06 | 2009-11-04 | Полипептиды, вариабельные домены антител и антагонисты |
JP2011536817A JP2012509852A (ja) | 2008-11-26 | 2009-11-04 | ポリペプチド、抗体可変ドメインおよびアンタゴニスト |
BRPI0921319A BRPI0921319A2 (pt) | 2008-11-26 | 2009-11-04 | composição, método para dispensação de uma composição, e para tratar, prevenir ou diagnosticar uma condição ocular, e, processo para produzir uma composição farmacêutica |
KR1020117014479A KR20110091777A (ko) | 2008-11-26 | 2009-11-04 | 폴리펩티드, 항체 가변 도메인 및 길항제 |
EP09748778A EP2356147A1 (fr) | 2008-11-26 | 2009-11-04 | Polypeptides, domaines variables d'anticorps et antagonistes |
CA2744055A CA2744055A1 (fr) | 2008-11-26 | 2009-11-04 | Polypeptides, domaines variables d'anticorps et antagonistes |
IL212333A IL212333A0 (en) | 2008-11-26 | 2011-04-14 | Polypeptides, antibody variable domains & antagonists |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011026948A1 (fr) * | 2009-09-03 | 2011-03-10 | Ablynx N.V. | Formulations stables de polypeptides et leurs utilisations |
JP2014501725A (ja) * | 2010-11-24 | 2014-01-23 | グラクソ グループ リミテッド | Hgfを標的とする多特異的抗原結合タンパク質 |
US9011861B2 (en) | 2010-02-25 | 2015-04-21 | Schepens Eye Research Institute | Therapeutic compositions for the treatment of dry eye disease |
WO2015071348A1 (fr) * | 2013-11-18 | 2015-05-21 | Formycon Ag | Composition pharmaceutique d'un anticorps anti-vegf |
US9265834B2 (en) | 2009-03-05 | 2016-02-23 | Ablynx N.V. | Stable formulations of polypeptides and uses thereof |
US9951130B2 (en) | 2012-11-08 | 2018-04-24 | Eleven Biotherapeutics, Inc. | IL-6 antagonists and uses thereof |
US10005830B2 (en) | 2009-03-05 | 2018-06-26 | Ablynx N.V. | Antigen binding dimer-complexes, methods of making/avoiding and uses thereof |
US11142571B2 (en) | 2014-11-07 | 2021-10-12 | Sesen Bio, Inc. | IL-6 antibodies |
US12048746B2 (en) | 2021-08-31 | 2024-07-30 | Hoffmann-La Roche Inc. | IL-6 antagonist formulations and uses thereof |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006003388A2 (fr) * | 2004-06-30 | 2006-01-12 | Domantis Limited | Compositions et procedes pour le traitement de troubles inflammatoires |
WO2007049017A2 (fr) * | 2005-10-24 | 2007-05-03 | Domantis Limited | Antagonistes du recepteur 1 du facteur de necrose des tumeurs servant a traiter des maladies respiratoires |
WO2007063308A2 (fr) * | 2005-12-01 | 2007-06-07 | Domantis Limited | Formats d'anticorps a domaine non competitif qui lient le recepteur type 1 d'interleukine 1 |
WO2007066106A1 (fr) * | 2005-12-06 | 2007-06-14 | Domantis Limited | Ligands presentant une specificite de liaison avec l'egfr et/ou le vegf et leurs procedes d'utilisation |
WO2007087673A1 (fr) * | 2006-02-01 | 2007-08-09 | Arana Therapeutics Limited | Produit d'assemblage d'anticorps à domaines |
WO2008149148A2 (fr) * | 2007-06-06 | 2008-12-11 | Domantis Limited | Polypeptides, domaines variables d'anticorps et antagonistes |
WO2008149146A2 (fr) * | 2007-06-06 | 2008-12-11 | Domantis Limited | Polypeptides, domaines variables d'anticorps et antagonistes |
WO2008149147A2 (fr) * | 2007-06-06 | 2008-12-11 | Domantis Limited | Polypeptides, domaines variables d'anticorps et antagonistes |
-
2009
- 2009-11-04 CA CA2744055A patent/CA2744055A1/fr not_active Abandoned
- 2009-11-04 AU AU2009319175A patent/AU2009319175A1/en not_active Abandoned
- 2009-11-04 BR BRPI0921319A patent/BRPI0921319A2/pt not_active Application Discontinuation
- 2009-11-04 MX MX2011005540A patent/MX2011005540A/es not_active Application Discontinuation
- 2009-11-04 WO PCT/EP2009/064654 patent/WO2010060768A1/fr active Application Filing
- 2009-11-04 EP EP09748778A patent/EP2356147A1/fr not_active Withdrawn
- 2009-11-04 KR KR1020117014479A patent/KR20110091777A/ko not_active Application Discontinuation
- 2009-11-04 CN CN2009801469998A patent/CN102224169A/zh active Pending
- 2009-11-04 JP JP2011536817A patent/JP2012509852A/ja active Pending
-
2011
- 2011-04-14 IL IL212333A patent/IL212333A0/en unknown
- 2011-05-16 ZA ZA2011/03588A patent/ZA201103588B/en unknown
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006003388A2 (fr) * | 2004-06-30 | 2006-01-12 | Domantis Limited | Compositions et procedes pour le traitement de troubles inflammatoires |
WO2007049017A2 (fr) * | 2005-10-24 | 2007-05-03 | Domantis Limited | Antagonistes du recepteur 1 du facteur de necrose des tumeurs servant a traiter des maladies respiratoires |
WO2007063308A2 (fr) * | 2005-12-01 | 2007-06-07 | Domantis Limited | Formats d'anticorps a domaine non competitif qui lient le recepteur type 1 d'interleukine 1 |
WO2007066106A1 (fr) * | 2005-12-06 | 2007-06-14 | Domantis Limited | Ligands presentant une specificite de liaison avec l'egfr et/ou le vegf et leurs procedes d'utilisation |
WO2007087673A1 (fr) * | 2006-02-01 | 2007-08-09 | Arana Therapeutics Limited | Produit d'assemblage d'anticorps à domaines |
WO2008149148A2 (fr) * | 2007-06-06 | 2008-12-11 | Domantis Limited | Polypeptides, domaines variables d'anticorps et antagonistes |
WO2008149146A2 (fr) * | 2007-06-06 | 2008-12-11 | Domantis Limited | Polypeptides, domaines variables d'anticorps et antagonistes |
WO2008149147A2 (fr) * | 2007-06-06 | 2008-12-11 | Domantis Limited | Polypeptides, domaines variables d'anticorps et antagonistes |
Non-Patent Citations (4)
Title |
---|
ENEVER C ET AL: "Next generation immunotherapeutics-honing the magic bullet", CURRENT OPINION IN BIOTECHNOLOGY, LONDON, GB, vol. 20, no. 4, 1 August 2009 (2009-08-01), pages 405 - 411, XP026688087, ISSN: 0958-1669, [retrieved on 20090824] * |
FERRARA NAPOLEONE ET AL: "Development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular age-related macular degeneration", RETINA, LIPPINCOTT WILLIAMS AND WILKINS, PHILADELPHIA, PA, US, vol. 26, no. 8, 1 October 2006 (2006-10-01), pages 859 - 870, XP009099777, ISSN: 0275-004X * |
HARMSEN M M ET AL: "Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER VERLAG, BERLIN, DE, vol. 72, no. 3, 1 September 2006 (2006-09-01), pages 544 - 551, XP002510773, ISSN: 0175-7598, [retrieved on 20060201] * |
HOLT L J ET AL: "Domain antibodies: proteins for therapy", TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, vol. 21, no. 11, 1 November 2003 (2003-11-01), pages 484 - 490, XP004467495, ISSN: 0167-7799 * |
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US10005830B2 (en) | 2009-03-05 | 2018-06-26 | Ablynx N.V. | Antigen binding dimer-complexes, methods of making/avoiding and uses thereof |
US10919954B2 (en) | 2009-03-05 | 2021-02-16 | Ablynx N.V. | Antigen binding dimer-complexes, methods of making/avoiding and uses thereof |
EP3725330A1 (fr) * | 2009-09-03 | 2020-10-21 | Ablynx N.V. | Formulations stables de polypeptides et leurs utilisations |
EP2805731A3 (fr) * | 2009-09-03 | 2015-04-01 | Ablynx N.V. | Formulations stables de polypeptides et leurs utilisations |
US9884117B2 (en) | 2009-09-03 | 2018-02-06 | Ablynx N.V. | Stable formulations of polypeptides and uses thereof |
WO2011026948A1 (fr) * | 2009-09-03 | 2011-03-10 | Ablynx N.V. | Formulations stables de polypeptides et leurs utilisations |
US9011861B2 (en) | 2010-02-25 | 2015-04-21 | Schepens Eye Research Institute | Therapeutic compositions for the treatment of dry eye disease |
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US9951130B2 (en) | 2012-11-08 | 2018-04-24 | Eleven Biotherapeutics, Inc. | IL-6 antagonists and uses thereof |
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ZA201103588B (en) | 2012-01-25 |
AU2009319175A1 (en) | 2010-06-03 |
CN102224169A (zh) | 2011-10-19 |
KR20110091777A (ko) | 2011-08-12 |
IL212333A0 (en) | 2011-06-30 |
CA2744055A1 (fr) | 2010-06-03 |
MX2011005540A (es) | 2011-06-21 |
BRPI0921319A2 (pt) | 2018-10-16 |
EP2356147A1 (fr) | 2011-08-17 |
JP2012509852A (ja) | 2012-04-26 |
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