NZ612839B2 - Ligands that bind tgf-beta receptor ii - Google Patents
Ligands that bind tgf-beta receptor ii Download PDFInfo
- Publication number
- NZ612839B2 NZ612839B2 NZ612839A NZ61283912A NZ612839B2 NZ 612839 B2 NZ612839 B2 NZ 612839B2 NZ 612839 A NZ612839 A NZ 612839A NZ 61283912 A NZ61283912 A NZ 61283912A NZ 612839 B2 NZ612839 B2 NZ 612839B2
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- New Zealand
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- seq
- variable domain
- tgfbetarii
- single variable
- polypeptide
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
Abstract
Disclosed is an anti-TGFbetaRII immunoglobulin single variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 267 having up to 5 amino acid substitutions, deletions or additions, in any combination; wherein the sequence is as disclosed in the specification. Also disclosed are methods of preparation of the disclosed domain and its use in treating tissue fibrosis. methods of preparation of the disclosed domain and its use in treating tissue fibrosis.
Description
LIGANDS THAT BIND TA RECEPTOR II
Background
Transforming Growth Factor-β (TFGbeta; TGFβ (TGF-β) is a signaling molecule that mediates signal
transduction into cells through binding to a TGFbeta receptor (TGFbetaR; TGFβR R)).
TGFbeta signaling activity regulates cell differentiation and growth, the nature of its effect, i.e. as
cell -promoter, growth-suppressor or inducer of other cell ons, being dependent on cell
type (see Roberts, et al., The orming growth factor-betas, e Growth Factors and Their
Receptors, Part I, ed. by Sporn, M.B. & s, A.B., Springer-Verlag, Berlin, 1990, p.419-472).
TGFbeta is produced by a wide variety of cell types, and its cognate receptors are expressed
in a wide variety of organs and cells (see Shi and Massague, Cell, Volume 113, Issue 6, 13 June
2003, Pages 685-700; Biol. Signals., Vol. 5, p.232, 1996 and Pulmonary Fibrosis, Vol. 80 of Lung
Biology in Health and Disease , ed. by Phan, et al., p.627, Dekker, New York, 1995). TGFbeta
receptors have been identified to fall into three types: TGFbetaRI (TGFβRI) (TGFbeta type I receptor
(Franzen et al., Cell, Vol. 75, No. 4, p. 681, 1993; GenBank Accession No: )); TGFbetaRII
(TGFβRII) ta type II receptor (Herbert et al., Cell, Vol. 68, No. 4, p. 775, 1992; GenBank
Accession No: M85079)) and TGFbetaRIII (TGFbeta type III or (Lopez-Casillas, Cell, Vol. 67,
No. 4, p. 785, 1991; GenBank Accession No: L07594)). TGFbetaRI and aRII have been
shown to be essential for the signal transduction of TGF-beta (Laiho et al., J. Biol. Chem., Vol. 265,
p. 18518, 1990 and Laiho et al., J. Biol. Chem., Vol. 266, p. 9108, 1991), while TGFbetaRIII is not
thought to be ial.
TGFbeta signaling is mediated through its binding to both TGFbetaRI and RII. When the
ligand binds to the ellular ligand binding domain, the two receptors are brought together,
allowing RII to phosphorylate RI and begin the signaling cascade through the phosphorylation of
Smad proteins (see Shi and Massague as referred to above).
Three isoforms of TGFbeta have been identified in mammals: TGFbeta1, TGFbeta2, and
TGFbeta3. Each isoform is multifunctional and acts in self-regulatory feedback mechanisms to
l bioavailability for developmental processes and to maintain tissue homeostasis (as reviewed
in ten Dijke and Arthur, Nature Reviews, Molecular Cell Biology, Vol. 8, Nov. 2007, p. 857-869).
Levels of TFGbeta are controlled by regulation through TGFbeta expression as well as through
binding to proteoglycan, i.e., the extracellular matrix (ECM).
Dysregulated TGFbeta signaling, such as excess TGFbeta ing and high levels of
ilable TGFbeta, is implicated in a number of pathologies, including fibroses of various tissues,
such as pulmonary fibrosis and cirrhosis, chronic hepatitis, rheumatoid arthritis, ocular disorders,
vascular restenosis, keloid of skin, and the onset of sclerosis.
Accordingly, there is a need to provide compounds that block or disrupt TGFbeta signaling in
a specific manner, such as through binding to the TGFbeta receptor II. Such compounds can be
used in therapeutics.
Summary
In a first , the invention provides an anti-TGFbetaRII immunoglobulin single variable
domain comprising an amino acid sequence as set forth in SEQ ID NO:267 having up to 5 amino
acid substitutions, deletions or additions, in any combination.
In a second aspect, the invention provides an ed polypeptide comprising an anti-
TGFbetaRII immunoglobulin single variable domain according to the first aspect of the ion,
wherein the isolated ptide binds to TGFbetaRII.
In a third aspect, the invention es a ceutical composition comprising an anti-
aRII immunoglobulin single variable domain or polypeptide according to the first or second
aspect of the invention.
In a further aspect, the invention provides the use of an anti-TGFbetaRII single variable
domain or polypeptide according to the first or second aspect of the invention in the manufacture of
a medicament for the treatment of a e associated with TGFbeta signalling selected from the
group of: tissue fibrosis, such as pulmonary fibrosis, including idiopathic pulmonary is; liver
fibrosis, including sis and chronic hepatitis; rheumatoid arthritis; ocular disorders; fibrosis of
the skin, including keloid of skin, and Dupuytren’s Contracture; kidney fibrosis such as nephritis and
nephrosclerosis, wound g; and a vascular condition, such as restenosis.
In further aspects, the ion provides an isolated nucleic acid encoding an anti-
TGFbetaRII globulin single variable domain or polypeptide according to the first or second
aspect of the invention, a vector comprising such a nucleic acid molecule, and a host cell comprising
such a nucleic acid or such a vector.
In still a further aspect, the invention provides a method of producing a polypeptide of the
invention, the method comprising maintaining a host cell of the invention under conditions suitable
for expression of the nucleic acid or vector, whereby a polypeptide sing an immunoglobulin
single variable domain is produced.
In a further aspect, the invention provides a kit comprising an anti-TGFbetaRII single
variable domain or polypeptide of the invention, and a device for applying the single variable domain
or polypeptide to the skin.
The invention is as defined above and in the claims. However, the disclosure which follows
also includes additional GFbetaRII globulins and other subject matter outside the
scope of the present claims. This disclosure is retained for its technical content.
The disclosure relates to an anti-TGFbetaRII immunoglobulin single variable domain.
Suitably, an anti-TGFbetaRII immunoglobulin single le domain in accordance with the
disclosure is one which binds to TGFbetaRII with an equilibrium dissociation constant (KD) in the
range of 10pM to 50nM, optionally 10pM to 10nM, optionally 100pM to 10nM. In one embodiment,
the anti-TGFbetaRII immunoglobulin single variable domain is one which binds TGFbetaRII with high
affinity (high y) and has an equilibrium dissociation constant of 10pM to 500pM. In one
embodiment, the anti-TGFbetaRII immunoglobulin single variable domain is one which binds
TGFbetaRII with an affinity (KD) of approximately 100pM. In one embodiment, the anti-TGFbetaRII
immunoglobulin single variable domain is one which binds TGFbetaRII with an affinity (KD) of less
than 100pM. In another embodiment, the anti-TGFbetaRII globulin single variable domain is
one which binds aRII with moderate affinity (low potency) and has an brium
dissociation constant of 500pM to 50nM, preferably 500pM to 10nM. In another aspect, the
disclosure provides an isolated polypeptide comprising an anti-TGFbetaRII globulin single
variable domain. Suitably, the isolated polypeptide binds to human TGFbetaRII. In another
embodiment, the isolated polypeptide also binds to TGFbetaRII derived from a different species
such as mouse, dog or monkeys, such as cynomolgus s . Suitably, the isolated
polypeptide binds to both mouse and human TGFbetaRII. Such cross reactivity between TGFbetaRII
from humans and other species allows the same dy construct to be used in an animal disease
model, as well as in humans.
In an aspect of the disclosure there is provided an anti-TGFbetaRII immunoglobulin single
variable domain having an amino acid sequence as set forth in any one of SEQ ID NO:1-38, 204,
206, 208, 214, 234, 236, 238, 240, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and
287, and having up to 5 amino acid alterations, wherein each amino acid tion is an amino acid
tution, deletion or addition i.e. up to 5 amino acid substitutions, deletions or additions, in any
combination. In a particular embodiment the amino acid substitutions are conservative substitutions.
In an embodiment, the anti-TGFbetaRII immunoglobulin single variable domain has the
amino acid sequence as set forth in SEQ ID NO: 234 or 279 and having up to 5 amino acid
alterations, wherein each amino acid tion is a an amino acid substitution, deletion or addition.
In a ular embodiment, the amino acid alteration(s) are not within CDR3, more specifically not
within CDR3 and CDR1, or CDR3 and CDR2, more ically not within any of the CDRs. In an
embodiment, the anti-TGFbetaRII immunoglobulin single variable domain consists of any one of the
following sequences: SEQ ID NO:1-38, 204, 206, 208, 214, 234, 236, 238, 240, 263, 265, 267, 269,
271, 273, 275, 277, 279, 281, 283, 285 and 287. In an embodiment the anti-TGFbetaRII
immunoglobulin single le domain consists of an amino acid sequence of SEQ ID NO: 234 or
236.
It is not intended to cover any specific anti-TGFbetaRII immunoglobulin single variable
domain sequence disclosed in WO 2011012609. For the avoidance of doubt each and every
sequence disclosed in WO 2011012609 may be disclaimed from the present invention. In particular
DOM23h-271 (SEQ ID NO:4) and DOM-23h-439 (SEQ ID NO:10) as disclosed in WO 2011012609
may be disclaimed. An anti-TGFbetaRII immunoglobulin single variable domain consisting of the
amino acid sequence as set forth in SEQ ID NO: 199 or 201 herein may be disclaimed.
An anti-TGFbetaRII globulin single variable domain according to the invention may
comprise one or more (e.g. 1, 2, 3, 4, or 5) C-terminal alanine residues. Alternatively, an anti-
TGFbetaRII immunoglobulin single variable domain may comprise a C-terminal peptide of up to 5
amino acids in length. In an embodiment, the C-terminal peptide comprises 1, 2, 3, 4, or 5 amino
acids.
A person d in the art is able to deduce from a given single variable domain ce,
e.g. one having a sequence as set out in any one of SEQ ID NO:1-38, 204, 206, 208, 214, 234, 236,
238, 240, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 which CDR
sequences are ned within them using the various methods outlined herein e.g. CDR sequences
as defined by reference to Kabat (1987), Chothia (1989), AbM or contact methods, or a combination
of these s. Suitably, CDR sequences are determined using the method of Kabat described
herein. In one embodiment, the CDR sequences of each sequence are those set out in tables 1, 2,
9, and 13.
Described herein is an anti-TGFbetaRII immunoglobulin single variable domain of the
disclosure having 90% or greater than 90% sequence identity to an amino acid ce ed
from the group consisting of SEQ ID NO:1-28.
Also described herein is an anti-TGFbetaRII immunoglobulin single variable domain of the
disclosure having an amino acid sequence ed from the group consisting of SEQ ID NO:1-28
with 25 or fewer amino acid changes. In a particular embodiment an anti-TGFbetaRII
immunoglobulin single variable domain of the disclosure has an amino acid sequence selected from
the group consisting of SEQ ID 8 with 20 or fewer, 15 or fewer, 10 or fewer, 9 or fewer, 8 or
fewer, 7 or fewer, 6 or fewer, or 5 or fewer amino acid s.
In an aspect of the disclosure there is provided an isolated polypeptide comprising an anti-
TGFbetaRII immunoglobulin single variable domain of the disclosure, in particular an anti-
TGFbetaRII immunoglobulin single variable domain identical to an amino acid sequence selected
from the group consisting of SEQ ID NO:1-38, 204, 206, 208, 214, 234, 236, 238, 240, 263, 265,
267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 n said isolated polypeptide binds
to TGFbetaRII.
In an aspect of the sure there is provided an isolated polypeptide encoded by a
tide sequence that is at least 80% identical to at least one nucleic acid sequence selected
from the group of: SEQ ID NOS:39-66, wherein said isolated polypeptide binds to TGFbetaRII.
An anti-TGFbetaRII globulin single variable domain or a polypeptide in accordance
with any aspect of the disclosure may comprise any of the following amino acids: R at position 39, I
at position 48, D at position 53, N at position 61, R at position 61, K at position 61, R at position 64,
F at position 64, D at position 64, E at position 64, Y at position 64, H at position 102, or S at
position 103 of the immunoglobulin single variable domain, said position being according to the
kabat numbering convention. In one embodiment, the globulin single variable domain or
polypeptide comprises a ation of these amino acids. In another embodiment, the
immunoglobulin single variable domain or polypeptide comprises amino acid N at 61 and R at 64. In
another embodiment, the immunoglobulin single variable domain or polypeptide comprises amino
acid R or K at position 61. In an embodiment, the anti-TGFbetaRII immunoglobulin single variable
domain comprises an I at position 48 in addition to any one of the aforementioned es at
position 61 and/or 64. In these ments, the amino acid ing is that of the
immunoglobulin single variable domain, as exemplified, for e, by those sequences given in
SEQ ID NOs:1-38, 204, 206, 208, 214, 234, 236, 238, 240, 263, 265, 267, 269, 271, 273, 275, 277,
279, 281, 283, 285 and 287.
An anti-TGFbetaRII immunoglobulin single variable domain or a polypeptide in accordance
with any aspect of the disclosure may comprise one of the following amino acid combinations
selected from the group: N at position 61 and R at position 64; R at on 61 and E at position
64; R at position 61 and M at position 64; R at position 61 and F at position 64; R at position 61 and
Y at position 64; and R at position 61 and D at position 64 of the immunoglobulin single variable
domain. In an embodiment, the anti-TGFbetaRII immunoglobulin single variable domain comprises
an I at position 48 in addition to any one of the entioned combination of residues at positions
61 and 64.
In another aspect, there is ed a ligand or binding moiety that has binding specificity
for TGFbetaRII and inhibits the g of an anti-TGFbetaRII immunoglobulin single variable
domain comprising an amino acid sequence selected from the group of SEQ ID NOs:1-28 to
TGFbetaRII.
In a further aspect of the disclosure, there is provided a fusion protein comprising an
globulin single variable , polypeptide or ligand in accordance with any aspect of the
disclosure.
In one embodiment, the immunoglobulin single variable domain, polypeptide, ligand or
fusion protein in accordance with the disclosure is one which neutralises TGFbeta activity. Suitably,
the immunoglobulin single variable domain or polypeptide in accordance with the disclosure inhibits
g of TGFbeta to TGFbetaRII. In another ment, the immunoglobulin single variable
domain or polypeptide in accordance with the disclosure ts TGFbeta signalling activity through
TGFbetaRII. In another embodiment, the immunoglobulin single variable domain or polypeptide in
accordance with the disclosure suppresses a activity, in particular, TGFbeta cell growth
activity and/or fibrogenic activity. Suitably, TGFbetaRII is human TGFbetaRII.
In one embodiment, the immunoglobulin single variable domain, polypeptide, ligand or
fusion protein in accordance with the disclosure is devoid of TGFbetaRII agonist activity at 15
micromolar (µM).
In another aspect, there is provided an immunoglobulin single variable domain, polypeptide,
ligand or fusion protein in accordance with any aspect of the disclosure further sing a half-life
extending moiety. ly, the half-life extending moiety is a polyethylene glycol moiety, serum
albumin or a fragment thereof, transferrin receptor or a transferrin-binding portion thereof, or an
antibody or antibody fragment comprising a binding site for a polypeptide that enhances half-life in
vivo. In one embodiment, the half-life extending moiety is an antibody or dy fragment
sing a g site for serum albumin or neonatal Fc receptor. In another ment, the
half-life extending moiety is a dAb, antibody or antibody fragment.
In another aspect, the disclosure provides an isolated or recombinant nucleic acid encoding
a polypeptide comprising an GFbetaRII immunoglobulin single variable domain, polypeptide,
ligand or fusion protein in accordance with any aspect of the disclosure.
In one embodiment, the isolated or recombinant nucleic acid molecule comprises or consists
of a nucleic acid molecule selected from the group of any of the nucleic acid molecules having the
sequences set out in SEQ ID NOS:39-76,203, 205, 207, 212, 233, 235, 237, 239, 262, 264, 266,
268, 270, 272, 274, 276, 278, 280, 282, 284, 286..
In one , the sure provides an isolated or recombinant nucleic acid, wherein the
c acid comprises a nucleotide sequence that is at least 80% identical to the nucleotide
ce of any of the nucleic acid molecules having the sequences set out in SEQ ID NOS:39-66,
and wherein the nucleic acid encodes a polypeptide comprising an immunoglobulin single variable
domain that specifically binds to TGFbetaRII.
In another aspect, there is provided a vector sing a c acid in accordance with
the disclosure.
In a further aspect, there is provided a host cell comprising a c acid or a vector in
accordance with the disclosure. In yet another aspect of the disclosure there is provided a method
of producing a polypeptide comprising an anti-TGFbetaRII immunoglobulin single variable domain or
a polypeptide or ligand or a fusion protein in accordance with the disclosure, the method comprising
maintaining a host cell in ance with the disclosure under conditions suitable for sion of
said nucleic acid or vector, whereby a polypeptide comprising an immunoglobulin single le
domain, polypeptide or ligand or fusion protein is produced. Optionally, the method further
comprises the step of isolating the polypeptide and ally producing a variant, e.g., a mutated
variant, having an ed affinity (Kd); or EC50 for TGFbeta neutralization in a standard assay
than the isolated polypeptide. Suitable assays for TGFbeta activity, such as a cell sensor assay, are
described herein, for example, in the Examples section.
In one aspect of the disclosure, the anti-TGFbetaRII immunoglobulin single variable domain,
polypeptide or ligand or fusion protein in accordance with the disclosure is for use as a medicament.
Accordingly, there is provided a composition comprising anti-TGFbetaRII immunoglobulin single
variable domain, polypeptide or ligand or fusion protein in accordance with the sure for use as
a medicament.
In one aspect of the disclosure, there is provided a use of an anti-TGFbetaRII
globulin single variable domain, polypeptide or ligand or fusion protein in accordance with
the disclosure for the manufacture of a medicament, particularly for use in treating disease
associated with TGFbeta signalling.
Suitably, the anti-TGFbetaRII immunoglobulin single variable domain, ptide or ligand
or fusion protein or composition in accordance with the disclosure is for treatment of a disease
associated with TGFbeta signaling. Suitably, the disease is a tissue fibrosis, such as pulmonary
fibrosis ing idiopathic pulmonary fibrosis; liver fibrosis, including cirrhosis and chronic
hepatitis; rheumatoid arthritis; ocular disorders; or fibrosis of the skin including keloid of skin;
Dupuytren’s Contracture; and kidney fibrosis such as nephritis and nephrosclerosis; or a vascular
condition such as restenosis. Other diseases associated with a signaling include vascular
diseases such as hypertension, pre-eclampsia, hereditary haemorrhagic telangtiectasia type I
(HHT1), HHT2, pulmonary arterial hypertension, aortic aneurysms, Marfan syndrome, familial
aneurysm disorder, Dietz syndrome, arterial tortuosity syndrome (ATS). Other es
associated with TGFbeta signaling include es of the musculoskeletal system, such as
Duchenne’s muscular phy and muscle is. Further diseases associated with TGFbeta
signaling include cancer, such as colon, c, and pancreatic cancer, as well as glioma and NSCLC.
In on, the disclosure provides methods for targeting cancer by modulating TGFbeta signaling in
tumour angiogenesis. Other diseases or ions include those related to tissue scarring. Other
diseases include pulmonary diseases such as COPD (Chronic obstructive pulmonary disease). An
GFbetaRII immunoglobulin single variable domain, polypeptide or ligand or fusion protein or
composition in ance with the disclosure may be used in wound healing and/or to prevent or
e the formation of scars. In one aspect, the disclosure provides the anti-TGFbetaRII single
variable domain, ligand or antagonist, composition or fusion n for intradermal delivery. In one
aspect, the disclosure provides the anti-TGFbetaRII single variable domain, ligand or antagonist or
fusion protein for delivery to the skin of a patient. In one aspect, the disclosure provides the use of
the anti-TGFbetaRII single variable domain, ligand or antagonist or fusion n in the
manufacture of a medicament for ermal delivery. In one aspect, the disclosure provides the
use of the anti-TGFbetaRII single variable domain or antagonist or fusion protein in accordance with
the disclosure in the manufacture of a medicament for delivery to the skin of a patient.
In one embodiment, the variable domain is substantially monomeric. In a particular
embodiment the variable domain is 65%-98% monomeric in solution as determined by SEC-MALS.
In another embodiment the variable domain is 65%-100%, 70%-100%, 75%-100%, 80%-100%,
85%-100%, 90%-100%, 95%-100% monomeric in solution as determined by SEC-MALS.
In r embodiment, the variable domain, , fusion protein or polypeptide as
disclosed herein, ularly when in a pharmaceutical ition, does not contain any one or
combination or all of the following post-translational modifications: deamidation, oxidation or
glycosylation. In a particular ment the variable domain, ligand, fusion protein or polypeptide
according to the disclosure does not deamidate.
Suitably, the composition is for therapy or prophylaxis of a TGFbeta-mediated ion in a
human.
Accordingly, in one embodiment, there is provided an anti-TGFbetaRII dAb for treating
fibrosis of the skin, in particular keloid disease or Dupuytren’s Contracture. Suitably, the anti-
TGFbetaRII dAb is provided as a substantially monomeric dAb for intradermal delivery, preferably
lacking any tag (i.e., untagged) such as a myc or another purification tag.
In one aspect, the composition is a pharmaceutical composition and further ses a
pharmaceutically acceptable carrier, excipient or diluent.
In another aspect, there is provided a method of treating and/or preventing an TGFbetamediated
condition in a human patient, the method comprising administering a composition
comprising an GFbetaRII immunoglobulin single variable , polypeptide or ligand in
accordance with the disclosure the to the patient.
In a further aspect, the disclosure provides an intradermal ry device containing a
composition in ance with the disclosure. Suitably, such a device is a microneedle or collection
of microneedles.
An a further , there is provided a kit comprising an anti-TGFbetaRII single variable
domain or polypeptide as disclosed herein and a device, such as an intradermal ry device, for
applying said single variable domain or polypeptide to the skin.
ed description
Within this specification, the disclosure has been described, with reference to embodiments, in a
way which enables a clear and concise specification to be n. It is intended and should be
appreciated that embodiments may be variously combined or separated without parting from the
disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same
g as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular
genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques
are used for molecular, genetic and biochemical methods (see generally, Sambrook, et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. and Ausubel, et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley &
Sons, Inc., which are incorporated herein by reference) and al methods.
Immunoglobulin: As used , “immunoglobulin” refers to a family of polypeptides which
retain the immunoglobulin fold characteristic of dy molecules, which contain two β sheets and,
usually, a conserved disulphide bond.
Domain: As used herein “domain” refers to a folded n ure which retains its
tertiary structure independently 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 on of the remainder of the protein and/or of the domain. By
single antibody variable domain or immunoglobulin single variable domain is meant a folded
polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore
includes complete antibody variable domains and modified variable domains, for example in which
one or more loops have been replaced by sequences which are not characteristic of antibody
variable domains, or antibody variable domains which have been truncated or se N- or C-
terminal extensions, as well as folded fragments of variable domains which retain at least in part the
binding activity and specificity of the full-length domain.
Immunoglobulin single variable domain: The phrase “immunoglobulin single variable
domain” refers to an antibody variable domain (VH, VHH, VL) or binding domain that specifically binds
an n or epitope independently of different or other V s or domains i.e. is monovalent.
An immunoglobulin single variable domain can be present in a format (e.g., homo- or multimer
) with other le regions or variable domains where the other regions or domains are
not ed for antigen binding by the single globulin variable domain (i.e., where the
immunoglobulin single variable domain binds antigen independently of the additional variable
domains). A n antibody” or “dAb” is an “immunoglobulin single variable domain” as the term
is used herein. A “single antibody variable domain” or an “antibody single le domain” is the
same as an “immunoglobulin single variable domain” as the term is used herein. An globulin
single variable domain is in one embodiment a human dy variable domain, but also includes
single antibody variable domains from other species such as rodent (for example, as sed in
WO 00/29004, the contents of which are incorporated herein by reference in their entirety), nurse
shark and Camelid VHH dAbs. Camelid VHH are immunoglobulin single variable domain
polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco,
which produce heavy chain dies naturally devoid of light chains. The VHH may be humanized.
In all aspects of the disclosure, the immunoglobulin single variable domain is independently
selected from antibody heavy chain and light chain single variable domains, e.g. VH, VL and VHH.
As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgE or a fragment (such as a
Fab, F(ab’)2, Fv, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphidelinked
scFv, diabody) whether derived from any species naturally producing an antibody, or created
by recombinant DNA technology; whether isolated from, for example, serum, B-cells, hybridomas,
transfectomas, yeast or bacteria.
Antibody : In one embodiment, the immunoglobulin single variable ,
polypeptide or ligand in accordance with the disclosure can be ed in any antibody . As
used herein, “antibody format” refers to any suitable polypeptide structure in which one or more
antibody variable s can be orated 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 dies, 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, VH, VHH, VL), 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 VHH). In one embodiment, alternative dy formats include alternative
scaffolds in which the CDRs of any molecules in accordance with the disclosure can be grafted onto
a suitable protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A
domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973,
089932, 2005/0164301) or an EGF domain. Further, the ligand can be nt
(heterobivalent) or multivalent (heteromultivalent) as described . In other ments, a
“Universal framework” may be used wherein “Universal framework” refers to a single dy
framework ce corresponding to the regions of an antibody conserved in sequence as defined
by Kabat (“Sequences of Proteins of logical Interest”, US Department of Health and Human
Services) or corresponding to the human germline immunoglobulin repertoire or structure as defined
by a and Lesk, (1987) J. Mol. Biol. 196:910-917. The disclosure provides for the use of a
single framework, or a set of such frameworks, which has been found to permit the tion of
virtually any binding specificity through variation in the hypervariable regions alone.
In embodiments of the disclosure described throughout this sure, instead of the use of
an anti-TGFbetaRII “dAb” in a peptide or ligand of the disclosure, it is contemplated that one of
ordinary skill in the art can use a polypeptide or domain that comprises one or more or all 3 of the
CDRs of a dAb of the disclosure that binds TGFbetaRII (e.g., CDRs grafted onto a suitable protein
scaffold or skeleton, e.g. an dy, an SpA scaffold, an LDL receptor class A domain or an EGF
domain). The disclosure as a whole is to be construed accordingly to provide disclosure of
polypeptides using such domains in place of a dAb. In this respect, see WO2008096158, the
disclosure of which is incorporated by reference.
In one embodiment, the anti-TGFbetaRII immunoglobulin single variable domain is any
suitable immunoglobulin variable domain, and optionally is a human variable domain or a variable
domain that comprises or is derived from a human framework region (e.g., DP47 or DPK9
framework regions).
Antigen: As described herein an “antigen” is a molecule that is bound by a binding domain
ing to the present disclosure. lly, antigens are bound by antibody ligands and are
e of raising an dy response in vivo. It may be, for example, a polypeptide, protein,
nucleic acid or other molecule.
Epitope: An “epitope” is a unit of structure tionally bound by an globulin
VH/VL pair. Epitopes define the minimum binding site for an antibody, and thus represent the target
of specificity of an antibody. In the case of a single domain dy, an epitope represents the unit
of ure bound by a le domain in isolation.
Binding: Typically, specific binding is indicated by a dissociation constant (Kd) of 50
nanomolar or less, optionally 250 picomolar or less. Specific binding of an antigen-binding protein
to an antigen or epitope can be determined by a suitable assay, including, for example, Scatchard
analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme
immunoassays such as ELISA and sandwich competition assays, and the different variants thereof.
g affinity: Binding affinity is optionally ined using surface plasmon resonance
(SPR) and BIACORE™ (Karlsson et al., 1991), using a E™ system (Uppsala, Sweden). The
BIACORE™ system uses surface plasmon nce (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1;
Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in
real time, and uses surface plasmon resonance which can detect s in the resonance angle of
light at the surface of a thin gold film on a glass support as a result of changes in the refractive
index of the surface up to 300 nm away. BIACORE™ analysis conveniently tes association
rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
Binding affinity is obtained by assessing the association and dissociation rate constants using a
BIACORE™ surface plasmon resonance system (BIACORE™, Inc.). A biosensor chip is activated for
covalent coupling of the target according to the manufacturer's (BIACORE™) instructions. The target
is then diluted and injected over the chip to obtain a signal in response units of immobilized
al. Since the signal in resonance units (RU) is proportional to the mass of immobilized
material, this represents a range of immobilized target densities on the matrix. Dissociation data are
fit to a one-site model to obtain koff +/- s.d. (standard deviation of measurements). -first
order rate constant (Kd’s) are calculated for each association curve, and plotted as a function of
protein concentration to obtain kon +/- s.e. (standard error of fit). brium iation constants
for binding, Kd's, are calculated from SPR measurements as koff/kon.
Another aspect of the disclosure provides an anti-TGFbetaRII immunoglobulin single variable
domain that specifically binds to human TGFbetaRII. In one embodiment, the variable domain binds
human TGFbetaRII with an equilibrium dissociation constant (KD) of about 50nM, 40nM, 30nM,
20nM, 10nM or less, optionally about 9, 8, 7, 6 or 5nM or less, optionally about 4 nM or less, about
3 nM or less or about 2 nM or less or about 1 nM or less, optionally about 500pM or less. ly,
where the variable domain has an equilibrium iation constant in the range of about 50nM to
500pM, it is particularly suitable for local administration to a tissue of interest such as the lung. In
this embodiment, a high concentration of such a “moderate affinity” binder can be provided as an
effective therapeutic. In r embodiment, the variable domain binds human TGFbetaRII with an
equilibrium iation constant (KD) of about 500pM or less, optionally about 450pM, 400pM,
350pM, 300pM, 250pM, 200pM, 150pM, 100pM, 50pM or less, optionally about 40pM, 30pM, 20pM,
10pM or less. Suitably, where the le domain has a dissociation constant in the range of about
500pM to 10pM, it is particularly suitable for systemic administration such that the amount in any
one tissue of interest is ient to provide an ive therapy. In this embodiment, a low
concentration of such a “high affinity” binder can be provided as an effective therapeutic.
In one embodiment, single variable domains of the present disclosure show cross-reactivity
between human TGFbetaRII and TGFbetaRII from another species, such as mouse TGFbetaRII. In
this embodiment, the variable domains specifically bind human and mouse TGFbetaRII. This is
particularly useful, since drug development lly requires testing of lead drug ates in
mouse systems before the drug is tested in humans. The provision of a drug that can bind human
and mouse species allows one to test s in these system and make side-by-side comparisons of
data using the same drug. This avoids the complication of g to find a drug that works against
a mouse TGFbetaRII and a separate drug that works against human TGFbetaRII, and also avoids
the need to compare results in humans and mice using non-identical drugs. Cross vity between
other species used in disease models such as dog or monkey such as cynomolgus monkey is also
envisaged.
Optionally, the binding ty of the immunoglobulin single variable domain for at least
mouse TGFbetaRII and the binding affinity for human TGFbetaRII differ by no more than a factor of
, 50 or 100.
CDRs: The immunoglobulin single variable domains (dAbs) described herein contain
complementarity determining regions (CDR1, CDR2 and CDR3). The locations of CDRs and frame
work (FR) regions and a numbering system have been defined by Kabat et al. (Kabat, E.A. et al.,
Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and
Human Services, U.S. Government Printing Office (1991)). The amino acid sequences of the CDRs
(CDR1, CDR2, CDR3) of the VH (CDRH1 etc.) and VL (CDRL1 etc.) (Vκ) dAbs disclosed herein will be
readily apparent to the person of skill in the art based on the well known Kabat amino acid
numbering system and definition of the CDRs. According to the Kabat ing system, the most
commonly used method based on sequence variability, heavy chain CDR-H3 have varying lengths,
insertions are ed between residue H100 and H101 with letters up to K (i.e. H100, H100A ...
H100K, H101). CDRs can alternatively be determined using the system of Chothia (based on location
of the structural loop regions) (Chothia et al., (1989) Conformations of immunoglobulin
hypervariable regions; Nature 342, p877-883), according to AbM (compromise n Kabat and
Chothia) or according to the t method (based on crystal structures and prediction of contact
residues with antigen) as follows. See http://www.bioinf.org.uk/abs/ for le methods for
determining CDRs.
Once each residue has been ed, one can then apply the following CDR definitions:
Kabat:
CDR H1: 31-35/35A/35B
CDR H2: 50-65
CDR H3: 95-102
CDR L1: 24-34
CDR L2: 50-56
CDR L3: 89-97
Chothia:
CDR H1: 26-32
CDR H2: 52-56
CDR H3: 95-102
CDR L1: 24-34
CDR L2: 50-56
CDR L3: 89-97
AbM:
(using Kabat numbering): (using Chothia numbering):
CDR H1: 26-35/35A/35B 26-35
CDR H2: 50-58 -
CDR H3: 95-102 -
CDR L1: 24-34 -
CDR L2: 50-56 -
CDR L3: 89-97 -
(using Kabat numbering): (using Chothia numbering):
CDR H1: 30-35/35A/35B 30-35
CDR H2: 47-58 -
CDR H3: 93-101 -
CDR L1: 30-36 -
CDR L2: 46-55 -
CDR L3: 89-96 -
(“-“ means the same ing as Kabat)
Accordingly, a person skilled in the art is able to deduce from a given single variable domain
sequence, e.g. one having a sequence as set out in any one of SEQ ID NO:1-38, 204, 206, 208,
214, 234, 236, 238, 240, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 which
CDR sequences are contained within them using the various methods outlined herein. For example,
for a given single variable domain sequence e.g. SEQ ID NO:1 a skilled person is able to ine
the CDR1 , CDR2 and CDR3 ces contained therein using any one or a combination of the CDR
definition methods mentioned above. When using the Kabat CDR definition, the d person is
able to determine that CDR1, CDR2 and CDR 3 sequences are those set forth in SEQ ID NO:77, 113
and 149 tively. ly, CDR sequences are determined using the method of Kabat described
herein. In one embodiment, the CDR sequences of each sequence are those set out in tables 1, 2, 9
and 13. In an embodiment a CDR1 sequence is a CDR1 sequence selected from SEQ ID NO:77-112,
241, 244, 247, and 250. In an embodiment a CDR2 sequence is a CDR2 sequence selected from
SEQ ID NO:113-148, 242, 245, 248, and 251. In an embodiment a CDR3 sequence is a CDR3
sequence selected from SEQ ID NO:149-184, 243, 246, 249, and 252.
A CDR variant or variant binding unit includes an amino acid sequence modified by at least
one amino acid, wherein said modification can be chemical or a partial alteration of the amino acid
sequence (for e by no more than 10 amino acids), which modification permits the variant to
retain the ical characteristics of the unmodified sequence. For example, the variant is a
onal variant which specifically binds to TGFbetaRII. A partial alteration of the CDR amino acid
ce may be by deletion or substitution of one to several amino acids, or by addition or
insertion of one to several amino acids, or by a combination thereof (for example by no more than
amino acids). The CDR t or binding unit variant may contain 1, 2, 3, 4, 5 or 6 amino acid
substitutions, additions or deletions, in any combination, in the amino acid sequence. The CDR
variant or g unit variant may contain 1, 2 or 3 amino acid substitutions, insertions or deletions,
in any combination, in the amino acid sequence. The substitutions in amino acid residues may be
conservative substitutions, for example, substituting one hydrophobic amino acid for an alternative
hydrophobic amino acid. For example leucine may be substituted with valine, or isoleucine.
TGFbetaRII: As used herein “TGFbetaRII” (transforming growth factor beta type II receptor;
TGFβRII) refers to naturally occurring or endogenous mammalian TGFbetaRII proteins and to
proteins having an amino acid ce which is the same as that of a naturally occurring or
endogenous corresponding mammalian TGFbetaRII protein (e.g., recombinant proteins, synthetic
proteins (i.e., produced using the methods of tic organic chemistry)). Accordingly, as defined
herein, the term includes mature TGFbetaRII protein, rphic or c variants, and other
isoforms of TGFbetaRII and modified or unmodified forms of the foregoing (e.g., lipidated,
glycosylated). Naturally occurring or endogenous TGFbetaRII includes wild type proteins such as
mature TGFbetaRII, polymorphic or allelic variants and other isoforms and mutant forms which
occur naturally in mammals (e.g., humans, man primates). Such proteins can be red
or isolated from a source which naturally expresses TGFbetaRII, for example. These proteins and
proteins having the same amino acid sequence as a naturally occurring or endogenous
corresponding aRII, are referred to by the name of the corresponding mammal. For
example, where the ponding mammal is a human, the protein is designated as a human
TGFbetaRII. Human TGFbetaRII is described, for e, by Lin, et al., Cell 1992, Vol. 68(4),
785 and GenBank Accession No. M85079.
Human TGFbetaRII is a transmembrane receptor consisting of 567 amino acids with an
ellular domain of approximately 159 amino acids, a transmembrane domain and a asmic
domain which comprises a protein kinase domain for signal transduction.
As used herein “TGFbetaRII” also includes a portion or fragment of TGFbetaRII. In one
embodiment, such a portion or fragment includes the extracellular domain of TGFbetaRII or a
portion thereof.
By “anti-TGFbetaRII” with reference to an immunoglobulin single variable domain,
polypeptide, ligand, fusion protein or so forth is meant a moiety which recognises and binds
TGFbetaRII. In one embodiment an “anti-TGFbetaRII” specifically recognises and/or specifically
binds to the protein TGFbetaRII, and, suitably, human aRII. In another embodiment, the
anti-TGFbetaRII immunoglobulin single le domain in accordance with the disclosure also binds
to mouse TGFbetaRII (GenBank accession number 575; described, for example in Massague
et al., Cell 69 (7), 1067-1070 ).
“TGFbeta” includes isoforms such as TGFbeta1, TGFbeta2 and TGFbeta3.
TGFbeta binds TGFbetaRII and, in a complex with TGFbetaRI initiates a ing pathway.
Accordingly, TGFbeta activity and inhibition or neutralization of TGFbeta activity can be ined
through any assay which measures an output of TGFbeta signaling. TGFbeta signaling is reviewed,
for example in Itoh, et al., Eur. J. Biochem 2000, Vol. 267, p.6954; Dennler, et al., Journal of
Leucocyte Biol. 2002, 71(5), p. 731-40. Thus, TGFbeta activity can be tested in a number of
different assays familiar to the person skilled in the art. “Inhibition” or “Neutralization” means that a
biological activity of TGFbeta is reduced either totally or partially in the presence of the
immunoglobulin single variable domain of the present disclosure in comparison to the ty of
TGFbeta in the absence of such immunoglobulin single variable domain.
In one embodiment, an inhibition or neutralisation of TGFbeta activity is tested in an IL-11
release assay. In this embodiment, the y of the immunoglobulin single variable domain in
accordance with the disclosure is tested for its ability to inhibit human TGFbeta1 (TGFbeta1; TGF-
β1) ated IL-11 e from cells such as A549 cells. TGFbeta1 (TGF-β1) binds directly to
TGFbetaRII (TGF-βRII) and induces the assembly of the TGFbetaRI/RII (TGF-βRI/II) complex.
TGFbetaRI (TGF-βRI) is phosphorylated and is able to signal through several pathways including the
Smad 4 pathway. Activation of the Smad 4 pathway results in the e of IL-11. The IL-11 is
secreted into the cell supernatant and is then measured by metric ELISA. Suitable IL-11
release assays are described herein, such as the Human IL-11 Quantikine ELISA assay kit supplied
by R & D systems (ref. D1100).
In another embodiment, TGFbeta ty is tested in an assay for the ability of the
immunoglobulin single variable domain in accordance with the sure to inhibit TGFbeta-induced
expression of CAGA-luciferase in MC3T3-E1 cells in a MC3T3-E1 luciferase assay. Three copies of a
TGFbeta-responsive sequence motif, termed a CAGA box are present in the human PAI-1 er
and specifically binds Smad3 and 4 ns. Cloning multiple copies of the CAGA box into a
luciferase er construct confers TGFbeta responsiveness to cells ected with the reporter
system. One le assay is bed herein and uses MC3T3-E1 cells (mouse osteoblasts) stably
transfected with a [CAGA]12-luciferase reporter construct (Dennler, et al., (1998) EMBO J. 17, 3091–
3100).
Other suitable assays include a human SBE beta-lactamase cell assay (INVITROGEN®, cell
sensor assay). Examples of suitable assays are described herein.
ly, the immunoglobulin single variable domain, polypeptide, ligand or fusion protein in
accordance with the disclosure does not, itself activate TGFbetaRII receptor signalling. Accordingly,
in one embodiment, the immunoglobulin single variable domain, polypeptide, ligand or fusion
protein in accordance with the disclosure is devoid of agonist activity at 10 µM. Agonist activity can
be determined by testing a compound of interest in a aRII assay as described herein in the
absence of TGFbeta. Where TGFbeta is absent, agonist activity of a compound of interest would be
detected by detecting TGFbetaRII signalling.
Homology: Sequences similar or homologous (e.g., at least about 70% sequence identity) to
the sequences disclosed herein are also part of the disclosure. In some embodiments, the sequence
identity at the amino acid level can be about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under
selective ization conditions (e.g., very high stringency hybridization conditions), to the
complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a
partially ed or substantially pure form.
As used herein, the terms “low stringency,” “medium stringency,” “high stringency,” or “very
high stringency” conditions describe conditions for c acid hybridization and washing. Guidance
for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. , 6.3.1-6.3.6, which is incorporated herein by reference in its entirety.
Aqueous and nonaqueous s are described in that nce and either can be used. Specific
hybridization conditions referred to herein are as s: (1) low stringency hybridization conditions
in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC,
0.1% SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low
stringency conditions); (2) medium stringency hybridization conditions in 6X SSC at about 45°C,
followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; (3) high stringency hybridization
conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at
65°C; and optionally (4) very high stringency hybridization ions are 0.5M sodium phosphate,
7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Very high
stringency conditions (4) are the preferred conditions and the ones that should be used unless
otherwise specified.
Calculations of ogy” or “sequence ty” or “similarity” between two sequences
(the terms are used hangeably herein) are performed as follows. The sequences are d
for optimal comparison es (e.g., gaps can be introduced in one or both of a first and a second
amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In one embodiment, the length of a reference sequence
aligned for comparison purposes is at least about 30%, ally at least about 40%, optionally at
least about 50%, optionally at least about 60%, and optionally at least about 70%, 80%, 85% 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the length of the reference
sequence. The amino acid residues or nucleotides at corresponding amino acid positions or
nucleotide positions are then compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position in the second sequence, then
the molecules are identical at that position (as used herein amino acid or nucleic acid “ homology” is
equivalent to amino acid or nucleic acid ity”). 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 are optionally prepared and determined using the algorithm BLAST 2 Sequences,
using default parameters (Tatusova, T. A. et al.., FEMS Microbiol Lett, 174:187-188 (1999)).
Alternatively, the BLAST algorithm (version 2.0) is employed for sequence ent, with
parameters set to default values. BLAST (Basic Local Alignment Search Tool) is the heuristic search
algorithm employed by the programs blastp, , blastx, tblastn, and tblastx; these programs
ascribe significance to their findings using the statistical methods of Karlin and Altschul, 1990, Proc.
Natl. Acad. Sci. USA 2264-8.
Ligand: As used herein, the term “ligand” refers to a compound that comprises at least one
peptide, polypeptide or protein moiety that has a binding site with binding icity for
TGFbetaRII. A ligand can also be referred to as a “binding moiety”.
The ligands or binding es ing to the disclosure optionally comprise
immunoglobulin variable s which have different binding specificities, and do not contain
variable domain pairs which together form a binding site for target compound (i.e., do not comprise
an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain
that together form a binding site for TGFbetaRII). Optionally, each domain which has a binding site
that has binding specificity for a target is an globulin single variable domain (e.g.,
immunoglobulin single heavy chain variable domain (e.g., VH, VHH), immunoglobulin single light
chain variable domain (e.g., VL)) that has binding specificity for a desired target (e.g., TGFbetaRII).
Thus, “ligands” include ptides that se two or more immunoglobulin single
variable domains wherein each globulin single variable domain binds to a different target.
Ligands also include ptides that comprise at least two immunoglobulin single le s
or the CDR sequences of the single variable domains that bind different targets in a suitable format,
such as an antibody format (e.g., IgG-like format, scFv, Fab, Fab’, F(ab’)2) or a suitable protein
scaffold or skeleton, such as an affibody, a SpA ld, an LDL receptor class A domain, an EGF
, avimer and dual- and multi-specific ligands as described herein.
The polypeptide domain which has a binding site that has binding specificity for a target
(e.g., TGFbetaRII) can also be a protein domain comprising a binding site for a desired target, e.g.,
a protein domain is selected from an affibody, a SpA domain, an LDL receptor class A domain, an
avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932,
2005/0164301). If desired, a “ligand” can further comprise one or more additional moieties that can
each ndently be a peptide, polypeptide or protein moiety or a non-peptidic moiety (e.g., a
polyalkylene glycol, a lipid, a carbohydrate). For example, the ligand can further comprise a half-life
extending moiety as described herein (e.g., a polyalkylene glycol moiety, a moiety comprising
albumin, an albumin fragment or albumin variant, a moiety comprising transferrin, a transferrin
fragment or transferrin variant, a moiety that binds albumin, a moiety that binds neonatal Fc
receptor).
Competes: As referred to herein, the term “competes” means that the binding of a first
target (e.g., TGFbetaRII) to its cognate target binding domain (e.g., immunoglobulin single variable
domain) is inhibited in the ce of a second binding domain (e.g., immunoglobulin single
variable domain) that is specific for said cognate target. For example, binding may be inhibited
sterically, for example by al 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 m competition ELISA and competition BIACORE™
experiments to determine competition between first and second binding domains, the details of
which are incorporated herein by reference to e explicit disclosure for use in the present
disclosure. The disclosure includes antigen g proteins, ically single variable domains,
polypeptides, ligands and fusion proteins, that e with any one of single variable domains of
SEQ ID NO:1-38. In a particular embodiment there is provided a TGFbetaRII binding protein which
competes with any one of single variable domains of SEQ ID NO:1-38 and also has a KD of 50nM or
less to TGFbetaRII. In a particular embodiment the KD is between 10pM and 50nM. In a particular
embodiment, the KD is between 10pM and 10nM. In a particular embodiment, the KD is between
100pM and 10nM. In a particular ment the KD is approximately 100pM.
TGFbeta signaling: Suitably, the single variable domain, polypeptide or ligand of the
disclosure can neutralize a signaling h TGFbetaRII. By “neutralizing”, it is meant that
the normal signaling effect of a is blocked such that the presence of TGFbeta has a neutral
effect on aRII signaling. Suitable s for ing a neutralizing effect include assays
for TGFbeta signaling as described herein. In one embodiment, neutralization is observed as a %
inhibition of TFGbeta activity in a TGFbeta signaling assay. In one embodiment, the single variable
domain or ptide binds to the extracellular domain of TGFbetaRII thereby inhibiting/blocking
the binding of TGFbeta to the extracellular domain of TGFbetaRII. Suitably, the single variable
domain or polypeptide is useful where there is an excess of bioavailable TGFbeta and the single
variable domain or polypeptide serves to inhibit the signaling activity of the bioavailable TGFbeta
through inhibiting binding or TGFbeta to its cognate receptor TGFbetaRII.
As used herein, the term onist of TGFbetaRII” or “anti-TGFbetaRII antagonist” or the
like refers to an agent (e.g., a molecule, a compound) which binds TGFbetaRII and can inhibit a
(i.e., one or more) function of TGFbetaRII. For example, an antagonist of TGFbetaRII can inhibit
the binding of TGFbeta to TGFbetaRII and/or inhibit signal transduction mediated through
TGFbetaRII. Accordingly, TGFbeta-mediated processes and cellular responses can be inhibited with
an antagonist of TGFbetaRII.
In one embodiment, the ligand (e.g., immunoglobulin single variable domain) that binds
TGFbetaRII inhibits binding of TGFbeta to a aRII receptor with an inhibitory concentration 50
(IC50) that is ≤ about 10 µM, ≤ about 1 µM, ≤ about 100 nM, ≤ about 50 nM, ≤ about 10 nM, ≤
about 5 nM, ≤ about 1 nM, ≤ about 500 pM, ≤ about 300 pM, ≤ about 100 pM, or ≤ about 10 pM.
In a particular embodiment, an anti-TGFbetaRII immunoglobulin single variable domain of the
disclosure has an IC50 of 15 µM or less. The IC50 is optionally ined using an in vitro TGFbeta
receptor binding assay, or cell assay, such as the assay described herein.
It is also contemplated that the ligand (e.g., immunoglobulin single variable domain)
optionally t TGFbetaRII induced functions in a suitable in vitro assay with a neutralizing dose
50 (ND50) that is ≤ about 10 µM, ≤ about 1 µM, ≤ about 100 nM, ≤ about 50 nM, ≤ about 10 nM,
≤ about 5 nM, ≤ about 1 nM, ≤ about 500 pM, ≤ about 300 pM, ≤ about 100 pM, ≤ about 10 pM,
≤ about 1 pM ≤ about 500 fM, ≤ about 300 fM, ≤ about 100 fM, ≤ about 10 fM. In a particular
embodiment, an anti-TGFbetaRII immunoglobulin single variable domain of the disclosure es
greater than 40% neutralisation of TGF-β.
“dual-specific ligand”: In one embodiment, the immunoglobulin single le ,
polypeptide or ligand in accordance with the disclosure can be part of a “dual-specific ligand” which
refers to a ligand sing a first antigen or epitope binding site (e.g., first immunoglobulin single
variable domain) and a second antigen or epitope binding site (e.g., second immunoglobulin single
variable domain), wherein the binding sites or variable domains are capable of binding to two
antigens (e.g., different antigens or two copies of the same n) or two epitopes on the same
antigen which are not normally bound by a monospecific immunoglobulin. For example, the two
es may be on the same antigen, but are not the same epitope or sufficiently adjacent to be
bound by a ecific ligand. In one embodiment, dual-specific ligands according to the
disclosure are composed of binding sites or variable domains which have different specificities, and
do not contain mutually complementary variable domain pairs (i.e. VH/VL pairs) which have the same
specificity (i.e., do not form a unitary binding site).
In one embodiment, a specific ligand” may bind to TGFbetaRII and to r target
molecule. For example, another target molecule may be a -specific target le such that
the dual-specific ligand of the disclosure enables an anti-TGFbetaRII polypeptide or immunoglobulin
single variable domain in accordance with the disclosure to be targeted to a tissue of st. Such
tissues e lung, liver and so forth.
Multispecific dAb multimers are also provided. This includes a dAb multimer comprising an
anti-TGFbetaRII immunoglobulin single variable domain according to any aspect of the disclosure
and one or more single le domains each of which binds to a different target (e.g. a target
other than TGFbetaRII). In an embodiment a bispecific dAb multimer is provided e.g. a dab
multimer comprising one or more anti-TGFbetaRII immunoglobulin single variable domains
according to any aspect of the disclosure and one or more dabs which bind to a second, different
target. In an embodiment a trispecific dAb multimer is provided.
The ligands of the disclosure (e.g., ptides, dAbs and antagonists) can be ted as
a fusion protein that contains a first immunoglobulin single variable domain that is fused directly to
a second immunoglobulin single variable domain. If desired such a format can further comprise a
half-life extending moiety. For example, the ligand can se 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.
Generally, the orientation of the polypeptide domains that have a binding site with binding
specificity for a target, and r the ligand comprises a linker, is a matter of design .
However, some orientations, with or without linkers, may provide better binding teristics than
other orientations. All orientations (e.g., dAb1-linker-dAb2; dAb2-linker-dAb1) are encompassed by
the disclosure are ligands that contain an orientation that provides desired g characteristics
can be easily identified by screening.
Polypeptides and dAbs according to the sure, including dAb monomers, dimers and
trimers, can be linked to an antibody Fc region, comprising one or both of CH2 and CH3 domains,
and optionally a hinge region. For example, vectors encoding ligands linked as a single nucleotide
sequence to an Fc region may be used to prepare such polypeptides. In an embodiment there is
provided a dAb-Fc fusion.
The disclosure moreover provides dimers, s and polymers of the aforementioned dAb
monomers.
Target: As used herein, the phrase “target” refers to a biological molecule (e.g., e,
polypeptide, protein, lipid, carbohydrate) to which a polypeptide domain which has a binding site
can bind. The target can be, for e, an intracellular target (e.g., an intracellular protein
target), a soluble target (e.g., a secreted), or a cell surface target (e.g., a membrane protein, a
receptor protein). In one embodiment, the target is TGFbetaRII. In another embodiment, the target
is TGFbetaRII extracellular domain.
Complementary: As used herein ementary” refers to when two immunoglobulin
domains belong to families of ures which form cognate pairs or groups or are derived from
such families and retain this feature. For e, a VH domain and a VL domain of an antibody are
complementary; two VH domains are not complementary, and two VL domains are not
complementary. Complementary domains may be found in other members of the immunoglobulin
superfamily, such as the Vα and Vβ (or γ and δ) s of the T-cell receptor. s which are
artificial, such as domains based on protein scaffolds which do not bind epitopes unless engineered
to do so, are non-complementary. Likewise, two domains based on (for example) an
immunoglobulin domain and a ectin domain are not complementary.
“Affinity” and “avidity” are terms of art that describe the strength of a binding interaction.
With respect to the ligands of the disclosure, avidity refers to the overall strength of binding
between the targets (e.g., first target and second target) on the cell and the ligand. Avidity is more
than the sum of the individual affinities for the individual targets.
Nucleic acid molecules, s and host cells: The disclosure also es isolated and/or
recombinant nucleic acid molecules encoding ligands (single variable domains, fusion proteins,
polypeptides, dual-specific ligands and multispecific ligands) as bed herein.
Nucleic acids referred to herein as "isolated" are nucleic acids which have been separated
away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it
exists in cells or in a mixture of nucleic acids such as a library), and include nucleic acids obtained
by methods described herein or other le methods, including ially pure nucleic acids,
nucleic acids produced by chemical synthesis, by combinations of ical and chemical methods,
and recombinant nucleic acids which are isolated (see e.g., Daugherty, B.L. et al., Nucleic Acids
Res., 19(9): 2471 2476 (1991); Lewis, A.P. and J.S. Crowe, Gene, 101: 297-302 (1991)).
Nucleic acids ed to herein as "recombinant" are nucleic acids which have been
produced by recombinant DNA methodology, ing those nucleic acids that are generated by
procedures which rely upon a method of artificial recombination, such as the polymerase chain
reaction (PCR) and/or cloning into a vector using restriction enzymes.
In certain embodiments, the isolated and/or recombinant nucleic acid comprises a nucleotide
sequence encoding an immunoglobulin single variable domain, polypeptide or ligand, as described
, wherein said ligand comprises an amino acid sequence that has at least about 80%, at least
about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%,
or at least about 99% amino acid sequence identity with the amino acid sequence of a dAb that
binds TGFbetaRII disclosed herein, e.g. amino acid ces set out in any of SEQ ID NOS: 1-38.
Nucleotide sequence identity can be determined over the whole length of the tide sequence
that encodes the selected anti-TGFbetaRII dAb. In an embodiment the nucleic acid sequence
comprises or consists of a nucleic acid sequence at least 80% identical to of any one of SEQ ID
NO:39-66. In an embodiment the nucleic acid sequence comprises or consists of a nucleic acid
sequence of any one of SEQ ID NO:39-76.
ments of the disclosure also provide codon optimized tide sequences encoding
polypeptides and variable domains as sed herein e.g. optimised for expression in bacterial,
mammalian or yeast cells.
The disclosure also provides a vector comprising a recombinant nucleic acid molecule of the
sure. In n embodiments, the vector is an sion vector comprising one or more
expression control elements or sequences that are operably linked to the recombinant nucleic acid
of the disclosure. The disclosure also provides a recombinant host cell sing a recombinant
nucleic acid molecule or vector of the disclosure. Suitable vectors (e.g., plasmids, phagemids),
expression control elements, host cells and methods for producing recombinant host cells of the
disclosure are well-known in the art, and examples are further described herein.
Suitable expression vectors can contain a number of components, for example, an origin of
ation, a selectable marker gene, one or more expression l elements, such as a
transcription control element (e.g., promoter, enhancer, terminator) and/or one or more translation
signals, a signal sequence or leader sequence, and the like. Expression control elements and a
signal sequence, if present, can be ed by the vector or other source. For example, the
transcriptional and/or translational control sequences of a cloned nucleic acid encoding an antibody
chain can be used to direct expression.
A promoter can be provided for expression in a desired host cell. Promoters can be
constitutive or inducible. For example, a er can be operably linked to a nucleic acid ng
an antibody, antibody chain or portion thereof, such that it directs transcription of the c acid.
A variety of suitable promoters for prokaryotic (e.g., lac, tac, T3, T7 promoters for E. coli) and
eukaryotic (e.g., Simian Virus 40 early or late promoter, Rous sarcoma virus long terminal repeat
promoter, cytomegalovirus promoter, irus late promoter) hosts are available.
In addition, expression vectors typically se a selectable marker for selection of host
cells carrying the vector, and, in the case of a replicable expression vector, an origin of replication.
Genes encoding products which confer antibiotic or drug resistance are common selectable markers
and may be used in prokaryotic (e.g., lactamase gene (ampicillin resistance), Tet gene for
tetracycline resistance) and eukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolic
acid), ampicillin, or hygromycin ance genes). Dihydrofolate reductase marker genes permit
selection with methotrexate in a variety of hosts. Genes encoding the gene product of ophic
markers of the host (e.g., LEU2, URA3, HIS3) are often used as selectable markers in yeast. Use of
viral (e.g., baculovirus) or phage s, and vectors which are capable of integrating into the
genome of the host cell, such as retroviral vectors, are also contemplated. Suitable expression
vectors for expression in mammalian cells and prokaryotic cells (E. coli), insect cells (Drosophila
Schnieder S2 cells, Sf9) and yeast (P. methanolica, P. pastoris, S. cerevisiae) are well-known in the
art.
Suitable host cells can be yotic, including bacterial cells such as E. coli, B. subtilis
and/or other le bacteria; eukaryotic cells, such as fungal or yeast cells (e.g., Pichia pastoris,
Aspergillus sp., romyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa), or
other lower eukaryotic cells, and cells of higher eukaryotes such as those from insects (e.g.,
Drosophila Schnieder S2 cells, Sf9 insect cells (WO 94/26087 (O’Connor)), s (e.g., COS
cells, such as COS-1 (ATCC Accession No. 50) and COS-7 (ATCC Accession No. CRL-1651),
CHO (e.g., ATCC Accession No. CRL-9096, CHO DG44 (Urlaub, G. and Chasin, LA., Proc. Natl. Acad.
Sci. USA, 77(7):4216-4220 (1980))), 293 (ATCC Accession No. CRL-1573), HeLa (ATCC ion
No. CCL-2), CV1 (ATCC Accession No. CCL-70), WOP (Dailey, L., et al., J. Virol., 54:739-749 (1985),
3T3, 293T (Pear, W. S., et al., Proc. Natl. Acad. Sci. U.S.A., 90:8392-8396 (1993)) NS0 cells, SP2/0,
HuT 78 cells and the like, or plants (e.g., tobacco). (See, for example, Ausubel, F.M. et al., eds.
Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc.
(1993). In some embodiments, the host cell is an isolated host cell and is not part of a multicellular
organism (e.g., plant or animal). In n embodiments, the host cell is a man host cell.
The disclosure also provides a method for producing a ligand (e.g., dual-specific ligand, multispecific
ligand) of the disclosure, comprising maintaining a recombinant host cell comprising a recombinant
nucleic acid of the disclosure under ions suitable for expression of the recombinant nucleic
acid, whereby the recombinant nucleic acid is expressed and a ligand is produced. In some
embodiments, the method further comprises isolating the ligand.
Reference is made to WO200708515, page 161, line 24 to page 189, line 10 for s of
disclosure that is applicable to ments of the present disclosure. This disclosure is hereby
incorporated herein by reference as though it appears itly in the text of the present disclosure
and relates to the embodiments of the present disclosure, and to provide explicit support for
disclosure to incorporated into claims below. This es disclosure presented in WO200708515,
page 161, line 24 to page 189, line 10 providing details of the ration of Immunoglobulin
Based Ligands”, “Library vector systems”, “Library Construction”, “Combining Single Variable
Domains”, “Characterisation of Ligands”, “Structure of Ligands”, “Skeletons”, “Protein Scaffolds”,
“Scaffolds for Use in Constructing Ligands”, “Diversification of the Canonical Sequence” and
“Therapeutic and diagnostic itions and uses”, as well as definitions of "operably linked",
“naive”, “prevention”, “suppression”, “treatment”, "allergic disease", “Th2-mediated disease”,
"therapeutically-effective dose" and “effective”.
The phrase, “half-life” refers to the time taken for the serum concentration of the
immunoglobulin single variable , polypeptide or ligand to reduce by 50%, in vivo, for
example due to ation of the ligand and/or clearance or sequestration of the ligand by natural
mechanisms. The ligands of the disclosure can be stabilized in vivo and their half-life increased by
binding to les which resist degradation and/or clearance or sequestration. Typically, such
molecules are lly ing proteins which themselves have a long ife in vivo. The halflife
of a ligand is sed if its functional activity ts, in vivo, for a longer period than a similar
ligand which is not specific for the half-life increasing molecule. Thus a ligand specific for HSA and a
target molecules is compared with the same ligand wherein the specificity to HSA is not present,
that is does not bind HSA but binds another molecule. Typically, the ife is increased by 10%,
%, 30%, 40%, 50% or more. Increases in the range of 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x or
more of the ife are possible. Alternatively, or in addition, increases in the range of up to 30x,
40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x of the half life are possible.
Formats: Increased half-life can be useful in in vivo applications of immunoglobulins,
especially antibodies and most especially antibody fragments of small size. Such fragments (Fvs,
hide bonded Fvs, Fabs, scFvs, dAbs) are generally rapidly cleared from the body. dAbs,
polypeptides or ligands in accordance with the disclosure can be adapted to provide increased half-
life in vivo and consequently longer persistence times in the body of the functional activity of the
ligand.
Methods for pharmacokinetic analysis and determination of ligand half-life will be familiar to
those skilled in the art. Details may be found in Kenneth, A et al: Chemical Stability of
Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinetic is: A
Practical Approach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D ,
published by Marcel Dekker, 2nd Rev. ex n (1982), which describes pharmacokinetic
parameters such as t alpha and t beta half lives and area under the curve (AUC).
Half lives (t½ alpha and t½ 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. In a first
phase (the alpha phase) the ligand is undergoing mainly distribution in the patient, with some
elimination. A second phase (beta phase) is the terminal phase when the ligand has been
distributed and the serum concentration is decreasing as the ligand is cleared from the patient. The
t alpha half life is the half life of the first phase and the t beta half life is the half life of the second
phase. Thus, in one embodiment, the present disclosure provides a ligand or a composition
comprising a ligand according to the disclosure having a tα half life in the range of 15 minutes or
more. In one ment, the lower end of the range is 30 minutes, 45 minutes, 1 hour, 2 hours,
3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, or
alternatively, a ligand or composition according to the disclosure will have a tα half life in the range
of up to and including 12 hours. In one embodiment, the upper end of the range is 11, 10, 9, 8, 7,
6 or 5 hours. An example of a suitable range is 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.
In one embodiment, the present disclosure es a ligand (polypeptide, dAb or
antagonist) or a composition comprising a ligand according to the disclosure having a tβ half life in
the range of about 2.5 hours or more. In one embodiment, the lower end of the range is about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 10 hours, about 11 hours,
or about 12 hours. In addition, or alternatively, a ligand or composition ing to the disclosure
has a tβ half life in the range of up to and including 21 days. In one embodiment, the upper end
of the range is about 12 hours, about 24 hours, about 2 days, about 3 days, about 5 days, about 10
days, about 15 days or about 20 days. In one embodiment a ligand or ition according to the
disclosure will have a tβ half life in the range about 12 to about 60 hours. In a further embodiment,
it will be in the range about 12 to about 48 hours. In a further embodiment still, it will be in the
range about 12 to about 26 hours.
In on, or alternatively to the above criteria, the present disclosure provides a ligand or
a composition comprising a ligand according to the disclosure having an AUC value (area under the
curve) in the range of about 1 mg•min/ml or more. In one embodiment, the lower end of the range
is about 5, about 10, about 15, about 20, about 30, about 100, about 200 or about 300 /ml.
In addition, or atively, a ligand or composition according to the disclosure has an AUC in the
range of up to about 600 mg•min/ml. In one embodiment, the upper end of the range is about 500,
about 400, about 300, about 200, about 150, about 100, about 75 or about 50 mg•min/ml. In one
embodiment a ligand according to the disclosure will have a AUC in the range selected from the
group consisting of the following: about 15 to about 150 mg•min/ml, about 15 to about 100
/ml, about 15 to about 75 mg•min/ml, and about 15 to about 50mg•min/ml.
Polypeptides and dAbs of the disclosure and antagonists comprising these can be formatted
to have a larger hydrodynamic size, for example, by attachment of a PEG group, serum albumin,
transferrin, transferrin receptor or at least the transferrin-binding portion thereof, an antibody Fc
region, or by conjugation to an antibody domain. For example, ptides dAbs and antagonists
formatted as a larger antigen-binding fragment of an antibody or as an dy (e.g., formatted as
a Fab, Fab’, , F(ab’)2, IgG, scFv).
As used herein, “hydrodynamic size” refers to the apparent size of a molecule (e.g., a
protein molecule, ligand) based on the diffusion of the molecule through an aqueous solution. The
diffusion or motion of a protein through solution can be processed to derive an apparent size of the
protein, where the size is given by the “Stokes radius” or “hydrodynamic radius” of the protein
particle. The “hydrodynamic size” of a n 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.
Hydrodynamic size of the ligands (e.g., dAb monomers and multimers) of the disclosure may
be determined using methods which are well known in the art. For example, gel filtration
chromatography may be used to determine the ynamic size of a ligand. Suitable gel filtration
es for determining the hydrodynamic sizes of ligands, such as cross-linked agarose matrices,
are well known and readily available.
The size of a ligand format (e.g., the size of a PEG moiety attached to a dAb monomer), can
be varied depending on the d application. For example, where ligand is intended to leave the
circulation and enter into peripheral tissues, it is desirable to keep the ynamic size of the
ligand low to facilitate extravazation from the blood stream. atively, where it is desired to
have the ligand remain in the ic circulation for a longer period of time the size of the ligand
can be increased, for example by ting as an Ig like protein.
Half-life extension by targeting an antigen or epitope that increases half-live in vivo: The
hydrodynamic size of a ligand and its serum half-life can also be increased by conjugating or
associating an TGFbetaRII binding polypeptide, dAb or ligand of the disclosure to a g domain
(e.g., antibody or antibody nt) that binds an antigen or epitope that increases ive in
vivo, as described herein. For example, the TGFbetaRII binding agent (e.g., polypeptide) 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 eonatal 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 ed from, but not limited to, the group consisting of CTLA-4,
lipocallin, SpA, an affibody, an avimer, GroEl and ectin (see WO2008096158 for disclosure of
these binding domains, which domains and their sequences are incorporated herein by reference
and form part of the disclosure of the present text). Conjugating refers to a composition comprising
polypeptide, dAb or antagonist of the sure that is bonded (covalently or noncovalently) to a
binding domain such as a binding domain that binds serum albumin.
Typically, a polypeptide that enhances serum ife in vivo is a polypeptide which occurs
naturally in vivo and which s degradation or removal by endogenous mechanisms which
remove unwanted material from the organism (e.g., human). For example, a polypeptide that
enhances serum half-life in vivo can be selected from proteins from the extracellular matrix, proteins
found in blood, proteins found at the blood brain barrier or in neural tissue, proteins localized to the
kidney, liver, lung, heart, skin or bone, stress proteins, disease-specific proteins, or proteins involved
in Fc transport. Suitable polypeptides are described, for example, in WO2008/096158.
Such an approach can also be used for targeted delivery of a single variable domain,
polypeptide or ligand in accordance with the sure to a tissue of interest. In one embodiment
targeted delivery of a high affinity single variable domain in accordance with the disclosure is
provided.
dAbs that Bind Serum Albumin: The disclosure in one embodiment provides a polypeptide
or nist (e.g., dual specific ligand comprising an anti-TGFbetaRII dAb (a first dAb)) that binds
to TGFbetaRII and a second dAb that binds serum albumin (SA), the second dAb binding SA. Details
of dual specific ligands are found in WO03002609, 3019, WO2008096158 and
WO04058821.
In particular embodiments of the s and antagonists, the dAb binds human serum
albumin and competes for binding to albumin with a dAb selected from the group ting of any
of the dAb sequences disclosed in WO2004003019 (which sequences and their nucleic acid
counterpart are incorporated herein by reference and form part of the disclosure of the present
text), any of the dAb sequences sed in WO2007080392 (which ces and their nucleic
acid counterpart are incorporated herein by reference and form part of the disclosure of the present
text), any of the dAb sequences disclosed in WO2008096158 (which sequences and their nucleic
acid counterpart are incorporated herein by nce and form part of the disclosure of the present
text).
In certain embodiments, the dAb binds human serum albumin and comprises an amino acid
sequence that has at least about 80%, or at least about 85%, or at least about 90%, or at least
about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about
99% amino acid sequence identity with the amino acid sequence of a dAb described in any of
WO2004003019, WO2007080392 or WO2008096158. For example, the dAb that binds human
serum albumin can comprise an amino acid sequence that has at least about 90%, or at least about
95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%
amino acid sequence identity with the amino acid sequence of any of these dAbs. In certain
embodiments, the dAb binds human serum n 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 the amino acid ce of any of these dAbs.
In more particular ments, the dAb is a Vκ dAb that binds human serum albumin. In
more particular embodiments, the dAb is a VH dAb that binds human serum albumin.
Suitable Camelid VHH that bind serum albumin include those disclosed in WO2004041862
(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). In certain
embodiments, the Camelid VHH 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 those ces disclosed in WO2007080392 or any one of SEQ ID
NOS:518-534, these sequence numbers corresponding to those cited in WO2007080392 or WO
2004041862.
In an alternative embodiment, the antagonist or ligand comprises a binding moiety specific
for TGFbetaRII (e.g., human TGFbetaRII), wherein the moiety comprises non-immunoglobulin
ces as described in WO2008096158, the disclosure of these binding es, their methods
of production and selection (e.g., from diverse libraries) and their sequences are incorporated herein
by reference as part of the disclosure of the present text).
Conjugation to a ife extending moiety (e.g., albumin): In one embodiment, a (one or
more) half-life extending moiety (e.g., albumin, transferrin and nts and analogues thereof) is
ated or associated with the TGFbetaRII-binding ptide, dAb or antagonist of the
disclosure. Examples of le albumin, albumin fragments or albumin variants for use in a
TGFbetaRII-binding format are described in WO2005077042, which disclosure is incorporated herein
by reference and forms part of the disclosure of the present text.
Further examples of suitable n, fragments and analogs for use in a TGFbetaRII-
g format are described in WO 03076567, which sure is incorporated herein by reference
and which forms part of the disclosure of the present text.
Where a (one or more) half-life extending moiety (e.g., albumin, transferrin and fragments
and analogues thereof) is used to format the TGFbetaRII-binding polypeptides, dAbs and
antagonists of the disclosure, it can be conjugated using any le method, such as, by direct
fusion to the TGFbetaRII-binding moiety (e.g., anti- TGFbetaRII dAb), 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 ife extending moiety located N- or C-terminally to the TGFbetaRII
binding . Alternatively, conjugation can be achieved by using a peptide linker between
moieties, e.g., a peptide linker as described in WO03076567 or WO2004003019 (these linker
disclosures being incorporated by reference in the present disclosure to provide es for use in
the present disclosure).
Conjugation to PEG: In other embodiments, the half-life extending moiety is a polyethylene
glycol moiety. In one ment, the antagonist ses (optionally consists of) a single
variable domain of the disclosure linked to a polyethylene glycol moiety (optionally, wherein said
moiety has a size of about 20 to about 50 kDa, optionally about 40 kDa linear or branched PEG).
Reference is made to WO04081026 for more detail on PEGylation of dAbs and binding moieties. In
one embodiment, the antagonist consists of a dAb monomer linked to a PEG, wherein the dAb
monomer is a single variable domain according to the disclosure.
In another embodiment, a single variable domain, ligand or polypeptide in accordance with
the disclosure may be linked to a toxin moiety or toxin.
Protease resistance: Single variable domains, polypeptides or ligands in accordance with the
disclosure can be modified to improve their resistance to se degradation. As used herein, a
peptide or polypeptide (e.g. a domain antibody (dAb)) that is “resistant to protease degradation” is
not substantially degraded by a protease when incubated with the protease under ions
suitable for protease activity. A polypeptide (e.g., a dAb) is not substantially degraded when no
more than about 25%, no more than about 20%, no more than about 15%, no more than about
14%, no more than about 13%, no more than about 12%, no more than about 11%, no more than
about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more
than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no
more that about 2%, no more than about 1%, or substantially none of the protein is degraded by
se 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.
Methods for generating dAbs with enhanced protease resistance are sed, for example,
in WO2008149143. In one embodiment, the single variable , polypeptide or ligand in
accordance with the disclosure is resistant to degradation by leucozyme and/or trypsin.
Polypeptides, immunoglobulin single variable domains and ligands of the disclosure may be resistant
to one or more of the following: serine protease, cysteine protease, aspartate ses, 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), nase (e.g., nase 1, proteinase 2, proteinase 3),
thermolysin, chymosin, enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4, caspase 5,
caspase 9, caspase 12, caspase 13), calpain, ficain, clostripain, actinidain, bromelain, and separase.
In particular embodiments, the protease is trypsin, elastase or leucozyme. The protease can also be
provided by a biological t, biological homogenate or biological preparation. ptides,
immunoglobulin single le domains and ligands as disclosed herein may be selected in the
presence of lung proteases, such that said polypeptides, immunoglobulin single variable domains
and ligands are ant to said lung proteases. In one embodiment, the protease is a protease
found in , mucus (e.g., gastric mucus, nasal mucus, ial mucus), bronchoalveolar
lavage, lung homogenate, lung extract, pancreatic extract, c fluid, saliva. In one ment,
the protease is one found in the eye and/or tears. Examples of such proteases found in the eye
include caspases, calpains, matrix metalloproteases, disintegrin, metalloproteinases (e.g. ADAMs – a
disintegrin and metalloproteinase) and ADAM with thrombospondin , the proteosomes, tissue
nogen activator, secretases, sin B and D, cystatin C, serine se PRSS1, ubiquitin
proteosome pathway (UPP). In one embodiment, the protease is a non bacterial protease. In an
embodiment, the protease is an animal, e.g., mammalian, e.g., human, protease. In an
embodiment, the protease is a GI tract protease or a pulmonary tissue protease, e.g., a GI tract
protease or a pulmonary tissue protease found in humans. Such protease listed here can also be
used in the methods described, for example, in WO2008149143, involving exposure of a repertoire
of library to a protease.
Stability: In one aspect of the disclosure, the polypeptides, single variable domains, dAbs,
ligands, compositions or formulations of the disclosure are ntially stable after incubation (at a
concentration of ptide or variable domain of ) at 37 to 50 °C for 14 days in Britton
Robinson or PBS buffer. In one embodiment, at least 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99% of the polypeptide, antagonist or variable domain etc. remains
egated after such incubation at 37 degrees C. In one embodiment, at least 65, 70, 75, 80,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the polypeptide or variable domain
remains monomeric after such incubation at 37 degrees C.
In one embodiment, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the polypeptide, antagonist or variable
domain remains unaggregated after such incubation at 50 degrees C. In one embodiment, at least
, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99% of the polypeptide or variable domain remains monomeric after such incubation
at 50 degrees C. In one embodiment, no aggregation of the polypeptides, variable domains,
antagonists is seen after any one of such incubations. In one embodiment, the pI of the polypeptide
or variable domain s unchanged or substantially unchanged after incubation at 37 s C
at a concentration of polypeptide or variable domain of 1mg/ml in n-Robinson buffer. In one
aspect of the disclosure, the ptides, le domains, antagonists, compositions or
formulations of the disclosure are substantially stable after incubation (at a concentration of
polypeptide or variable domain of 100mg/ml) at 4 °C for 7 days in Britton Robinson buffer or PBS at
a pH of 7 to 7.5 (e.g., at pH7 or pH7.5). In one ment, at least 95, 95.5, 96, 96.5, 97, 97.5,
98, 98.5, 99 or 99.5% of the polypeptide, antagonist or variable domain remains egated after
such incubation. In one embodiment, at least 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5% of
the polypeptide or variable domain remains monomeric after such incubation. In one embodiment,
no aggregation of the polypeptides, le domains, antagonists is seen after any one of such
incubations.
In one aspect of the disclosure, the ptides, variable s, antagonists,
compositions or formulations of the disclosure are substantially stable after nebulisation (e.g. at a
concentration of polypeptide or variable domain of 40mg/ml) e.g., at room temperature, 20 degrees
C or 37°C, for 1 hour, e.g. jet nebuliser, e.g. in a Pari LC+ cup. In one embodiment, at least 65, 70,
75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5% of
the polypeptide, antagonist or variable domain remains unaggregated after such nebulisation. In
one embodiment, at least 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5,
97, 97.5, 98, 98.5, 99 or 99.5% of the polypeptide or variable domain remains monomeric after
such sation. In one embodiment, no aggregation of the polypeptides, variable domains,
antagonists is seen after any one of such nebulisation.
ric form: In one embodiment, the dAb of the present disclosure is identified to be
preferentially monomeric. Suitably, the disclosure provides a (substantially) pure monomer. In one
embodiment, the dAb is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% pure or 100%
pure monomer. To determine whether dAbs are monomeric or form higher order oligomers in
solution, they can be analysed by SEC-MALLS. SEC MALLS (size exclusion chromatography with
multi-angle-LASER-light-scattering) is a non-invasive technique for the characterizing of
macromolecules in solution, that is familiar to any skilled in the art. Briefly, proteins (at
concentration of 1mg/mL in buffer Dulbecco’s PBS) are separated according to their hydrodynamic
properties by size exclusion chromatography (column: 0; S200). Following separation, the
propensity of the protein to scatter light is measured using a multi-angle-LASER-light-scattering
(MALLS) detector. The intensity of the scattered light while n passes through the detector is
measured as a function of angle. This measurement taken together with the protein tration
determined using the refractive index (RI) detector allows ation of the molar mass using
appropriate equations (integral part of the analysis re Astra v.5.3.4.12).
Therapeutic use: The disclosure provides a method for treating, suppressing or preventing
diseases associated with TGFbeta signaling. In one embodiment, such disease may be caused or
contributed to by dysregulated TGFbeta signaling, by pression of TGFbeta or by high levels of
bioavailable TGFbeta. Diseases ated with TGFbeta signaling include diseases relating to
fibroses of various tissues, such as ary fibrosis including idiopathic pulmonary is (IPF)
and other interstitial lung disease such as acute respiratory ss syndrome (ARDS), fibrosis of the
liver including cirrhosis and chronic hepatitis, rheumatoid arthritis, ocular disorders, vascular
conditions such as restenosis, fibrosis of the skin including keloid of skin and scarring following
wound healing and Dupuytren’s Contracture, and kidney such as nephritis, kidney fibrosis and
nephrosclerosis or a vascular condition such as restenosis. Other diseases associated with TGFbeta
signaling include vascular diseases such as hypertension, pre-eclampsia, hereditary haemorrhagic
telangtiectasia type I (HHT1), HHT2, pulmonary arterial hypertension, aortic aneurysms, Marfan
syndrome, al sm disorder, Loeys-Dietz syndrome, arterial tortuosity syndrome (ATS).
Other es associated with TGFbeta signaling include es of the musculoskeletal system
such as Duchenne’s muscular dystrophy and muscle fibrosis. Further diseases associated with
TGFbeta signaling include cancer such as colon, gastric and atic cancer as well as glioma and
NSCLC. In addition, the disclosure provides s for targeting cancer by, for example,
modulating TGFbeta signaling in the tumour angiogenesis or h treatment of the cancer
stroma. Other diseases or ions e those related to tissue scarring. Other diseases include
pulmonary diseases such as COPD (Chronic obstructive pulmonary disease), liver es such as
liver failure (e.g. viral hepatitis, alcohol, obesity, autoimmune, metabolic, obstructive), kidney
diseases including renal failure (e.g. diabetes, hypertension), hypertrophic myopathy,
transplant rejection (lung/liver/kidney) and hypertrophic and keloid scarring.
“Fibrosis” is the result of excess deposition of extracellular matrix components such as
collagen causing overgrowth, scarring and/or hardening of tissues.
“Skin Fibrosis”: cutaneous fibrosis covers a variety of human disorders with differing
aetiology, but with a common dysregulation of tive tissue metabolism, particularly of dermal
fibroblasts. Specific examples of cutaneous fibrosis include keloid e, hypertrophic scars (HS)
and scleroderma. Keloid disease and hypertrophic scars, although not ups of the same
condition are both resultant from scarring following wound g, with Keloids spreading beyond
the original wound site whilst hypertrophic scar is constrained within the margins of the original
wound. Scleroderma, however is used to describe fibrosis of the skin in systemic sclerosis which is a
systemic condition resulting in fibrosis of multiple organs. In an embodiment, the variable domain,
ligand, fusion protein or polypeptide as disclosed herein is used to prevent or treat keloid disease,
hypertrophic scars or scleroderma.
“Keloids” are fibrous overgrowths at sites of cutaneous injury that form as a result of an
al healing process in genetically susceptible individuals and, unlike normal scars, do
not regress. Predominantly observed in patients with darkly pigmented skin, “Keloid disease” is a
benign dermal roliferative tumor unique to humans that never becomes malignant.
“Dupuytren's contracture” is a localized formation of scar tissue beneath the skin of the palm
of the hand. The scarring accumulates in a tissue (fascia) that ly covers the tendons that pull
the s to grip. As Dupuytren contracture progresses, more of the fascia becomes thickened and
shortened, ing in flexion contracture of the hand where the fingers bend towards the palm and
cannot be fully extended ghtened), resulting in extreme cases to loss of use of the hand.
Scarring occurs following, surgery, injury or trauma to tissues or organs within the body.
They are a consequence of repair mechanisms that te extracellular matrix to replace missing
normal tissue. The skin represents the most frequently injured tissue resulting in dermal scarring,
which can result in adverse consequences including: loss of on; contracture; and, poor
aesthetics which may have cause psychological effects to the sufferer. Scars can be defined ‘a
macroscopic bance of the normal structure and function of the skin architecture, resulting from
the end product of wound healing’ (Fergusson et al., 1996). Currently no therapies exist to prevent
or improve scarring effectively.
The role of TGFbeta in pulmonary fibrosis has been observed (Wynn et al., J. Pathology
2008, 214, p.199-210; Sime et al. J. Clinical Immunology 1997, 0, p. 768-776). A shift to
increased production of Th2 cytokines and decreased production of Th1 cytokines is observed as a
result of unknown lung . Overexpression of TGFbeta stimulates angiogenesis, last
activation, deposition of ECM, and fibrogenesis. Animal models (e.g. TGFbeta overexpression,
SMAD3 KO, inhibition of TGFbetaR signaling) show that TGFbeta is a key mediator for the
development of pulmonary fibrosis.
“Idiopathic pulmonary fibrosis (IPF)” is a chronic and progressive e resulting in
abnormal and excessive deposition of fibrotic tissue in the pulmonary interstitium without a known
cause. There is an incidence of approximately 10-20 cases per 100,000 in U.S per year. The
prevalence increases y with age, reaching 175 cases per 100,000 over the age of 75 with
onset usually occurring between 50 and 70 yrs. The five year survival rate is 20% with a mean
survival of 2.8 years. Symptoms include a dry cough and progressive breathlessness, abnormal
chest x-ray or HRCT and reduced lung volumes. Current treatments include corticosteroids
(Prednisone), immunosuppressives (cyclophosphamide) or transplantation although none of the
currently available therapies have a proven efficacy. In one embodiment, the single variable domain
or polypeptide of the present disclosure provides a treatment for IPF.
ly, a successful treatment for Idiopathic pulmonary fibrosis (IPF) will show any one of
a decrease in lung fibroblast proliferation, an increase in lung fibroblast apoptosis, a decrease in
excessive extracellular matrix synthesis and tion, an increase in extracellular matrix
own and remodelling or will show some protection against ongoing tissue injury and
restoration of normal histopathology.
Suitably, a successful treatment would decelerate disease progression.
The cy of a treatment for IPF can be trated in the bleomycin induced
pulmonary is model. In one embodiment, the immunoglobulin single variable domain of the
present disclosure cross reacts with mouse TGFbetaRII such that its cy can be tested in the
mouse model.
TGFbeta is an important cell signaling molecule in the modulation of cell behaviour in ocular
tissues. Overactivation of TGFbeta is implicated in the pathogenesis of fibrotic diseases in eye tissue
which can be wound healing-related and lead to impaired vision and ocular tissue homeostasis
(reviewed, for example, by Saika, Laboratory Investigation , 86, 106-115).
ingly, in one embodiment, diseases associated with TGFbeta signaling include ocular
disorders such as fibrotic diseases of the eye tissue. Fibrotic e of the eye may occur in the
cornea, conjunctiva, lens or retina. Ocular disorders e proliferative vitreoretinopathy (PVR), a
er of post-retinal detachment and retinal fibrosis, diabetic retinopathy, glaucoma, such as
open-angle glaucoma, angle-closure, congenital and -exfoliation syndrome, wound healing
reactions in the lens, such as post al or thermal burn, or Stevens-Johnson’s syndrome, and
post-cataract surgery complications. a also has a role in cataract development (Wormstone et
al. Exp Eye Res; 83 1238-1245, 2006). A number of ocular disorders occur as a result of fibrosis
post surgery. In addition, over activity of a2 (transforming growth factor β2) is believed to
cause scarring in and around the eye after glaucoma filtration surgery. TGFbeta2 is the predominant
isoform involved in pathological scarring of ocular tissues including the , retina, conjunctiva
and trabelular meshwork. Scarring or fibrosis of the trabelular meshwork can lead to occlusion of
the normal aqueous outflow pathway g to raised intraocular pressure and risk of glaucoma
development. TGFbeta 2 has been shown to be a pathological agent in pre-clinical models of
glaucoma disease. TGFbeta2 levels are elevated in patients with glaucoma, in vitro treatment of
huTM cells with TGFbeta-2 leads to phenotypic changes and lation of ECM modulating
proteins (MMP-2, PAI-I) (Lutjen-Drecol (2005), Experimental Eye Research, Vol. 81, Issue 1, pages
1-4; Liton (2005), Biochemical and Biophysical Research Communications Vol. 337, issue 4, p.1229-
1236; ofer et al (2003), Experimental Eye ch, Vol. 77, issue 6, p. 757-765; Association
for Research in Vision and Ophthalmology (ARVO) conference poster #1631 2009). Moreover,
overexpression of a in the eye leads to glaucoma-like ogy in mice (ARVO conference
poster #5108 2009) and delivery of TGFbeta-2 using AAV has been shown to inhibit retinal ganglion
cell loss in a rat model of glaucoma (ARVO conference poster #5510 2009). More recently,
oxidative stress induction in ed human optic nerve head astrocytes has been shown to
increase TFGbeta2 secretion (Yu et al (2009) . Ophthalmol. Vis. Sci. 50: 1707-1717). This all
indicates that reduction of TGFbeta 2 levels might minimize the characteristic optic nerve head
changes seen in glaucoma. However, TGFbeta is also known to have an immunosuppressive role
and so in some aspects can be protective so a reduction in elevated levels of TGFbeta2 rather than
a complete knock down may be preferred in ent of chronic ocular conditions such as
glaucoma. Accordingly, diseases which can be treated using the dAbs and compositions etc. in
accordance with the disclosure include scarring post glaucoma filtration surgery.
Accordingly, in one aspect there is provided a method for treating, suppressing or ting
a disease associated with TGFbeta signaling and, in particular, dysregulated TGFbeta ing,
comprising stering to a mammal in need thereof a therapeutically-effective dose or amount of
a polypeptide, fusion protein, single variable domain, antagonist or composition according to the
disclosure.
In another aspect, the disclosure provides an immunoglobulin single variable domain,
polypeptide, ligand or fusion protein in accordance with the disclosure for use as a medicament.
Suitable a medicament may se an immunoglobulin single le domain etc. in accordance
with the disclosure ted as described herein.
Suitably, the medicament is a pharmaceutical composition. In a further aspect of the
disclosure, there is ed a composition (e.g., pharmaceutical composition) comprising a
polypeptide, single variable domain, ligand, composition or nist ing to the disclosure
and a physiologically or pharmaceutically acceptable carrier, diluent or excipient. In one
embodiment, the composition comprises a e for delivery. In particular embodiments, the
polypeptide, fusion protein, single variable domain, antagonist or composition is administered via
pulmonary delivery, such as by inhalation (e.g., intrabronchial, intranasal or oral inhalation,
intranasal such as by drops) or by systemic delivery (e.g., parenteral, intravenous, intramuscular,
eritoneal, intraarterial, intrathecal, intraarticular, subcutaneous, vaginal or rectal
administration). In another embodiment, the polypeptide, single variable domain, ligand or fusion
protein or compositions in accordance with the disclosure is administered to the eye e.g. by topical
administration, as eye drops, particulate polymer system, gel or t, or by intraocular injection
e.g. into the vitreous humour. Delivery can be targeted to particular s of the eye such as the
surface of the eye, or the tear ducts or lacrimal glands or to the anterior or posterior chambers of
the eye such as the vitreous humour). It can also be useful if the immunoglobulin single le
domain, composition etc. is delivered to the eye along with an ocular penetration enhancer e.g.
sodium caprate or with a viscosity enhancer e.g. Hydroxypropylmethylcellulose (HPMC). In further
ments, the polypeptide, fusion protein, single variable , antagonist or composition is
administered to the skin; by topical delivery to the surface of the skin and/or ry to a region(s)
within the skin e.g. intradermal delivery.
Although the most accessible organ of the body for delivery, the skin’s outermost barrier,
the stratum corneum (SC), acts as a rate limiting barrier for drug delivery. Traditionally, intradermal
injection has been required to circumvent the SC allowing delivery of drug to site of action in deeper
skin layers. Delivery however, maybe achieved through other transdermal delivery approaches.
Formulation methodologies maybe utilised for delivery, including: chemical enhancers to alter the
lipid structure of the SC; peptide facilitators enabling transfollicular transport; and encapsidation in
particles ing, liposome’s, niosomes, ethosomes and transfersomes, which are believed to aid
local fluidisation of the lipids and formation of depots for prolonged effect. Iontophoresis, involving
the application of a small electrical potential across the skin, has been used for localised drug
delivery. Iontophoresis allows for both the delivery of charged and neutral molecules by
electromigration and electroosmosis respectively. Microneedles, can be employed to create micronsized
channels in the skin to overcome the SC, ng ns to pass through these channels to
the lower epidermis. Microneedles can be broadly classified into solid and hollow microneedles. Solid
microneedles, maybe used to disrupt the SC, prior to drug administration, coated to allow delivery
as drug dissolves from the needles, or soluble allowing drug e as the needles dissolve in situ.
Hollow microneedles allow for infusion of a liquid formulation of drug substance. oporation,
unlike iontophoresis es higher voltages >50V, to alter skin permeability in order to enhance
drug penetration. Thermal and radiofrequency ablation methodologies allow for disruption of the SC
h localised heating and ablation of the SC. In heat ablation this results following application of
high ature for short periods of time, whereas radiofrequency ablation involves use of
radiofrquencies, to vibrate microelectrodes on the skin, resulting in sed heating. Disruption of
the SC can also be achieved through Laser abrasion, ation of low frequency ultrasound waves
(sonophoresis) and jet injectors ing high ties to propel drug through the SC.
Moreover, the present disclosure provides a method for the treatment of e using a
polypeptide, single le domain, composition, ligand or antagonist according to the present
disclosure. In one embodiment the disease is a tissue fibrosis such as keloid disease or Dupuytren’s
cture.
In an aspect of the disclosure, the polypeptide, single variable domain, ligand, composition
or antagonist is provided for therapy and/or prophylaxis of a e or condition associated with
TGFbeta ing in a human. In another , there is provided the use of the polypeptide,
single variable domain, composition or antagonist, in the manufacture of a medicament for therapy
or prophylaxis of a disease or condition associated with a signaling in a human. In another
, there is provided a method of treating and/or preventing a disease or condition associated
with TGFbeta signaling in a human t, the method comprising administering the polypeptide,
single variable domain, composition or nist to the patient. The disclosure also relates to
therapeutic methods that comprise administering a therapeutically effective amount of a ligand of
the disclosure (e.g., antagonist, or single variable domain) to a subject in need thereof.
In other embodiments, the disclosure relates to a method for treating idiopathic ary
fibrosis comprising administering to a subject in need thereof a therapeutically effective amount of a
ligand of the disclosure (e.g., antagonist, or single variable domain).
The disclosure also s to a drug delivery device comprising the composition (e.g.,
pharmaceutical ition) of the disclosure. In some embodiments, the drug delivery device
comprises a plurality of therapeutically effective doses of ligand.
In other embodiments, the drug delivery device is selected from the group consisting of
parenteral delivery , intravenous delivery , uscular delivery device, eritoneal
delivery device, transdermal or ermal delivery device, pulmonary delivery device, intraarterial
delivery device, intrathecal delivery device, intraarticular delivery device, subcutaneous delivery
device, intranasal delivery device, ocular delivery device, vaginal delivery device, rectal delivery
device, syringe, a transdermal delivery device, an intradermal delivery device, a capsule, a tablet, a
nebulizer, an inhaler, an atomizer, an aerosolizer, a mister, a dry powder inhaler, a metered dose
inhaler, a metered dose sprayer, a metered dose mister, a metered dose atomizer, and a catheter.
In an embodiment the drug delivery device is a transdermal or intradermal delivery device.
Suitably, the disclosure provides a pulmonary delivery device ning a polypeptide, single
variable domain, composition or antagonist ing to the disclosure. The device can be an
inhaler or an intranasal administration device. ly, the pulmonary delivery device enables
delivery of a therapeutically effective dose of a ligand etc. in accordance with the disclosure.
In r embodiment, the disclosure provides an ocular delivery device containing a
polypeptide, single le domain, composition or antagonist according to the disclosure. Suitably,
the ocular delivery device enables delivery of a therapeutically effective dose of a ligand etc. in
accordance with the disclosure.
As used herein, the term “dose” refers to the quantity of ligand administered to a subject all
at one time (unit dose), or in two or more administrations over a defined time interval. For
example, dose can refer to the quantity of ligand (e.g., ligand comprising an immunoglobulin single
variable domain that binds TGFbetaRII) stered to a t 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 al n doses can be any
desired amount of time. In a particular embodiment, the single variable domain or ptide of
the invention is administered into the skin by ion, in particular by intradermal delivery, weekly
or fortnightly or every 7-10 days, for example every 7, 8, 9 or 10 days.
In one embodiment, the single variable domain of the disclosure is provided as a dAb
monomer, optionally unformatted (e.g., not PEGylated or half-life extended) or linked to a PEG,
optionally as a dry powder formulation, optionally for delivery to a patient by inhalation (e.g.,
pulmonary delivery), optionally for treating and/or preventing a lung condition (e.g., Idiopathic
pulmonary fibrosis).
The ligands of the disclosure provide several advantages. For example, as described herein,
the ligand can be tailored to have a desired in vivo serum half-life. Domain antibodies are much
smaller than conventional antibodies, and can be administered to achieve better tissue ation
than conventional antibodies. Thus, dAbs and ligands that comprise a dAb provide advantages over
conventional antibodies when administered to treat e, such as TGFbeta-signaling-mediated
disease. In particular, pulmonary delivery of a dAb of the present disclosure to treat idiopathic
ary fibrosis enables specific local delivery of an inhibitor of TGFbeta signaling.
Advantageously, an unformatted dAb monomer which specifically binds to and ts TGFbetaRII is
small enough to be absorbed into the lung through ary delivery.
The examples of WO2007085815 are incorporated herein by reference to provide details of
relevant assays, formatting and experiments that can be equally applied to ligands of the present
disclosure.
All publications, including but not limited to patents and patent applications, cited in this
ication are herein incorporated by reference as though fully set forth.
The disclosure is further described, for the purposes of illustration only, in the following
examples.
EXAMPLES
Example 1. ion of dAbs which bind TGFbetaRII
Selection of dAbs which bind mouse TGFbetaRII
Naïve Selections: 4G and 6G naïve phage libraries, phage libraries displaying dy single
variable domains expressed from the GAS1 leader sequence (see WO2005093074) for 4G and
onally with heat/cool preselection for 6G (see WO04101790), were used. The DOM23 leads
were isolated by panning pools of VH and VK libraries ified as 4G H11-19 and 6G VH2-4 (VH
dAbs) and 4G κ1, 4G κ2 and 6G κ (Vκ dAbs) against the recombinant mouse and human TGF-β
RII/Fc chimera protein. These chimeric proteins were made by expression of a DNA sequence
encoding the amino acids residues 24 to 159 of the extracellular domain of human TGF-β or
Type II (Lin, et al., 1992, Cell 68:775-785) fused to the Fc region of human IgG1 in a human
embryonic kidney cell line, HEK-F.
The recombinant mouse and human TGF-β RII/Fc chimera proteins were biotinylated using
EZ-LINK™ Sulfo-NHS-LC-Biotin reagent (Pierce, Rockford, USA) (Henderikx, et al., 2002, Selection of
antibodies against biotinylated antigens. Antibody Phage Display: Methods and protocols, Ed.
O'Brien and Atkin, Humana Press). The phage libraries were pooled into six groups; 4G κ1 and κ2,
6G κ, 4G H11-13, 4G H14-16, 4G H17-19 and 6G VH2-4. 1x1011 phage per library were pooled.
The phage were blocked in 2 % MARVEL™ milk powder in phosphate buffered saline (MPBS)
with the addition of 10 uM Human IgG Fc fragment (Native IgG Fc fragment derived from human
myeloma plasma IgG, Calbiochem, California, US, cat. no. 401104) for one hour. 200 nM
biotinylated mouse TGF-β RII/Fc was incubated with the blocked phage and Fc nt mixture for
1 hour at room temperature and then captured on streptavidin ads™ (Dynal, UK) for five
minutes. The beads were washed seven times with 1 ml phosphate ed saline/0.1% TWEEN™
(PBST), followed by a wash with 1 ml phosphate buffered saline (PBS). The biotinylated mouse TGF-
β RII/Fc -bound phage were eluted in 500 ul 1mg/ml trypsin in PBS for 10 minutes and then used to
infect 1.75 ml of log-phase Escherichia coli TG1 for 30 minutes. Cells were plated on 2 x TYE
(Trypton Yeast Extract) agar plates supplemented with 15 ug/ml tetracycline. For subsesquent
rounds of selections, cells were d from the plates and used to inoculate 50 ml 2 x TY on
Yeast) + 15 ug/ml tetracycline cultures that were grown overnight at 37⁰C for phage ication.
Amplified phage was red by centrifugation of the overnight culture for 10 minutes at
4566 g. 40 ml of supernatant containing the amplified phage was added to 10ml of PEG/NaCl (20%
v/w PEG 8000 + 2.5M NaCl) and incubated on ice for 45 to 60 minutes. The samples were
centrifuged for 30 s at 4566 g to pellet the precipitated phage. The supernatant was
ded and the phage pellet was resuspended in 2ml 15% v/v glycerol/PBS. The phage sample
was transferred to 2 ml Eppendorf tubes and centrifuged for 10 minutes at g to remove any
remaining bacterial cell debris. The phage was used as input phage for the second round of
selection. The second round of selection was performed as described for the first round, except
approximately 1 x 1010 phage were added, and either 200 nM human TGF-β RII/Fc or 20 nM mouse
TGF-β RII/Fc was used in the selections.
Second round outputs were cloned from the fd-phage vector, pDOM4 into pDOM10. Vector
pDOM4, is a tive of the fd phage vector in which the gene III signal peptide sequence is
replaced with the yeast glycolipid anchored surface protein (GAS) signal peptide. It also contains a
c-myc tag between the leader ce and gene III, which puts the gene III back in frame. This
leader sequence functions well both in phage y vectors but also in other prokaryotic
sion vectors and can be universally used. pDOM10 is a plasmid vector designed for soluble
sion of dAbs. It is based on pUC119 vector, with expression under the control of the LacZ
promoter. sion of dAbs into the supernatant was ensured by fusion of the dAb gene to the
universal GAS leader signal peptide (see WO2005093074) at the N-terminal end. In addition, a
FLAG-tag was ed at the C-terminal end of the dAbs.
Subcloning of the dAb genes was performed by isolating pDOM4 DNA from the cells infected
by the ed splaying fd-phage using a P™ Spin MINIPREP™ kit in accordance with
the manufacturer’s instructions (cat. no. 27104, Qiagen). The DNA was ied by PCR using
biotinylated oligonucleotides DOM57 (5’ TTGCAGGCGTGGCAACAGCG-3’ (SEQ ID NO:197) and DOM6
(5’-CACGACGTTGTAAAACGACGGCC-3’ (SEQ ID NO:198)), digested with SalI and NotI restriction
endonucleases and ligated with pDOM10 digested with SalI and NotI. The ligation products were
transformed by electroporation into E. coli HB2151 cells and plated on TYE plates (Trypton Yeast
Extract) supplemented with 100 µg/ml of carbenicillin (TYE-carb). Individual clones were picked and
expressed in overnight express auto-induction medium (high-level protein sion system,
Novagen), supplemented with 100 µg/ml carbenicillin. in 96-well plates, grown with shaking at
either 30°C or 37°C. These expression plates were then centrifuged at 1800g for 10 minutes. dAb
clones that bound mouse and/or human TGF-β RII/Fc were identified by an ELISA and BIACORE™
(GE HEALTHCARE™) screen or by MSD (Meso Scale ery) binding assay screen. For the ELISA,
96-well MaxisorpTM immuno plates (Nunc, Denmark) were coated with either human or mouse TGF-
β RII/Fc overnight at 4oC. The wells were washed three times with PBST and then blocked with 1%
TWEEN™ in PBS (1%TPBS) for 1 hour at room temperature. The block was removed and a 1:1
mixture of 1%TPBS and dAb supernatant was added for 1 hour at room temperature. The plate was
washed three times with PBST and the detection antibody (Monoclonal anti-FLAG oxidase
antibody, Sigma-Aldrich, UK) was added and incubated for 1 hour at room temperature. The plates
were developed using a colourimetric substrate (SUREBLUE™ 1-component TMB Microwell
Peroxidase solution, KPL, Maryland, USA) and the optical density (OD) measured at 450 nM, the
OD450 being proportional to the amount of bound ion antibody. For BIACORE™, atants
were d 1:1 in HBS-EP buffer and screened on BIACORE™ for binding to biotinylated human
and mouse TGF-β RII/Fc (SA chip coated with 1500 Ru biotinylated hRII-Fc and 1550 Ru
biotinylated mRII-Fc in accordance with the manufacturer’s recommendations) (BIACORE™, GE
HEALTHCARE™). Samples were run on BIACORE™ at a flow rate of 50 µl / min.
Naïve human selections and ing
Selection of dAbs which bind human TGFbetaRII
Naïve selections were performed as described for mouse TGFbRII but using 150 and 15 nM
biotinylated human TGFbRII/Fc at round one and two, respectively. A third round was performed
using the same method as for round two, but with 1.5 nM biotinylated human TGFbRII/Fc.
The third round outputs were cloned from the fd-phage vector, pDOM4 into pDOM10.
Subcloning of the dAb genes was performed by isolating pDOM4 DNA from the cells infected by the
selected dAb-displaying fd-phage using a P™ Spin MIDIPREP™ kit in accordance with the
manufacturer’s instructions (cat. no. 27104, Qiagen). The plasmid DNA was digested with SalI and
NotI restriction endonucleases and the dAb gene insert ligated with pDOM10 digested with SalI,
NotI and PstI restriction endonucleases. The ligation products were transformed by electroporation
into E. coli HB2151 cells and plated on TYE plates (Trypton Yeast Extract) mented with 100
µg/ml of carbenicillin (TYE-carb). Individual clones were picked and sed in 96-well plates at
250 rpm, 30°C 72 hours, in 1 ml/well overnight express auto-induction medium (Novagen)
supplemented with 100 µg/ml carbenicillin. These plates were then centrifuged at 1800g for 10
minutes. The soluble dAb supernatants were screened for antigen g in the I MSD
binding assay combined with the fluorescent polarization concentration ination assay. The
number of human TGFbRII binders was high and there were too many clones to take forward for
further characterization. Therefore, a subset of clones was sequenced and those with unique
ces were further characterized.
TGFβRII MSD binding assay
This assay was used to determine the binding activity of anti-TGFbRII dAbs. TGFbRII-Fc antigen
was coated onto a MSD plate, which was subsequently blocked to prevent non-specific binding.
Serially diluted supernatants containing soluble FLAG-tagged dAb were added. After incubation, the
plate was washed and only dAbs that bound specifically to TGFBRII-Fc remained bound to the plate.
Bound dAbs were detected with a ruthenylated anti-FLAG tagged antibody and MSD read buffer. If
the concentration of the dAbs in the supernatant dilutions was determined using the Fluorescent
Polarisation Concentration Determination assay, then tration binding curves were plotted.
0.5 ul per well of either 60 µg/ml human TGFbRII-Fc, 60 µg/ml mouse TGFbRII-Fc or 60
ug/ml human IgG1 Fc (R&D systems, catalogue number 110-HG) was d onto 384 well MSD
high bind plates (Meso Scale Discovery). The plates were air-dried at room temperature for a
m of four hours and no longer than overnight. The plates were blocked with 50 ul per well of
% MARVEL™ in Tris buffered saline (TBS) + 0.1 % TWEEN™ 20 for either 1 hour at room
temperature or overnight at 4°C. The blocking reagent was removed from the wells by flicking the
plates. A 1:3 dilution series of the the dAb supernatants was prepared in 2xTY medium. The dAbs
were expressed in the pDOM10 sion vector so were the dAb n was expressed as a FLAG
fusion protein. The blocking reagent was removed and 10 ul per well of the diluted dAb
supernatants were transferred to the blocked MSD plates. The dAbs supernatants were screened as
either 4 point curves or as 11 point curves. In addition to the d dAb supernatants, two controls
were ed in each plate, one low control (normalised to 0% binding), with no TGFbRII binding
specificity and a high control (normalised to 100% binding) with high TGFbRII binding specificity,
data not shown.
The plates were incubated with the dAb supernatants and the control samples for one hour
at room temperature and then washed three times with 50 ul per well of TBS + 0.1% TWEEN™. 15
l of ruthenylated anti-FLAG antibody was added to the plates and incubated for one hour at
room temperature. The anti-FLAG antibody (anti-FLAG M2 monoclonal antibody, Sigma, UK,
catalogue number F3165) was conjugated to ruthenium II tris-bipyridine N-hydroxy succinimide
following the manufacturer’s instructions (Meso Scale Discovery, catalogue number R91BN-1). The
ylated LAG antibody was added to all wells except to the mouse anti-human IgG1 Fc
antibody control wells. d, 15 ul /well of anti-Mouse MSD tag (Meso Scale ery, catalogue
number R31AC-1) were added. The anti-Mouse MSD tag was diluted in 2% MARVEL™ in TBS +
0.1% TWEEN™ 20 to a final concentration of 750 ng/ml. The plates were incubated at room
ature for one hour and washed three times with 50 ul per well of TBS + 0.1% TWEEN™. 35
ul 1x MSD read buffer (Meso Scale Discovery) was added to each well and the plates were read on a
MSD Sector 6000 reader (Meso Scale Discovery).
Data were analysed using XC50 ty Base. All data was normalised to the mean of the
high and low control wells on each plate, with the low control normalised to 0% binding and the
high control normalised to 100% binding. A four parameter curve fit was applied to the normalised
data and concentration binding curves using dAb concentrations calculated using the Fluorescent
Polarisation Concentration Determination of dAbs in supernatants assay, were plotted.
The four parameter fit used was as follows:
= + , where a is the minimum, b is the Hill slope, c is the XC50 and d is the
maximum.
Fluorescence Polarisation Concentration Determination of dAbs in Supernatants Assay
This assay allows the concentration of soluble FLAG-tagged dAbs expressed in atants to be
determined. A fluorescently labelled-FLAG peptide was mixed with an anti-FLAG antibody. The
fluorescent molecules were excited with polarised light at a ngth of 531 nM and the emitted
polarised light was read at a wavelength of 595 nM. The addition of a FLAG-tagged dAb resulted in
the displacement of the fluorescent peptide from the anti-FLAG antibody which in turn resulted in
reduced polarisation of the emission signal. A standard curve of known trations of purified
agged VH dummy dAb was ed and was used to back calculate the concentration of the
soluble dAbs in the supernatants. The concentration data was combined with binding activity data,
allowing tration binding curves to be plotted for dAb supernatants.
The dAb supernatants were serially diluted 1:2 in 2xTY medium (1:2, 1:4, 1:8 and 1:16),
followed by a 1:10 dilution in phosphate buffered saline (PBS). The d supernatants were
transferred to a black 384 well plate. A standard curve was set up by serially ng purified VH
Dummy dAb 1:1.7 in 10% v/v 2xTY medium in PBS. The highest dAb concentration was 10 uM and
there were 16 dilutions in total. 5 ul of each dilution was transferred to the 384 well plate. A mixture
of 5 nM FLAG peptide labelled at the c-terminus with Cy3b, 100 mM anti-FLAG M2 monoclonal
antibody (Sigma, catalogue number F3165), 0.4 mg/ml bovine serum albumin (BSA) in 2 mM CHAPs
buffer was prepared. 5 ul of the mixture was transferred to the wells containing the diluted dAbs
(both supernatants and standard curve wells). The plate was centrifuged at 1000 rpm (216 g) for 1
minute and then incubated in the dark at room temperature for 15 minutes. The plates were read
on an ENVISION™ reader (Perkin Elmer) fitted with the ing filters;
Excitation filter: BODIPY TMR FP 531
Emission filter 1: BODIPY TMR FP P pol 595
Emission filter 2: BODIPY TMR FP P pol 595
Mirror: BODIPY TMR FP Dual Enh
The rd curve was plotted and used to back calculate the concentrations of the soluble
dAbs in the supernatants.
Mouse and human TGF-β RII/Fc-binding dAbs identified in the ELISA, BIACORE™ and MSD
binding assays were expressed in overnight express autoinduction medium (ONEX™, Novagen) at
either 30°C for 48 to 72 hours. The cultures were centrifuged (4,600 rpm for 30 minutes) and the
supernatants were incubated with STREAMLINE™-protein A beads ham Biosciences, GE
HEALTHCARE™, UK. Binding capacity: 5 mg of dAb per ml of beads), either ght at 4°C or at
room temperature for at least one hour. The beads were packed into a chromatography column
and washed with either 1x or 2xPBS, ed by 10 or 100 mM Tris-HCl pH 7.4 (Sigma, UK). Bound
dAbs were eluted with 0.1 M glycine-HCl pH 2.0 and lized with 1M Tris pH 8.0. The OD at 280
nm of the dAbs was measured and protein concentrations were determined using extinction
coefficients calculated from the amino acid compositions of the dAbs.
The amino acid and nucleic acid sequences of the anti-human and urine TGFRII dAb
naive leads are given below.
Dom23h 802 amino acid sequence (SEQ ID NO:1)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSEGTMWWVRQAPGKGLEWVSAILAAGSNTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKKRQERDGFDYWGQGTLVTVSS
Dom23h 802 nucleic acid sequence (SEQ ID NO:39)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTAGTGAGGGGACGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGCTATTTTGGCTGCTGGTTCTAATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAAAAGAGGCAGGAGCGGGATGGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
Dom23h 803 amino acid sequence (SEQ ID NO:2)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAGRMWWVRQAPGKGLEWVSAINRDGTRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKHDDGHGNFDYWGQGTLVTVSS
Dom23h 803 nucleic acid sequence (SEQ ID NO:40)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTAGTGCTGGGCGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGCGATTAATCGGGATGGTACTAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACATGATGATGGTCATGGTAATTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
-813 amino acid sequence (SEQ ID NO:3)
EVQLLESGGGLVQPGGSLRLSCAASGSTFTDDRMWWVRQAPGKGLEWVSAIQPDGHTTYYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAEQDVKGSSSFDYWGQGTLVTVSS
DOM23h-813 nucleic acid ce (SEQ ID NO:41)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGATGATAGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGCTATTCAGCCTGATGGTCATACGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGGAACAGGATGTTAAGGGGTCGTCTTCGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCG
AGC
DOM23h-815 amino acid sequence (SEQ ID NO:4)
EVQLLESGGGLVQPGGSLRLSCAASGFTFAEDRMWWVRQAPGKGLEWVSAIDPQGQHTYYADSVKGRFTISRD
YLQMNSLRAEDTAVYYCAKQSTGSATSDYWGQGTLVTVSS
DOM23h-815 nucleic acid sequence (SEQ ID NO:42)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGCGGAGGATCGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGCTATTGATCCTCAGGGTCAGCATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACAGTCTACTGGGTCTGCTACGTCTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
DOM23h-828 amino acid sequence (SEQ ID NO:5)
EVQLLESGGGLVQPGGSLRLSCAASGFTFMSYRMWWVRQAPGKGLEWVSAISPSGSDTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQVVEYSRTHKGVFDYWGQGTLVTVSS
DOM23h-828 nucleic acid sequence (SEQ ID NO:43)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTATGAGTTATAGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGCTATTTCTCCGAGTGGTAGTGATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACAGGTGGTGGAGTATTCGCGTACTCATAAGGGTGTGTTTGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGC
DOM23h-830 amino acid sequence (SEQ ID NO:6)
EVQLLESGGGLVQPGGFLRLSCAASGFTFEGYRMWWVRQAPGKGLEWVSAIDSLGDRTYYADSVKGRFTISRD
YLQMNSLRAEDTAVYYCAKQGLTHQSPSTFDYWGQGTLVTVSS
DOM23h-830 nucleic acid sequence (SEQ ID NO:44)
CAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTTCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGAGGGGTATAGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
CTATTGATTCTCTGGGTGATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACAGGGGCTTACGCATCAGTCTCCGAGTACTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCG
TCTCGAGC
DOM23h-831 amino acid sequence (SEQ ID NO:7)
EVQLLESGGGLVQPGGSLRLSCAASGFTFEAYKMTWVRQAPGKGLEWVSYITPSGGQTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKYGSSFDYWGQGTLVTVSS
DOM23h-831 nucleic acid sequence (SEQ ID NO:45)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGAGGCGTATAAGATGACGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTGGAGTGGG
TCTCATATATTACGCCGTCTGGTGGTCAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAATATGGTTCGAGTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
-840 amino acid sequence (SEQ ID NO:8)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGDGRMWWVRQAPGKGLEWVSAIEGAGSDTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQASRNSPFDYWGQGTLVTVSS
DOM23h-840 nucleic acid sequence (SEQ ID NO:46)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGGGGATGGTCGTATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
CTATTGAGGGGGCGGGTTCGGATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACAGGCGTCGCGGAATTCGCCGTTTGACTACTGGGGTCAGGGGACCCTGGTCACCGTCTCGAGC
DOM23h-842 amino acid sequence (SEQ ID NO:9)
EVQLLESGGGLVQPGGSLRLSCAASGFTFDDSEMAWARQAPGKGLEWVSLIRRNGNATYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKVTKDRSVLFDYWGQGTLVTVSS
DOM23h-842 nucleic acid sequence (SEQ ID NO:47)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGATGATAGTGAGATGGCGTGGGCCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCACTTATTCGGCGTAATGGTAATGCTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAAGTTACGAAGGATCGTTCTGTGCTTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGA
DOM23h-843 amino acid sequence (SEQ ID NO:10)
EVQLLESGGGLVQPGGSLRLSCAASGFTFDQDRMWWVRQAPGKGLEWVSAIESGGHRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQNESGRSGFDYWGQGTLVTVSS
DOM23h-843 c acid sequence (SEQ ID NO:48)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGATCAGGATCGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGCTATTGAGAGTGGTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACAGAATGAGTCGGGGCGTTCGGGTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCG
DOM23h-850 amino acid sequence (SEQ ID NO:11)
EVQLLESGGGLVQPGGSLRLSCAASGFTFDAARMWWARQAPGKGLEWVSAIADIGNTTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQSGSEDHFDYWGQGTLVTVSS
DOM23h-850 c acid sequence (SEQ ID NO:49)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGATGCGGCTAGGATGTGGTGGGCCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGCGATTGCGGATATTGGTAATACTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACAGTCTGGTTCGGAGGATCATTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
DOM23h-854 amino acid sequence (SEQ ID NO:12)
EVQLLESGGGLVQPGGSLRLSCAASGFTFAQDRMWWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQDLHGTSSLFDYWGQGTLVTVSS
DOM23h-854 nucleic acid ce (SEQ ID NO:50)
GAGGTGCAGCTGTTGGAGTCCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGCTCAGGATCGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACAGGATTTGCATGGTACTAGTTCTTTGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCT
CGAGC
DOM23h-855 amino acid sequence (SEQ ID NO:13)
EVQLLESGGGLVQPGGSLRLSCAASGFTFENTSMGWVRQAPGKGLEWVSRIDPKGSHTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQRELGKSHFDYWGQGTLVTVSS
DOM23h-855 nucleic acid sequence (SEQ ID NO:51)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGAGAATACGAGTATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCACGTATTGATCCTAAGGGTAGTCATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAATACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACAGCGTGAGTTGGGTAAGTCGCATTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCG
DOM23h-865 amino acid sequence (SEQ ID NO:14)
EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYEMTWVRQAPGKGLEWVSKIDPSGRFTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGRTDLQLFDYWGQGTLVTVSS
DOM23h-865 nucleic acid sequence (SEQ ID NO:52)
CAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
ATTCACCTTTCGTAGTTATGAGATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGT
CTCAAAGATTGATCCTTCGGGTCGTTTTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCG
CGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTG
TGCGAAAGGTCGGACGGATCTTCAGCTTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
DOM23h-866 amino acid sequence (SEQ ID NO:15)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYWMRWARQAPGKGLEWVSYITPKGDHTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAESLHNERVKHFDYWGQGTLVTVSS
DOM23h-866 nucleic acid sequence (SEQ ID NO:53)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTTCGAATTATTGGATGCGGTGGGCCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCATATATTACTCCTAAGGGTGATCATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCG
CGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTG
TGCGGAATCGCTTCATAATGAGCGTGTTAAGCATTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTC
GAGC
-874 amino acid sequence (SEQ ID NO:16)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTSYRMWWVRQAPGKGLEWVSVIDSTGSATYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQQAGSAMGEFDYWGQGTLVTVSS
DOM23h-874 nucleic acid sequence (SEQ ID NO:54)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTACTAGTTATCGTATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGT
CTCAGTTATTGATTCTACTGGTTCGGCTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCG
CGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTG
TGCGAAACAGCAGGCTGGGAGTGCGATGGGGGAGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCT
CGAGC
DOM23h-883 amino acid sequence (SEQ ID NO:17)
EVQLLESGGGLVQPGGSLRLSCAASGFTFVNYRMWWVRQAPGKGLEWVSAISGSGDKTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKHGLSFDYWGQGTLVTVSS
DOM23h-883 nucleic acid sequence (SEQ ID NO:55)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGTTAATTATCGTATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGT
CTCAGCTATTAGTGGTAGTGGTGATAAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCG
CGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTG
TGCGAAACATGGGCTGTCGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
DOM23h-903 amino acid ce (SEQ ID NO:18)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNDMRMWWVRQAPGKGLEWVSVINADGNRTYYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKDGLPFDYWGQGTLVTVSS
DOM23h-903 nucleic acid sequence (SEQ ID NO:56)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTAATGATATGAGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGTGATTAATGCTGATGGTAATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAAGATGGGCTGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
DOM23m-4 amino acid sequence (SEQ ID NO:19)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTTYGMGWVRQAPGKGLEWVSWIEKTGNKTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKAGRHIKVRSRDFDYWGQGTLVTVSS
DOM23m-4 nucleic acid sequence (SEQ ID NO:57)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
ATTCACCTTTACGACTTATGGTATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCATGGATTGAGAAGACGGGTAATAAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAAGCGGGGAGGCATATTAAGGTGCGTTCGAGGGATTTTGACTACTGGGGTCAGGGAACCCTGGTC
ACCGTCTCGAGC
DOM23m-29 amino acid sequence (SEQ ID NO:20)
EVQLLESGGGLVQPGGSLRLSCAASGFTFKRYSMGWVRQAPGKGLEWVSVINDLGSLTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGNISMVRPGSWFDYWGQGTLVTVSS
DOM23m-29 nucleic acid sequence (SEQ ID NO:58)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTAAGAGGTATTCTATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGTTATTAATGATCTGGGTAGTTTGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAAGGGAATATTAGTATGGTGAGGCCGGGGAGTTGGTTTGACTACTGGGGTCAGGGAACCCTGGTC
ACCGTCTCGAGC
DOM23m-32 amino acid sequence (SEQ ID NO:21)
EVQLLESGGGLVQPGGSLRLSCAASGFTFFEYPMGWVRQAPGKGLEWVSVISGDGQRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKSHTGTVRHLETFDYWGQGTLVTVSS
DOM23m-32 nucleic acid sequence (SEQ ID NO:59)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTTTTGAGTATCCTATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGT
CTCAGTTATTAGTGGGGATGGTCAGCGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCG
CGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTG
AAGTCATACGGGGACTGTGAGGCATCTGGAGACGTTTGACTACTGGGGTCAGGGAACCCTGGTCA
CCGTCTCGAGC
DOM23m-62 amino acid sequence (SEQ ID NO:22)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGQESMYWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRD
YLQMNSLRAEDTAVYYCAKSGTRIKQGFDYWGQGTLVTVSS
DOM23m-62 nucleic acid sequence (SEQ ID NO:60)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGGTCAGGAGAGTATGTATTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAAAGTGGTACGCGGATTAAGCAGGGTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCG
DOM23m-71 amino acid sequence (SEQ ID NO:23)
EVQLLESGGGLVQPGGSLRLSCAASGFTFMDYRMYWVRQAPGKGLEWVSGIDPTGLRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKIKWGEMGSYKTFDYWGQGTLVTVSS
DOM23m-71 nucleic acid sequence (SEQ ID NO:61)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTATGGATTATAGGATGTATTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGT
CTCAGGGATTGATCCTACTGGTTTGCGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCG
CGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTG
TGCGAAAATTAAGTGGGGGGAGATGGGGAGTTATAAGACTTTTGACTACTGGGGTCAGGGAACCCTGGTCA
CCGTCTCGAGC
DOM23m-72 amino acid sequence (SEQ ID NO:24)
EVQLLESGGGLVQPGGSLRLSCAASGFTFMDYDMSWVRQAPGKGLEWVSMIREDGGKTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKARVPYRRGHRDNFDYWGQGTLVTVSS
DOM23m-72 nucleic acid sequence (SEQ ID NO:62)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTATGGATTATGATATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGT
CTCAATGATTCGTGAGGATGGTGGTAAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCG
CGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTG
TGCGAAAGCGAGGGTGCCTTATCGGCGTGGGCATAGGGATAATTTTGACTACTGGGGTCAGGGAACCCTGG
TCACCGTCTCGAGC
DOM23m-81 amino acid sequence (SEQ ID NO:25)
EVQLLESGGGLVQPGGSLRLSCAASGFTFEPVIMGWVRQAPGKGLEWVSAIEARGGGTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKPGRHLSQDFDYWGQGTLVTVSS
DOM23m-81 c acid ce (SEQ ID NO:63)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
ATTCACCTTTGAGCCGGTTATTATGGGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAGCTATTGAGGCGCGGGGTGGGGGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCC
CGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTAC
TGTGCGAAACCTGGGCGGCATCTTAGTCAGGATTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCG
DOM23m-99 amino acid sequence (SEQ ID NO:26)
EVQLLESGGGLVQPGGSLRLSCAASGFTFDRYRMMWVRQAPGKGLEWVSTIDPAGMLTY
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRLASRSHFDYWGQGTLVTVSS
DOM23m-99 nucleic acid sequence (SEQ ID NO:64)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGATCGGTATCGTATGATGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG
TCTCAACGATTGATCCTGCTGGTATGCTTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAAAGGCTGGCTTCGCGGAGTCATTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
DOM23m-101 amino acid sequence (SEQ ID NO:27)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYDMAWVRQAPGKGLEWVSRIRSDGVRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKDRAKNGWFDYWGQGTLVTVSS
DOM23m-101 nucleic acid sequence (SEQ ID NO:65)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGCGCAGC
CTCCGGATTCACCTTTTCTGAGTATGATATGGCTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTTGAGTGGGT
CTCACGGATTCGTTCTGATGGTGTTAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCG
CGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTG
AGATCGTGCTAAGAATGGTTGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
DOM23h-352 amino acid sequence (SEQ ID NO:28)
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYKMAWVRQAPGKGLEWVSLIFPNGVPTYYANSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKYSGQGRDFDYWGQGTLVTVSS
DOM23h-352 nucleic acid sequence (SEQ ID NO:66)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGATAAGTATAAGATGGCTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTGGAGTGGG
TCTCACTTATTTTTCCGAATGGTGTTCCTACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCCG
CGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTG
ATATAGTGGTCAGGGGCGGGATTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
The CDRs as defined by Kabat of these uman and anti-murine TGFRII dAb naive leads
are shown in Tables 1 and 2, below, respectively.
Table 1. CDR ces of anti-human TGFβRII dAbs
Clone CDR1 CDR2 CDR3
DOM23h-802 SEGTMW AILAAGSNTYYADSVKG GFDY
(SEQ ID NO:77) (SEQ ID NO:113) (SEQ ID NO:149)
DOM23h-803 SAGRMW AINRDGTRTYYADSVKG HDDGHGNFDY
(SEQ ID NO:78) (SEQ ID NO:114) (SEQ ID NO:150)
DOM23h-813 TDDRMW AIQPDGHTTYYADSVKG EQDVKGSSSFDY
(SEQ ID NO:79) (SEQ ID NO:115) (SEQ ID NO:151)
DOM23h-815 AEDRMW AIDPQGQHTYYADSVKG QSTGSATSDY
(SEQ ID NO:80) (SEQ ID NO:116) (SEQ ID NO:152)
DOM23h-828 MSYRMW AISPSGSDTYYADSVKG QVVEYSRTHKGVFDY
(SEQ ID NO:81) (SEQ ID NO:117) (SEQ ID NO:153)
DOM23h-830 EGYRMW AIDSLGDRTYYADSVKG QGLTHQSPSTFDY
(SEQ ID NO:82) (SEQ ID NO:118) (SEQ ID NO:154)
DOM23h-831 EAYKMT YITPSGGQTYYADSVKG YGSSFDY
(SEQ ID NO:83) (SEQ ID NO:119) (SEQ ID NO:155)
-840 GDGRMW AIEGAGSDTYYADSVKG QASRNSPFDY
(SEQ ID NO:84) (SEQ ID NO:120) (SEQ ID NO:156)
DOM23h-842 DDSEMA LIRRNGNATYYADSVKG VTKDRSVLFDY
(SEQ ID NO:85) (SEQ ID NO:121) (SEQ ID NO:157)
DOM23h-843 DQDRMW AIESGGHRTYYADSVKG QNESGRSGFDY
(SEQ ID NO:86) (SEQ ID NO:122) (SEQ ID )
-850 DAARMW AIADIGNTTYYADSVKG HFDY
(SEQ ID NO:87) (SEQ ID NO:123) (SEQ ID )
DOM23h-854 AQDRMW GSTYYADSVKG QDLHGTSSLFDY
(SEQ ID NO:88) (SEQ ID NO:124) (SEQ ID NO:160)
DOM23h-855 ENTSMG RIDPKGSHTYYADSVKG QRELGKSHFDY
(SEQ ID NO:89) (SEQ ID NO:125) (SEQ ID NO:161)
DOM23h-865 RSYEMT KIDPSGRFTYYADSVKG GRTDLQLFDY
(SEQ ID NO:90) (SEQ ID NO:126) (SEQ ID NO:162)
DOM23h-866 SNYWMR YITPKGDHTYYADSVKG SLHNERVKHFDY
(SEQ ID NO:91) (SEQ ID NO:127) (SEQ ID NO:163)
DOM23h-874 TSYRMW VIDSTGSATYYADSVKG QQAGSAMGEFDY
(SEQ ID NO:92) (SEQ ID NO:128) (SEQ ID NO:164)
DOM23h-883 VNYRMW AISGSGDKTYYADSVKG HGLSFDY
(SEQ ID NO:93) (SEQ ID NO:129) (SEQ ID )
-903 NDMRMW VINADGNRTYYADSVKG DGLPFDY
(SEQ ID NO:94) (SEQ ID ) (SEQ ID NO:166)
Table 2. CDR Sequences of anti-murine TGFβRII dAbs
Clone CDR1 CDR2 CDR3
DOM23m-4 TTYGMG WIEKTGNKTYYADSVKG AGRHIKVRSRDFDY
(SEQ ID NO:95) (SEQ ID NO:131) (SEQ ID NO:167)
DOM23m-29 KRYSMG VINDLGSLTYYADSVKG GNISMVRPGSWFDY
(SEQ ID NO:96) (SEQ ID NO:132) (SEQ ID NO:168)
DOM23m-32 FEYPMG VISGDGQRTYYADSVKG SHTGTVRHLETFDY
(SEQ ID NO:97) (SEQ ID NO:133) (SEQ ID NO:169)
DOM23m-62 GQESMY AISGSGGSTYYADSVKGR SGTRIKQGFDY
(SEQ ID NO:98) (SEQ ID NO:134) (SEQ ID NO:170)
DOM23m-71 MDYRMY LRTYYADSVKG IKWGEMGSYKTFDY
(SEQ ID NO:99) (SEQ ID NO:135) (SEQ ID NO:171)
DOM23m-72 MDYDMS MIREDGGKTYYADSVKGR ARVPYRRGHRDNFDY
(SEQ ID NO:100) (SEQ ID NO:136) (SEQ ID NO:172)
DOM23m-81 EPVIMG AIEARGGGTYYADSVKG PGRHLSQDFDY
(SEQ ID NO:101) (SEQ ID NO:137) (SEQ ID NO:173)
DOM23m-99 DRYRMM TIDPAGMLTYYADSVKG RLASRSHFDY
(SEQ ID NO:102) (SEQ ID NO:138) (SEQ ID NO:174)
DOM23m-101 SEYDMA VRTYYADSVKG DRAKNGWFDY
(SEQ ID NO:103) (SEQ ID NO:139) (SEQ ID )
DOM23h-352 DKYKMA LIFPNGVPTYYANSVKG YSGQGRDFDY
(SEQ ID NO:104) (SEQ ID NO:140) (SEQ ID NO:176)
Example 2. DSC (differential scanning calorimetry) – naive clones
dAbs thermal stability was determined using Differential Scanning Calorimetry (DSC). dAbs were
dialysed overnight into PBS to a final tration of 1mg/ml. The dialysis buffer was used as a
reference for all s. DSC measurements were performed using the GE CARE™-
AL™VP-DSC capillary cell microcalorimeter, at a heating rate of 180˚C/hour. A typical scan
range was from 20-90˚C for both the reference buffer and the protein . A rescan was
performed each time in order to assess the extent of protein refolding under these experimental
conditions. After each protein sample scan, the capillary cell was cleaned with a solution of 5%
DECON™ (Fisher-Scientific) in water followed by a PBS scan. Resulting data traces were analyzed
using Origin 7.0 software. The DSC trace obtained from the reference buffer scan was subtracted
from that of the protein sample scan. The precise molar concentration of the protein sample was
entered into the data analysis routine to yield values for melting temperature (Tm), py (∆H)
and Van’t Hoff enthalpy (∆Hv) values. Data were fitted to a nonstate model (N2M). The best fit
was obtained with either 1 or 2 transition events. The Tm values obtained for the dAbs described in
this patent range from 52.1˚C to 73.3˚C. Tm values and percentage of refolding are shown in Table
Table 3
dAb Name DSC Apparent Tm ˚C % refolding
1-transition N2M 2-transition N2M
Tm Tm1 Tm2
DOM23h-802 - 56.28 57.54 0
DOM23h-803 - 61.19 64.59 23
DOM23h-813 52.11 - - 100
DOM23h-815 65.13 - - 93
DOM23h-828 - 60.86 59.40 0
-830 - 57.01 58.15 0
DOM23h-831 - 55.29 57.19 0
DOM23h-840 63.70 - - 100
DOM23h-842 63.08 - - 27
DOM23h-843 60.15 - 850 58.27 - - 60
DOM23h-854 - 55.31 58.20 30
-855 70.32 - - 88
DOM23h-865 63.02 - - 0
DOM23h-866 - 52.88 55.77 18
DOM23h-874 - 58.83 60.15 0
DOM23h-883 - 66.78 59.14 0
DOM23h-903 - 59.11 61.98 24
DOM23m-4 - 57.1 61.3 0
DOM23m-29 68 - 32 - 70.4 73.3 25
DOM23m-62 - - - -
DOM23m-71 63 - - 0
DOM23m-72 - - - -
DOM23m-81 - - - -
DOM23m-99 - 58.5 59 0
DOM23m-101 64 - - 30
DOM23m-352 66 - - 50
All molecules maintain tertiary structure up to at least 52°C upon heating.
Example 3. SEC-MALS (size exclusion chromatography with multi-angle-LASER-light
scattering) – naive clones
To determine whether dAbs are monomeric or form higher order ers in solution, they
were analyzed by SEC-MALLS (Size Exclusion Chromatography with Multi-Angle-LASER-Light-
Scattering). Agilent 1100 series HPLC system with an autosampler and a UV detector olled by
Empower re) was connected to Wyatt Mini Dawn Treos (Laser Light Scattering (LS) detector)
and Wyatt Optilab rEX DRI rential Refractive Index (RI) detector). The detectors were
connected in the following order -UV-LS-RI. Both RI and LS instruments operate at a wavelength of
658nm; the UV signal was monitored at 280nm and 220nm. Domain antibodies (100 microliters
ion at a concentration of 1mg/mL in PBS) were separated according to their hydrodynamic
properties by size exclusion chromatography using a GE HEALTHCARE™ 10/300 Superdex 75
column. The mobile phase was PBS plus 10% ethanol. The intensity of the red light while
n passed through the detector was measured as a function of angle. This measurement taken
together with the protein concentration ined using the RI detector allowed calculation of the
molar mass using appropriate equations (integral part of the analysis software Astra v.5.3.4.14). All
the dAbs described herein have a monomeric content ranging from 65% to 98%. Data is shown in
Table 4.
Table 4
dAb name Monomer by SEC-MALLS (%)
DOM23h-802 92.5
DOM23h-803 96.4
DOM23h-813 96.6
-815 98
DOM23h-828 80
DOM23h-830 65
DOM23h-831 72
DOM23h-840 91
DOM23h-842 91.6
DOM23h-843 90.2
DOM23h-850 97.7
DOM23h-854 83.4
DOM23h-855 96.3
DOM23h-865 83
DOM23h-866 92.4
DOM23h-874 92.6
-883 93.5
DOM23h-903 96.5
DOM23m-4* 93
DOM23m-29* 95
-32* 92
DOM23m-62 Not determined
DOM23m-71* 88
DOM23m-72 Not determined
DOM23m-81 Not ined
DOM23m-99 79
DOM23m-101 77.4
DOM23m-352 93
*These dAbs were run using the same SEC-MALLS set up as bed above except that
the HPLC used was a Shimadzu LC-20AD Prominence system. These dAbs were also run on a
Superdex75 column but the mobile phase buffer was PBS.
The molecules listed in the tables 3 and 4 were chosen on the basis of Solution State (propensity for
monomer) content and Thermal stability. All molecules show a ≥65% propensity for monomerisation
and maintain tertiary structure up to at least 52°C upon heating.
Example 4. Assays for TGFbetaRII inhibition (naive )
MC3T3-E1 luciferase assay – method m1:
The MC3T3-E1 luciferase assay es the ability of dAbs to inhibit TGFβ-induced expression of
CAGA-luciferase in MC3T3-E1 cells. Three copies of a esponsive ce motif, termed a
CAGA box are present in the human PAI-1 promoter and specifically bind Smad3 and 4 proteins.
g multiple copies of the CAGA box into a luciferase reporter construct confers TGFβ
responsiveness to cells transfected with the reporter system. This assay uses MC3T3-E1 cells
(mouse osteoblasts) stably transfected with a [CAGA]12-luciferase reporter construct (Dennler, et al.
(1998) EMBO J. 17, 3091–3100).
e dAbs were tested for their ability to block TGF-β1 signaling via the Smad3/4
pathway.
The protocol used to generate the data which appears as method m1 in table 5, is as
s. Briefly, 2.5 x 104 MC3T3-E1 cells per well in assay medium (RPMI medium (Gibco,
Invitrogen Ltd, Paisley, UK), 10 % heat inactivated foetal calf serum, and 1 %
penicillin/streptomycin) were added to a tissue culture 96 well plate (Nunc), followed by the dAb
and TGF-β1 (final concentration 1 ng/ml) and incubated for six hours at 37°C, 5% CO2. dAbs were
dialysed into PBS prior to being tested in the assay. BRIGHTGLOW™ rase reagent (Promega,
UK) was added to the wells and incubated at room temperature for two minutes to allow the cells to
lyse, and the resulting luminescence measured on a luminometer.
The assay was performed multiple times to obtain an average and range of maximum %
inhibitions values which are summarised in Table 5. This method has been modified and is described
below.
Modified MC3T3-E1 luciferase assay - method m2.
MC3T3-E1 cells were added to 96 well plates (Nunc 13610) at 1.25 x 104 per well in “plating
medium” lpha + Ribonucleosides, + Deoxyribonucleosides (Invitrogen 22571), 5% Charcoal
stripped FCS (Perbio Sciences UK Ltd; 8.03), 1/100 Sodium Pyruvate (Invitrogen11360),
250µg/ml of Geneticin 50mg/ml (Invitrogen, 10131027), and incubated overnight at 37°C, 5% CO2.
The media from the cells was replaced with “assay media” (DMEM (Invitrogen 31966021,) 25mM
Hepes (Invitrogen)), and purified dAbs in PBS at 4x final assay concentration were titrated in “assay
media” and added to the cell plates, followed by TGF-β1 (R&D, 240B) at 4x the EC80. The plates
were incubated for six hours at 37°C, 5% CO2. STEADYLITE™ luciferase reagent (PerkinElmer
6016987) was added to the wells and ted at room temperature for 30 s, and the
resulting scence measured on a the ENVISION™ plate reader.
Each dAb was titrated in duplicate in an assay and a maximum % inhibition determined
(n=2). The assay was performed multiple times to obtain an average and range of maximum %
inhibitions values which are summarised in Table 5. The assay QC parameters were met; in-house
small molecule showing an IC50 range from 100 to 900nM for the mouse assays. Also, the robust Z
factors were greater than 0.4 and the TGF-β EC80 was within 6 fold of the concentration added to
the assay.
A549 IL-11 release assay – h1
The A549 Interleukin-11 (IL-11) release assay measures the y of dAbs to inhibit human TGF-β1
stimulated IL-11 e from A549 cells. TGF-β1 binds directly to TGF-βRII and induces the
assembly of the TGF-βRI/II complex. TGF-βRI is phosphorylated and is able to signal through
several pathways ing the Smad4 pathway. Activation of the Smad4 pathway results in the
release of IL-11. The IL-11 is secreted into the cell supernatant and is then measured by
colourmetric ELISA.
e dAbs were tested for their ability to block TGF-β1 ling via the Smad4 pathway.
Briefly, 1x105 A549 cells per well in “assay medium” (DMEM high e medium (GibcoTM,
Invitrogen Ltd, Paisley, UK), 10 % heat inactivated foetal calf serum (PAA, Austria), 10 mM HEPES
(Sigma, UK) and 1 % penicillin/streptomycin (PAA, Austria)) were added to a tissue culture 96 well
plate (Nunc), followed by the dAb and TGF-β1 (final concentration 3 ng /ml) (R&D s,
Abingdon, UK) and incubated overnight at 37°C, 5% CO2. dAbs were dialysed into PBS prior to
being assayed. The concentration of IL-11 released into the supernatant was measured using a
Human IL-11 DUOSET™ (R&D systems, on, UK), in accordance with the manufacturer’s
ctions.
The A549 IL-11 release assay is referred to in tables 5 and 6 as assay method h1. The assay
was performed multiple times to obtain an average and range of maximum % inhibitions values
which are summarised in Table 5. The assay QC parameters were met; in-house small molecule
showing an IC50 range from 50 to 500nM for the human .
SBE-bla HEK 293T Cell Sensor assay – h2:
Members of the Smad family of signal transduction molecules are components of an intracellular
pathway that transmits TGF-β signals from the cell e to nucleus. TGF-β1 binds directly to TGF-
βRII and induces the assembly of the TGF-βRI/II complex. Smad2 and Smad3 are then
phosphorylated by TGF-βRI, and subsequently form a heteromeric complex with the co-smad family
member Smad4. These complexes are translocated to the nucleus where they bind DNA and
regulate gene transcription.
Cell Sensor SBE-bla HEK 293T cells contain a actamase reporter gene under control of
the Smad binding element (SBE) which was stably integrated into HEK 293T cells (Invitrogen, UK).
The cells are responsive to TGF-βI and can be used to detect agonists / antagonists of the Smad2/3
signaling pathway.
Soluble dAbs were tested for their ability to block TGF-β1 signaling via this pathway
following the method below, which was based on an optimised method from Invitrogen, UK, (cell
line K1108).
The assay was med direct from frozen cells which had been grown for at least 4
passages in growth media (DMEM high glucose, Invitrogen 21068028, 10% Dialysed U.S. FBS.
Invitrogen 26400-044, 0.1mM (1/100) Non essential amino acids. Invitrogen 11140-050, 25mM
(1/40) HEPES buffer. Sigma H0887, 1mM (1/100) Sodium pyruvate. Invitrogen 11360-070, 1%
GLUTAMAX™. (200mM Invitrogen 35050038), 5µg/ml of Blasticidin. ogen R21001) and frozen
in house (at 4x107/ml). The cells were plated at 20,000 cells per well in cell culture plates r
3712) in plating media (as above with 1% FCS and no blasticidin). After incubating the cells
overnight, the purified dAbs were diluted in “assay media” (DMEM (Invitrogen 31966021,) 25mM
Hepes (Invitrogen) and added to the cells at 4x final assay concentration. After a 1 hour incubation
at 37°C, TGF-β (R&D Systems; 240B) was added at 4x EC80 and incubated for a further 5 hours.
The LIVEBLAZER™ substrate (Invitrogen K1030), was made up according to the manufacturer’s
instructions and added at 8x the volume. The plates were incubated in the dark at room
temperature for 16 hours and read on the ENVISION™ plate reader according to the Invitrogen
protocol.
The a HEK CELLSENSOR™ assay is referred to in tables 5 and 6 as method h2. Each
dAb was titrated in duplicate in an assay and an IC50 determined and maximum % inhibition
determined (n=2). Due to the difficulty of ing full curves in the mouse assay, only %
inhibitions are quoted in table 5. The assay was med le times to obtain an e and
a range of values which are summarised in Tables 5 and 6. The arithmetric mean IC50 was
calculated using pIC50’s (-log of IC50), and the range calculated adding and subtracting the log
standard deviation from mean pIC50, and then transforming back to IC50. The assay QC
parameters were met; se small molecule showing an IC50 range from 50 to 500nM for the
human assays. Also, the robust Z factors were greater than 0.4 and the TGF-β EC80 was within 6
fold of the concentration added to the assay
The s are shown in Tables 5 and 6. .
Table 5. Cell Functional assay data for mouse specific clones plus VH Dummy dAb.
Human IL-11 release (h1) or
Mouse 3T3 cell assay SBE-bla HEK CELLSENSOR™ assay (h2)
max % inhibition max % inhibition
Assay Assay
Method Average SD range n Method Average SD range n
DOM23m- 68.8 -
04 m1 73.3 8.4 83 3 h1 70.7 6.4 67 - 78 3
DOM23m-
04 h2 69.0 1
DOM23m- 50.5 -
29 m1 54.2 5.2 57.9 2
DOM23m- 36.9 -
32 m1 39.7 4.0 42.5 2
DOM23m-
62 m1 78.6 1 h1 79.0 1
DOM23m- 38.3 -
71 m1 44.9 9.3 51.4 2 h1 -2.0 1
DOM23m
72 m1 17.5 24.7 34.9 2 h1 1.7 1
DOM23m
81 m2 30.3 11.5 47 4
DOM23m- 26.7 -
99 m2 46.5 28.0 93.5 6
DOM23m- 22.0 -
101 m2 48.0 19 74.1 12 h2 59.8 27.0 17 - 81 5
DOM23h- 16.8 - 5.7 -
352 m2 48.0 23.9 78.9 16 h2 46.7 36.5 86 5
21 -
VHDUM-2 m2 21.4 13.2 33.9 15 h2 46.0 29.6 17 - 84 6
VHDUM-2 m1 22.5 0 22.5 2
Table 6. Cell Functional data for human specific clones plus VH Dummy dAb.
IC50 nM
Assay IC50 range
method dAb Mean (+/- log SD) n
h2 DOM23h-802 > 11062 6592 - 18562 6
h2 DOM23h-803 > 11619 5890 - 22922 6
h2 -813 > 9328 4301 - 20230 6
h2 DOM23h-815 7122 3026 - 16764 4
h2 DOM23h-828 7 4441 - 22065 4
h2 DOM23h 830 6299 5442 - 7291 4
h2 DOM23h-831 > 3126 534 - 18291 8
h2 DOM23h 840 2915 650 - 13081 7
h2 DOM23h 842 2042 2223 - 18704 4
h2 DOM23h-843 > 9007 3396 -23894 8
h2 DOM23h-850 5350 2358 - 12137 6
h2 DOM23h-854 > 9551 3085 - 29569 8
h2 DOM23h-855 > 4467 1088 - 18339 8
h2 DOM23h 865 5559 1070 - 28893 4
h2 DOM23h 866 > 1762 195 - 15900 6
h2 DOM23h 874 > 925 89 - 9591 6
h2 DOM23h 883 10123 60 - 17344 6
h2 DOM23h 903 1048 492 - 223 5
25000-
h2 VHDummy-2 > 25119 250000 12
The mouse clones were selected on the basis that they showed greater than 40% neutralisation of
TGF-β in several assays. The only exception to this was DOM23m-72. The clones also showed good
neutralisation curves (data not shown). The human clones were selected on the basis that the
e IC50’s were less than 15µM.
Example 5. Error Prone Affinity tion of Naive Clones (from Example 1)
Error-prone mutagenesis was performed to improve the affinity of the dAbs identified as active with
suitable biophysical characteristics (described above).
Phage Library Construction: Error prone libraries of DOM23h-843, DOM23h-850, DOM23h-854,
DOM23h-855, DOM23h-865, DOM23h-866, DOM23h-874, DOM23h-883, DOM23h-439 and DOM23h-
903, were made using GENEMORPH™ II Random Mutagenesis kit (Stratagene, Cat No 200550). The
target dAb genes were amplified by PCR using Taq DNA polymerase and oligonucleotides DOM008
(5’-AGCGGATAACAATTTCACACAGGA-3’ (SEQ ID NO:185)) and DOM009 (5’-
CGCCAGGGTTTTCCCAGTCACGAC-3’ (SEQ ID NO:186)), followed by re-amplification of the diluted
PCR product with oligonucleotides DOM172 (5’ TTGCAGGCGTGGCAACAGCG-3’ (SEQ ID NO:187))
and DOM173 (5’-CACGACGTTGTAAAACGACGGCC-3’ (SEQ ID NO:188)), and MUTAZYME™ II DNA
polymerase, according to manufacturer’s instructions. This PCR product was further amplified using
Taq DNA polymerase and oligonucleotides DOM172 and DOM173, to increase the DNA product yield.
The PCR product was digested with Sal I and Not I restriction endonucleases. Undigested product
and digested ends were removed from the digested product using streptavidin beads (Dynal
Biotech, UK). For the anti-human error prone selections digested product was ligated into pDOM4
phage vector digested with Sal I and Not I restriction endonucleases and used to transform E. coli
TB1 cells. The ormed cells were plated on 2xTY agar supplemented with 15 µg/ml tetracycline,
yielding library sizes of >1×107 transformants.
Human TGFbetaRII specific dAb Error-prone selections: Three rounds of selection were performed
with the DOM23h-843, DOM23h-850, DOM23h-854, DOM23h-855, DOM23h-865, -866,
DOM23h-874, DOM23h-883, DOM23h-903, and -439 libraries. Round one was med
using 1 nM ylated human TGFbetaRII/Fc (N13241-57). Two different methods were followed
for rounds two and three, method 1 using the dimeric TGFbetaRII/Fc form of the n and
method two using the e, monomeric form of TGFbetaRII. Method 1: Round two was
med with 1 nM biotinylated human TGFbetaRII/Fc with 1 uM non-biotinylated human
TGFbetaRII/Fc itor. Round three was performed with 100 pM biotinylated human
aRII/Fc with 1 uM non-biotinylated human TGFbetaRII/Fc (N12717-4). Method 2: Round two
was performed with 1 nM biotinylated human TGFbetaRII with 1 uM non-biotinylated human
TGFbetaRII competitor. Round three was performed with 100 pM biotinylated human TGFbetaRII
with 1 uM otinylated human TGFbetaRII competitor.
Second and third round selection outputs were subcloned into the pDOM13 vector, as described
above. dual clones were picked and expressed in 96 well plates at 850 rpm, 37°C for 24
hours, 90% humidity in 0.5 ml/well overnight s auto-induction medium supplemented with
100 μg/ml carbenicillin. Plates were then centrifuged at 1800g for 10 minutes. Supernatants were
diluted either 1/5 or 1/2 in HBS-EP buffer and screened on E™ for binding to biotinylated
human TGF-β RII/Fc (SA chip coated with 1000 Ru biotinylated hRII-Fc in accordance with the
manufacturer’s recommendations) (BIACORE™, GE HEALTHCARE™). Samples were run on
E™ at a flow rate of 50 μl/min. Clones that bound with a high number of resonance units
(RUs) or with an improved off-rate compared to the parent clone were sed in 50ml overnight
express autoinduction medium at 30°C for 48 to 72 hours and centrifuged at 4,600 rpm for 30
minutes. The supernatants were incubated overnight at 4°C with Streamline-protein A beads. The
beads were then packed into drip s, washed with 5 column volumes of 2xPBS, followed by
one bed volume of 10 mM Tris-HCl pH 7.4 and bound dAbs were eluted in 0.1 M glycine-HCl, pH 2.0
and neutralised with 1 M Tris-HCl, pH 8.0. The OD at 280 nm of the dAbs was measured and protein
concentrations were determined using extinction coefficients calculated from the amino acid
compositions of the dAbs.
In vitro analysis of off rate improved error prone selections: Purified dAbs were subjected to the
same tests as those from the naïve selections, namely, e, a HEK 293T Cell Sensor assay
(h2), DSC, and SEC-MALS. Examples of clones improved over parent are shown in table 6A. IC50
values are a mean of ‘n’ number of experiments.
Table 6A
DOM23h On-rate Off-rate Affinity ka Fold kd Fold KD Fold Mean IC50
ka1 (1/Ms) kd1 (1/s) KD improvement ement improvement (nM)*
439 2.08E+06 5.02E-02 2.42E-08 3570 (3)
439-20 4.43E+06 3.54E-03 7.99E-10 2.1 14.2 30.3 48 (10)
843 05 3.43E-01 3.78E-07 1947 (3)
843-13 5.11E+06 2.11E-02 4.13E-09 5.6 16.2 91.7 540 (4)
855 3.35E+05 3.15E-01 9.41E-07 >25000 (3)
855-21 1.86E+06 3.36E-02 1.80E-08 5.6 9.4 52.3 18580 (6)
* number of experiments for calculation of mean IC50s provided in parenthesis
ty Matured Sequences
DOM23h21 nucleic acid sequence (SEQ ID NO: 203)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTC
TCCTGTGCAGCCTCCGGATTCACCTTTGAGAATACGAGTATGGGTTGGGTCCGCCAGGCT
CCAGGGAAGGGTCTAGAGTGGGTCTCACGTATTGATCCTAAGGGTAGTCATACATACTAC
ACAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAATACGCTGTAT
CTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACAGCGT
GAGTTGGGTAAGTCGTATTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
DOM23h21 amino acid sequence (SEQ ID NO: 204)
EVQLLESGGGLVQPGGSLRLSCAASGFTFENTSMGWVRQAPGKGLEWVSRIDPKGSHTYY
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQRELGKSYFDYWGQGTLVTVSS
DOM23h13 nucleic acid sequence (SEQ ID NO: 205)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCCTGGTACAGCCTGGGGGGTCCCTGCGTCTC
TCCTGTGCAGCCTCCGGATTCACCTTTGATCAGGATCGGATGTGGTGGGTCCGCCAGGCC
AAGGGTCTAGAGTGGGTCTCAGCTATTGAGAGTGGTGGTCATAGGACATACTAC
GCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTAT
CTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAATCAGAAT
AAGTCGGGGCGTTCGGGTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
DOM23h13 amino acid sequence (SEQ ID NO: 206)
EVQLLESGGGLVQPGGSLRLSCAASGFTFDQDRMWWVRQAPGKGLEWVSAIESGGHRTY
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANQNKSGRSGFDYWGQGTLVTVS
DOM23h20 nucleic acid sequence (SEQ ID NO: 207)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTG
TCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACGGCATGCGGCTGGGGTTTCGGGTACTTATTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
GTCTCGAGC
DOM23h20 amino acid sequence (SEQ ID NO: 208)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKRHAAGVSGTYFDYWGQGTLVTVSS
Example 6. Affinity tion of DOM23h7 lineage
DOM23h-271 amino acid sequence (SEQ ID NO:199)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKQIPGRKWTANSRFDYWGQGTLVTVSS
DOM23h-271 nucleic acid sequence (SEQ ID NO:200)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
CGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGATTCCGGGGCGTAAGTGGACTGCTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGC
7 amino acid sequence (SEQ ID )
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKQIPGRKWTANSRFDYWGQGTLVTVSS
DOM23h7 nucleic acid sequence (SEQ ID NO:202)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGATTCCGGGGCGTAAGTGGACTGCTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGC
Domain antibody DOM23h-271(SEQ ID No:199) had been isolated from the phage libraries in a
previous selection campaign and variant DOM23h7 (SEQ ID NO:201) was isolated following
error prone affinity maturation, both as described in DOM23h7 (SEQ ID
NO:201) was selected for further affinity maturation based on its binding kinetics, sequence and
biophysical our. Affinity maturation was performed using degenerative mutagenesis to rediversify
the CDRs, and improved leads were identified using DNA display or phage display. Two
types of libraries were constructed to re-diversify the CDRs, and these are referred to as triplet and
doped libraries. To make the triplet libraries oligonucleotide s were designed to cover each
CDR, and within each primer the codons for three amino acids were replaced with NNS codons, so
that three positions were diversified. Multiple oligonucleotides were used to cover all targeted amino
acids within each CDR: 2 for CDR1, 3 for CDR2 and 6 for CDR3. Complementary ucleotide
primers were used to amplify a sequence fragment containing each mutated CDR, and also an
overlapping ce fragment ng the rest of the dAb coding sequence. These fragments
were mixed and led by splice extension overlap PCR to produce the full length dAb coding
sequence. This product was PCR amplified using primers DOM172 (SEQ ID NO:187) and DOM173
(SEQ ID NO:188), digested with SalI and NotI, and ligated into similarly cut pDOM4 (described
above ) for phage selections, or pIE2A2 (described in WO2006018650) for DNA Display selections.
The doped libraries were constructed using a similar method, ially as described in
WO2006018650. A single degenerate oligonucleotide primer was used to cover all mutations within
each CDR. Within each primer the amino acids to be diversified were specified using degenerate
codons to y multiple amino acids. Five amino acids were diversified in CDR1, 7 in CDR2, and
13 in CDR3. In the primers the following degenerate coding is used: 'a' = 91 %A + 3 %T + 3 %G
+ 3 %C; 'g' = 91 %G + 3 %T + 3 %C + 3 %A; 'c' = 91 %C + 3 %T + 3 %G + 3 %A; 't' = 91
%T + 3 %A + 3 %G + 3 %C; ‘S’ = 50% G and 50% C. Capital letters indicate 100% of the
specified nucleotide. The primers used were:
271-7R1deg CDR1
(GCAGCCTCCGGATTCACCTTTacSgaStatagSATGtgSTGGGTCCGCCAGGCTCCGGGG) (SEQ ID NO:189);
271-7R2deg CDR2
(GGGTCTCGAGTGGGTCTCAgcSATTgaSccSatSggSaaScgSACATACTACGCAGACTCCGTG)
(SEQ ID );
271-7R3deg CDR3:
(GCGGTATATTACTGTGCGAAAcaSatSccSggScgSaaStgSacSgcSaaStcScgSttSGACTACTGGGGTCAGGG)
(SEQ ID NO:191).
The degenerate y primers were used in the same way as the triplet s. Each diversified
CDR was amplified separately and then combined with a parental sequence fragment using splice
ion overlap. The fragments were subcloned to pDOM4 and pIE2A2 using SalI and NotI.
DNA Display
Selections were performed using in vitro compartmentalisation in emulsions and DNA display using
the scArc DNA binding protein essentially as described in WO2006018650. Briefly, TGFbRII-FC
antigen was biotinylated using a 5:1 molar ratio of Biotin and the EZ-LINK™ Sulpho-NHS-LC-Biotin
kit (Thermo #21327). A DNA fragment containing the Arc operator sequences and expression
cassette containing the diversified dAb library was PCR amplified from the pIE2A2 vector using
flanking primers. The product was ed from an eGel (Invitrogen) and diluted to 1.7 or 0.85nM in
1mg/ml BSA. For ion of improved binders the doped and Triplet ies were processed
tely under slightly different conditions. The Triplet CDR libraries were combined to give
pooled CDR 1, 2 or 3 libraries. Ten rounds of ion were used for each type of library. For both
methods, after 2 selection cycles, the diversified CDR’s were amplified and recombined by splice
overlap extension PCR to produce a 4th library with mutations in all 3 CDRs.
For the doped libraries 5x108 copies of DNA were mixed with 50ul of SWAY™ In vitro
translation mix rogen). Each reaction ned 10.0µl SLYD™ extract;10.0µl 2.5x reaction
buffer; l 2x feed buffer; 1.0µl Methionine (75 mM); 1.25µl Amino Acid mix (50 mM); 15µl
H2O; 0.5µl T7 Polymerase; 0.25 µl anti-HA mAb 3F10 (Roche, cat. 1 867 423); and 1.5µl
Glutathione (100 mM) (Sigma). This was added to 800µl of hydrophobic phase (4.5% SPAN™-80,
(Fluka) + 0.5% Triton X-100 (Sigma) in Light white mineral oil (Sigma)) in a 4ml glass vial
(CHROMACOL™ 4SV P837) and stirred at 2000rpm for 4-5 minutes. The tubes were sealed and
ted for 3 hours at 30°C. All subsequent steps were done at room temperature. To extract the
DNA-protein complexes 200µl of C+ buffer (10 mM Tris, 0.1 M KCl, 0.05% -20, 5 mM
MgCl2, 1% BSA, pH 7.4) and 500µl of Hexane was added to the vial, mixed and transferred to a
microtube and centrifuged at 13000g for 1 minute. The organic phase was removed and the
aqueous phase re-extracted with 800µl of Hexane 3-5 more times until the ace was almost
clear. For the first 5 rounds of selections the biotinylated TGFbRII-FC was pre-bound to Streptavidin
DYNAbeads™ (Invitrogen) and added to the extracted complexes to give an n concentration
lent to 40, 40, 10, 5 and 5 nM antigen (rounds 1, 2, 3, 4 and 5 respectively). T1 beads were
used for selections 1-3, and C1 for selections 4 and 5. After 30 minutes incubation the beads were
washed 3-5 times with C+ buffer. The DNA complexes ing bound to the beads were then
recovered by PCR with flanking primers. Selection rounds 6-10 were referred to as ‘soluble’
selections, where the Biotinylated TGFbRII-FC was added directly to the complexes after extraction
to give a concentration of 5, 5, 4, 5 and 5 nM (rounds 6, 7, 8, 9 and 10 respectively), and incubated
for 30 minutes to allow binding to be established. Non-biotinylated TGFbRII-FC was then added as a
competitor to 74, 750, 400, 250 and 250 nM (rounds 6, 7, 8, 9 and 10 respectively), and incubated
for 15, 15, 30, 30 and 30 minutes (rounds 6, 7, 8, 9 and 10 respectively). In rounds 9 and 10 a
double stranded oligonucleotide containing the ARC operator sequence was included at 50nM in the
C+ buffer used in the hexane extractions to reduce cross-reactions between any non-complexed
DNA and excess protein released from the emulsions. After the competition period 10µl of C1
Streptavidin DYNAbeads™ were added. After 10 minutes the beads were washed 5 times with Buffer
C+ and the bound complexes red by PCR with flanking primers as previously described. The
PCR product was purified on an eGel and used for the next selection cycle. Following the 10th
selection the recovered t was cut with SalI and NotI enzymes, and cloned into similarly cut
pDOM13 for expression.
The triplet ies were selected using a similar method, except that 1x109 DNA copies
were used in the first round selection, and 5x108 thereafter. Also, the incubation time for protein
sion in the emulsion was reduced to 2 hours. Soluble Biotinylated TGFbRII FC was used in all
ten selection rounds at 25, 10, 5, 5, 5, 5, 2.5, 2.5, 2.5, 2.5, 2.5, 2.5nM respectively. The Biotinylated
target was incubated with the extracted complexes for 30 minutes. In selection rounds 5-10 the
non-biotinylated TGFbRII-FC competitor was added to a final concentration of 250nM for 15, 30, 60,
60, 75, and 90 minutes respectively before addition of C1 streptavidin DYNAbeads™. In round 5
competition was at room temperature, but from round 6 the competition temperature was increased
to 30°C. The Arc Operator decoy oligo was included in selection rounds 1-4 to reduce cross
complexing of defective DNA.
ing selections the dAb encoding inserts were excised from the DNA display sion
cassettes using SalI and NotI, and cloned into the pDOM13 bacterial expression vector. The dAbs
were sequenced and expressed in TB ONEX™ medium and supernatants were screened by
BIACORE™ to identify clones with improved off-rates when compared to parent. Clones with
improved off-rates were sed and purified, and were assessed for affinity by BIACORE™ and
potency in the cell sensor assay. Clones giving poor kinetic profiles, containing unfavourable
sequence motifs, or giving very low yields were not pursued. Three were selected to be of further
interest. Clones DOM23h21 (SEQ ID NO:29) and DOM23h22 (SEQ ID NO:30) were
isolated from doped library selections. Clone DOM23h27 (SEQ ID NO:31) was isolated from a
triplet library selection. The affinity of the selected clones for human TGFbRII-FC is shown in table
Table 7
Ka(M-1.s-1) Kd (s-1) KD (nM)
DOM23h7* 5.37E+6 5.10E-2 9.49
DOM23h21 6 4 0.147
DOM23h22 6 9.19E-4 0.286
DOM23h27 2.17E+6 1.26E-3 0.578
*N.B. values in the above table are for ranking purposes only since the fitting for DOM23h-
271-7 to the 1:1 model was poor, although the affinity matured samples fitted well to this model.
Phage y:
Triplet or doped libraries in separate CDR1, CDR2 and CDR3 pools were subjected to rounds of
phage selection as described above against either biotinylated human TGF-β RII/Fc antigen over 4
rounds in concentrations of 10 nM, 1 nM, 100 pM and 20 pM respectively, or two rounds of selection
using 20pM followed by 2pM n. Inserts from phage selections were cloned into the pDOM10
expression vector and supernatants with off rates improved over parent were ed for further
study. Domain antibodies were expressed and purified their affinity and ivity against human
TGF-β RII/Fc antigen tested on the BIACORE™ T100 and in the Cell sensor assay described above
(data not . The affinity of the selected clones for human TGFbRII-FC is shown in table 8.
Table 8
Sample Ka (M-1.s-1) Kd (s-1) KD (M)
271-101 3.73E+06 0.02014 5.40E-09
271-102 9.35E+06 0.01531 1.64E-09
271-105a* 3.29E+06 0.00747 2.27E-09
271-105b* 3.07E+06 0.007977 09
271-106a* 7.39E+06 0.02333 3.16E-09
271-106b* 6.77E+06 0.01947 2.88E-09
271-114 1.04E+07 0.07084 6.80E-09
271-7a* 3.04E+06 0.04193 1.38E-08
* 2.42E+06 8 1.84E-08
* The designation “a” and “b” refer to separate supernatants resulting from different colonies of
the numbered clones.
The sequence of the selected clones with improved activity was determined and the full
sequences and CDR ces are shown below.
DOM23h21 amino acid ce (SEQ ID NO:29)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKQMPGRKWTAKFRWDYWGQGTLVIVSS
DOM23h21 c acid sequence (SEQ ID NO:67)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTACCGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGATGCCGGGCCGGAAGTGGACGGCCAAGTTCCGCTGGGACTACTGGGGTCAGGGAACCCTG
GTCATCGTCTCGAGC
DOM23h22 amino acid sequence (SEQ ID NO:30)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKQMPGQKWMAKSRFDYWGQGTLVTVSS
DOM23h22 nucleic acid sequence (SEQ ID NO:68)
CAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGATGCCCGGCCAGAAGTGGATGGCCAAGTCCCGCTTCGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGC
DOM23h27 amino acid ce (SEQ ID NO:31)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGQKTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQIPGRKWTANSRFDYWGQGTLVIVSS
DOM23h27 c acid sequence (SEQ ID NO:69)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCAGAAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGATTCCGGGGCGTAAGTGGACTGCTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTG
GTCATCGTCTCGAGC
DOM23h101 amino acid sequence (SEQ ID NO:32)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKQIPGRKWTANGRKDYWGQGTLVTVSS
DOM23h101 nucleic acid sequence (SEQ ID NO:70)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGATTCCGGGGCGTAAGTGGACTGCTAATGGTCGTAAGGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGC
DOM23h102 amino acid sequence (SEQ ID NO:33)
EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQIPGRKWTANSRFDYWGQGTLVTVSS
DOM23h102 nucleic acid sequence (SEQ ID NO:71)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGATTCCGGGGCGTAAGTGGACTGCTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGC
DOM23h105 amino acid sequence (SEQ ID NO:34)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTY
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQIPGQRWTGNSRFDYWGQGTL
VTVSS
DOM23h105 c acid sequence (SEQ ID NO:72)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGATTCCGGGGCAGCGGTGGACTGGTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGC
106 amino acid sequence (SEQ ID NO:35)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKQFPGRKWTANSRSDYWGQGTLVTVSS
DOM23h106 nucleic acid ce (SEQ ID NO:73)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGTTTCCGGGGCGTAAGTGGACTGCTAATTCGCGGTCTGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGC
DOM23h114 amino acid sequence (SEQ ID NO:36)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKQIPGRKGTANSRFDYWGQGTLVTVSS
DOM23h114 nucleic acid sequence (SEQ ID NO:74)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGATTCCGGGGCGTAAGGGAACTGCTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGC
Table 9. CDR sequences of 271 ty matured clones with improved activity
CDR1 (Kabat 26- CDR2 (Kabat 50-65) CDR3 (Kabat 95-
) 102)
DOM23h- YRMW AIEPIGNRTYYADSVKG QMPGRKWTAKFRWDY
271-21 (SEQ ID NO:105) (SEQ ID NO:141) (SEQ ID NO:177)
DOM23h- GFTFTEYRMW AIEPIGNRTYYADSVKG QMPGQKWMAKSRFDY
271-22 (SEQ ID NO:106) (SEQ ID NO:142) (SEQ ID NO:178)
DOM23h- YRMW AIEPIGQKTYYADSVKG QIPGRKWTANSRFDY
271-27 (SEQ ID NO:107) (SEQ ID NO:143) (SEQ ID NO:179)
- GFTFTEYRMW NRTYYADSVKG QIPGRKWTANGRKDY
271-101 (SEQ ID NO:108) (SEQ ID NO:144) (SEQ ID NO:180)
DOM23h- GSTFTEYRMW AIEPIGHRTYYADSVKG WTANSRFDY
2 (SEQ ID NO:109) (SEQ ID NO:145) (SEQ ID NO:181)
DOM23h- GFTFTEYRMW AIEPIGNRTYYADSVKG QIPGQRWTGNSRFDY
271-105 (SEQ ID NO:110) (SEQ ID NO:146) (SEQ ID NO:182)
DOM23h- GFTFTEYRMW AIEPIGNRTYYADSVKG QFPGRKWTANSRSDY
271-106 (SEQ ID NO:111) (SEQ ID NO:147) (SEQ ID NO:183)
DOM23h- GFTFTEYRMW AIEPIGNRTYYADSVKG QIPGRKGTANSRFDY
271-114 (SEQ ID NO:112) (SEQ ID ) (SEQ ID NO:184)
N.B CDR2 and CDR3 are as defined by Kabat. CDR1 is defined by a combination of the Kabat and
Chothia methods.
Generic method for binding kinetics – T100
BIACORE™ is was carried out using a capture surface on a CM4 chip. Anti-human IgG was
used as the capturing agent and coupled to a CM4 biosensor chip by y amine coupling. The
Antigen molecule fused to the human Fc was captured on this immobilised surface to a level from
250 to 300 resonance units and defined concentrations of Domain antibodies diluted in running
buffer were passed over this captured surface. An injection of buffer over the captured antigen
surface was used for double ncing. The ed surface was regenerated, after each domain
antibody injection using 3M magnesium chloride solution; the ration removed the captured
antigen but did not significantly affect the ability of the surface to capture antigen in a subsequent
cycle. All runs were carried out at 25°C using HBS-EP buffer as running buffer. Data were generated
using the BIACORE™ T100 and fitted to the 1:1 binding model inherent to the software. When non-
specific binding was seen at the top concentration, the binding curve at this concentration was
d from the analysis set.
Further diversification of CDR3
The DOM23h7 derivatives with the t affinity ned methionines in on 96 and
100B. These positions, along with positions 99, 100D, 100E and 100G were diversified using NNK
mutagenesis to determine whether substitutions could be made. The NNK library was constructed as
described above using primer PEPF to introduce diversity at the selected positions in DOM23h-
271-22 or DOM23h102 background. DOM23h102 contains mutations at position 27 and
55 that confer improved affinity over DOM23h7.
PEPF (SEQ ID NO: 209)
GCGGTATATTACTGTGCGAAACAGNNSCCCGGCNNSAAGTGGNNSGCCNNSNNSCGCNNSGACTACTGGGG
TCAGGGAACC
DNA display libraries were ucted and selected on biotinylated hTGFbRII-FC as described above
using concentrations of 5nM; 0.5nM; 0.1nM; 0.1nM; 0.1nM; and 0.1nM in successive rounds. In
selection rounds 4-6 the non-biotinylated TGFbRII-FC competitor was added to a final concentration
of 100nM for 60, 90, and 90 minutes respectively before addition of C1 streptavidin DYNAbeads™.
Following selection the dAb inserts were PCR amplified using primers PelB NcoVh and PEP011, cut
with NcoI and EcoRI, and cloned into the bacterial expression vector pC10.
PelB NcoVh (SEQ ID NO: 210) CCGGCCATGGCGGAGGTGCAGCTGTTGGAGTCTGGG
PEP011 (SEQ ID NO: 211) GAATTCGCGGCCGCCTATTAGCTCGAGACGGTGACCAGGG
The cloned products were expressed and screened by Biacore. Clones with off-rates similar or better
than DOM23h22 were sequenced, purified, and assessed for affinity by BIACORE™ and
potency in the cell sensor assay. Clones giving poor kinetic profiles, ning unfavourable
ce motifs, or giving very low yields were not d. DOM23h50 was selected for
further affinity maturation.
DOM50 nucleic acid sequence (SEQ ID NO: 212)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
AACAGGCGCCCGGCGAGAAGTGGCTCGCCCGGGGCCGCTTGGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGC
DOM50 amino acid sequence (SEQ ID NO: 213 and duplicate entry SEQ ID NO:214)
EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWLARGRLDYWGQGTLVTVSS
Example 7. Introduction of mutations into TGFbRII dAb sequences (DOM23h-271
lineage) at positions 61 and 64
The D61N and K64R double mutations have previously been introduced into various TGFRβII dAb
lineages and have been shown to improve potency (WO2011/012609). These mutations were
introduced into TGFbRII dAb DOM23h-271 lineages to see if a similar improvement in y could
be achieved. Alternative mutations at this position were explored to ine whether further
enhancements in potency could be achieved.
Mutations were introduced into the DOM23h-271 (SEQ ID NO:199) backbone by overlap
extension using the polymerase chain reaction (PCR) (Ho, et al., Gene 1989 77(1)); complementary
oligonucleotide primers were used to generate two DNA fragments having overlapping or
complementary ends. These fragments were combined in a subsequent assembly PCR in which the
overlapping ends , allowing the 3' overlap of each strand to serve as a primer for the 3'
extension of the complementary strand and the resulting fusion product to be amplified further by
PCR. ic alterations in the nucleotide sequence were introduced by incorporating nucleotide
changes into the overlapping oligonucleotide primers. The target dAb gene fragments were
amplified by two separate PCRs using SUPERTAQ™ polymerase (HT Biotechnology). DOM23h-271
was selected as it has a low affinity for TGFbRII so that improvements in affinity were y
measurable using BIACORE™. The mutations at positions 61 and 64 were d using a
degenerate oligo-nucleotide to mutate both positions in combination, with the intension of using
affinity selection to enrich for mutants with improved binding affinity. As the NR on is known
to dominate this mutation was avoided in the library by employing a restricted codon, NNG, at
position 61. This codon encodes 13 amino acids but does not include the amino acids F, Y, C, H, I, N
or D. Free Cys was not ed within a dAb, and asparagine was also not wanted at this position,
so only 5 desirable amino acids combinations were excluded. At on 64 an NNK codon was
used to provide the full range of possible amino acids.
The oligonucleotide pairs used to introduce the mutations were PE008 (flanks the 5’ start of
the dAb gene: 5’- GCGTGGCAACAGCGTCGACAGAGGTGCAGCTGTTGGAG-3’) (SEQ ID
) with 271-6164 R (5’-GCGTAGTATGTACGATTACCAATCGG-3’) (SEQ ID NO:193) and mutated
64 deg-F ed residues underlined: 5’-
GGTAATCGTACATACTACGCANNGTCCGTGNNKGGCCGGTTCACCATCTCCCGC-3’) (SEQ ID NO:194)
with AS1309 (flanks the 3’ end of the dAb gene: 5’-
TGTGTGGCGGCCGCGCTCGAGACGGTGACCAGGGTTCCCTGACCCCA-3’) (SEQ ID ).
The two PCR fragments were recombined in an assembly PCR using AQ™ DNA polymerase
without the addition of primers. The fusion t was then amplified by the addition of flanking
s PE008 and AS1309 to the PCR reaction. The d dAb was digested with Sal I and Not I
restriction endonucleases, ligated into similarly cut pDOM13 expression vector and transformed into
E.Coli HB2151 cells. The D61N, K64R mutation was also introduced into -271 in the same
way, but substituting primer 271-6164 NR-F (5’-
GGTAATCGTACATACTACGCAAACTCCGTGCGCGGCCGGTTCACCATCTCCCGC-3’) (SEQ ID ) for
271-6164 deg-F. The mutated dAbs were identified by sequencing. 129 clones with novel
combinations of mutations at position 61 and 64 were identified, though 11 carried additional
mutations from PCR . These are shown in table 11. N.B. Clones with enhanced affinity are
ined. Clones with additional mutations are indicated by sks
To ine the effect of the mutations the selected clones were expressed in TB ONEX™
in 96 well plates for 72 hours at 30°C or 24 hours at 37°C. The atants were clarified by
centrifugation and ed through a 0.22µm filter before te evaluation by BIACORE™.
BIACORE™ A100 analysis was carried out using a capture surface on a CM5 chip. Antihuman
IgG was used as the capturing agent and coupled to a CM5 biosensor chip by primary amine
coupling. The Antigen molecule fused to the human Fc was captured on this immobilised surface to
a level from 200 to 300 resonance units and supernatants or purified domain antibodies were
passed over this captured surface. An injection of media or buffer over the captured antigen surface
was used for double referencing. On each flow cell, a protein A spot allowed to confirm the presence
of domain antibody on each sample injected. The surfaces were regenerated, after each domain
antibody injection using 3M magnesium chloride solution; the regeneration removed the captured
antigen but did not significantly affect the ability of the surface to capture antigen in a subsequent
cycle. All runs were carried out at 25°C using HBS-EP buffer as running buffer. Data were generated
using the BIACORE™ A100 and analyzed using its inherent software. Kinetics from purified samples
were fitted to the 1:1 binding model while supernatant off-rates were analyzed using the 1:1
dissociation model and / or using the binding level and the stability level of each sample in
comparison to the parent molecule.
Of clones tested 46 were identified with improved off-rate by comparison to the parent
DOM23h-271 dAb, as indicated in table 11. Mutations D61R and D61K were found to enhance
binding independent of mutations at position 64. A number of combinations appeared better than
the original D61N, K64R mutations, and these included RE, RM, RF and RY.
A selection of mutants with improved off-rate were purified and subjected to full BIACORE™
A100 kinetic analysis to determine their affinity (Table 10). The thermal stability of the mutants was
also determined by generation of melting profiles in the presence of SYPRO™ Orange (Invitrogen).
Briefly, the purified dAbs were diluted to 50 and 100ug/ml in a 1:500 dilution of SYPRO™ Orange in
PBS, and subjected to a 30-90°C melt curve in a Mini OPTICON™ PCR machine (BioRad). The Tm of
the major ve transition was determined at each concentration and used to calculate an
ted Tm value at 1mg/ml as an indication of the melting temperature for comparison (Table
10).
The mutations D61R, K64D and D61R, K64F were selected as they had a good ement
in affinity for a reduced impact in Tm. These mutations were transferred to an ty matured
DOM23h-271 derivative DOM23h22 (SEQ ID NO:30) to make dAbs DOM23h39 (SEQ ID
NO:37) and DOM23h40 (SEQ ID NO:38). These mutations were found to give an enhancement
in affinity by BIACORE™ including cross reactivity with murine TGFbRIIFC, and enhanced potency by
cell sensor assay. However the mutations were also found to reduce the Tm as ed by DSC,
and increase aggregation of the dabs in PBS, as measured by SEC MALLS. These assays were
carried out as described above, except that the buffer for SEC MALLS was 0.4M Nacl, 0.1M Sodium
Phosphate and 10% isopropanol pH7. These results are summarized in table 12 (mean values are
given for the human cell sensor assay and the mouse 3T3 luciferase .
Sequences of dabs referred to in this example are given below:
DOM23h39 amino acid sequence (SEQ ID NO:37)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYARSVDGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQMPGQKWMAKSRFDYWGQGTLVTVSS
DOM23h39 nucleic acid sequence (SEQ ID NO:75)
CAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCC
GGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCG
ATTGAGCCGATTGGTAATCGTACATACTACGCACGCTCCGTGGACGGCCGGTTCACCATCTCCCGCGACAATTCCA
AGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATGCC
CGGCCAGAAGTGGATGGCCAAGTCCCGCTTCGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
DOM23h40 amino acid ce (SEQ ID NO:38)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYARSVFGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKQMPGQKWMAKSRFDYWGQGTLVTVSS
DOM23h40 nucleic acid sequence (SEQ ID NO:76)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCC
GGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCG
ATTGAGCCGATTGGTAATCGTACATACTACGCACGCTCCGTGTTCGGCCGGTTCACCATCTCCCGCGACAATTCCA
AGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATGCC
CGGCCAGAAGTGGATGGCCAAGTCCCGCTTCGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC
Table 10
Description on Est. Tm 1mg/ml KD (nM)
1A2 RA 53.08 2.24
1D9 RD 52.35 1.17
3F11 RE 53.08 3.27
1G11 RM 51.81 0.97
3C9 RF 54.08 1.39
1E6 RY 49.62 1.17
1B11 RV 53.41 1.88
3D9 KG 48.69 4.34
1D5 KF 48.09 3.69
3D4 KT 54.74 4.26
1H11 LW 53.68 4.17
1B2 VW 52.68 1.79
2C12 NR 51.49 3.34
271 DK 58.21 N/D
N.B. the mutation corresponds to ‘XY’ wherein X is position 61 and Y is position 64
Table 11. Mutations inserted into DOM23h-271 at positions 61 and 64.
61\64 A R N D C Q E G H I L K M F P S T W Y V
A 1D6* 1C10 3B11 3C10* 3B10 1A12 3G3*
R 1A2 1A3 3F4 1D9 2C4 1A8 3F11 3G8 1A4 3C2 1G11 3C9* 2F5 1H1 1H2 1E6 1B11
N 2C12
D 3H6 (wt)
Q 1B6* 1E8 1B10 1H12 1D11 1C8 3F7 2C10
E 2A3 1F12 2G4 3H7 1F10 2B11 3D6
G 1D1 1B5 3B12 1E5 3G11 3D12 2B6 2G5 2D5
L 1D2 3E11 2A9 1B4 1H3 1A9 1F1 1G9 1H10 1C9 2C9 1H5 1G2 1E4 1H11
K 2E5 2B4 3D9 3E12 1D5 1G7 2E9* 3D4 1B1 1F2
M 1G6 2D3* 1E7 2E6 3C12 2F6 3H1 3B5* 1A6 1E11 3B8*
P 2A8 1A5 2C11 3H2 1G5 3G12
S 3C3 3E7 3E5 1H4 2B10 1C11 1B7 2C8 3B1 3D5
T 1E3 1D10* 2C1 1C1 2A5 3G9 2H10 2A4 3D11
W 2F10 1E12 2C2 2A2 2A6 1D4 3B2 2B1
V 1C12 2B9 1H6 1D3* 3C1 2G2 2F7 1F7 2H4 3C7 1B2 2G6
Clones with improved off-rate are underlined. Clones with onal mutations are indicated by asterisks
Table 12
Mutation Human Cell Mouse 3T3 Luciferase Human Cyno Mouse Tm Aggregation
sensor assay I TGFbRII TGRbRII (DSC) (SEC-MALS)
IC50 (nM) IC50 (nM) KD (pM) KD (pM) KD (pM)
DOM23h - 39.01 >17780 202.00 225.00 - 60 91.8% Monomer
22 8.2% Dimer
DOM23h RD 1.17 11120 14.90 24.00 1780.00 57.5 51.6% M/D Eq
39 38.5% D/T Eq
+ aggregates
DOM23h RF 0.53 1200 9.99 11.25 548.50 55.6 91.6% M/D Eq
40 8.4% Trimer /
oligomer
Example 8 Affinity maturation by CDR diversification of lineages DOM23h
, DOM23h13, 21 and DOM23h50
Domain antibodies DOM23h21(SEQ ID NO:204) and DOM23h13 (SEQ ID NO:206)
were isolated from the error prone ty maturation carried out on naïve clones DOM23h-855
(SEQ ID NO:13) and DOM23h-843 (SEQ ID NO:10) respectively, as detailed in example 5. Domain
antibody DOM23h-439 was isolated from phage libraries in a previous selection gn described
in and variant DOM23h20 (SEQ ID ) was subsequently isolated by
error prone affinity tion as detailed in example 5. Domain antibody 50 (SEQ ID
NO:214) was generated by CDR3 re-diversification of CDR-directed affinity matured variant
DOM23h22 (SEQ ID NO:30) as detailed in Example 6. DOM23h21, DOM23h13,
DOM23h20 and DOM23h50 were all selected for further affinity tion based on their
binding kinetics, potency in the SBE-bla HEK 293T cell sensor assay, sequence and sical
behaviour. Affinity tion was performed using degenerative mutagenesis to re-diversify the
CDRs, and improved leads were identified using phage display. Diversity was introduced into the
CDRs by construction of either doped or NNK libraries.
DOM23h50 was affinity matured using NNK libraries (saturation mutagenesis),
oligonucleotide primers were designed to cover each CDR and within each primer the codons for 5
amino acids were replaced with NNK codons so that 5 positions are diversified simultaneously.
Single or le oligonucleotides were used to cover all ed amino acids within each CDR; 1
for CDR1, 2 for CDR2 and 3 for CDR3. CDR-directed affinity maturation was achieved using
polymerase chain reaction (PCR); Complementary oligonucleotide primers were used to amplify a
sequence fragment ning each mutated CDR, and also an overlapping sequence fragment
covering the rest of the dAb coding sequence. These fragments were mixed and assembled by splice
ion overlap PCR to produce the full length dAb coding ce. This t was PCR
amplified using primers PelB NcoVh (SEQ ID NO:210) and PEP044 (5’-
GGAACCCTGGTCACCGTCTCGAGCGCGGCCGCATAATAAGAATTCA-3’ SEQ ID NO:215), digested with
NcoI and NotI, and ligated into NotI and NcoI digested pDOM4-gene3-pelB hybrid vector. pDOM4-
gene3-pelB hybrid vector is a modified version of the pDOM4 vector described above but has been
modified to replace the GAS leader sequence with the pelB (pectate lyase B) signal peptide.
The domain antibodies DOM23h21, DOM23h13 and DOM23h20 were ty
matured using doped libraries. The doped libraries were ucted essentially as described above
and in WO2006018650. A single degenerate oligonucleotide primer was used to cover all mutations
within each CDR. Within each primer the amino acids to be diversified were specified using
degenerate codons to encode multiple amino acids. The following degenerate coding was used: 'a' =
85 %A + 5 %T + 5 %G + 5 %C; 'g' = 85 %G + 5 %T + 5 %C + 5 %A; 'c' = 85 %C + 5 %T + 5
%G + 5 %A; 't' = 85 %T + 5 %A + 5 %G + 5 %C; ‘S’ = 50% G and 50% C. Capital letters
indicate 100% of the specified nucleotide. For the DOM23h20 doped library five amino acids
were diversified in CDR1, 6 in CDR2 and 11 in CDR3 to e the phenyalanine at position 100. For
the DOM23h21 doped library five amino acids were diversified in CDR1, 7 in CDR2 and 8 in
CDR3. For the DOM23h13 doped library five amino acids were diversified in CDR1, 6 in CDR2
and 8 in CDR3, position 94 before the CDR3 was also diversified by introducing the codon VRK. For
each of the dAbs, 4 doped libraries were constructed, one to diversify CDR1, one to diversify CDR2,
one to diversify CDR3 and a fourth where all CDRs were diversified. The degenerate library primers
were used in the same way as the triplet primers. Each ified CDR was amplified separately and
then combined with a parental sequence fragment using splice ion overlap, the full length
product was amplified using primers PelB NcoVh (SEQ ID NO:210) and DOM173 (SEQ ID NO:188).
The fragments were subcloned to pDOM4-gene3-pelB hybrid vector using NcoI and NotI.
Degenerate primer sequences:
23h20 CDRH1 (SEQ ID NO:216)
5’-GCAGCCTCCGGATTCACCTTTggSacSgagcagATGtggTGGGTCCGCCAGGCTCCAGGG-3’
9-20 CDRH2 (SEQ ID NO:217)
’-AAGGGTCTAGAGTTTGTCTCAcgSATTgattcSccSGGTggScgSACATACTACGCAGACTCCGTG-3’
23h20 CDRH3 (SEQ ID NO:218)
GTATATTACTGTGCGAAAcgScatgcSgcSggSgtStcSggSacStaYtttGACTACTGGGGTCAGGGAACC-3’
23h13 CDRH1 (SEQ ID NO:219)
’-GCAGCCTCCGGATTCACCTTTgatcaggatcgSATGtggTGGGTCCGCCAGGCCCCAGGG-3’
23h13 CDRH2 (SEQ ID NO:220)
’-AAGGGTCTAGAGTGGGTCTCAgcSATTgagtcSggSGGTcatcgSACATACTACGCAGACTCCGTG-3’
23h13 CDRH3 (SEQ ID )
5’-ACCGCGGTATATTACTGTGCGVRKcagaataagtcSggScgStcSggSTTTGACTACTGGGGTCAGGGA-3’
23h21 CDRH1 (SEQ ID NO:222)
’-GCAGCCTCCGGATTCACCTTTgagaatacStcSATGggSTGGGTCCGCCAGGCTCCAGGG-3’
23h21 CDRH2 (SEQ ID NO:223)
AAGGGTCTAGAGTGGGTCTCAcgSATTgatccSaagGGTtcScatACATACTACacSGACTCCGTGAAGGGCCGGT
TCACC-3’
23h21 CDRH3 (SEQ ID NO:224)
GTATATTACTGTGCGAAAcagcgSgagctSggSaagtcStaYTTTGACTACTGGGGTCAGGGA-3’
H143 R (SEQ ID NO:225)
’-GCAGCCTCCGGATTCACCTTTNNKNNKNNKNNKATGNNKTGGGTCCGCCAGGCTCCGGGGAAGGGTCTC-
3’
H2p143F (SEQ ID NO:226)
CCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCANNKATTNNKNNKNNKGGTNNKCGTACATACTACG
CAGACTCCG-3’
H2p243 F (SEQ ID NO:227)
CCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGNNKNNKNNKNNKNNKACATACTACG
CAGACTCCG-3’
H3p143 F (SEQ ID NO:228)
ACCGCGGTATATTACTGTGCGAAANNKNNKNNKNNKNNKAAGTGGATGGCCGTGGGCCGCTTGGACTACTG
GGG-3’
H3p243 F (SEQ ID NO:229)
ACCGCGGTATATTACTGTGCGAAACAGAAGCCCNNKNNKNNKNNKNNKGCCGTGGGCCGCTTGGACTACTG
GGGTCAGGG-3’
H3p343 F (SEQ ID NO:230)
ACCGCGGTATATTACTGTGCGAAACAGAAGCCCGGCCAGAAGTGGNNKNNKNNKNNKNNKTTGGACTACTG
GGGTCAGGG-3’
Phage Display:
NNK or doped libraries in separate CDR1, CDR2, CDR3 and combined CDR1, 2 and 3 pools were
subjected to at least 6 rounds of selection against 100nM, 10 nM, 1 nM, 1 nM,0.1 nM and 0.1 nM
(rounds 1, 2, 3, 4, 5 and 6 respectively) ylated monomeric human TGF-β RII antigen as
described in example 1 with the following deviation to the block step; the human IgG Fc fragment
which was previously added in example 1 was omitted and the block step performed for a minimum
of 20 minutes. In rounds 4 and 6 of selection competition was introduced by incubation with 100nM
non-biotinylated antigen for 30 min (rounds 4 and 6) following the incubation step with the labelled
antigen. For DOM 23h20 (CDR1, CDR3 and ed pools) and DOM 23h21 (CDR3) a
seventh round of selection was carried out t 0.1 nM ylated monomeric human TGF-β RII
antigen and 100 nM competition for 120 min or 20 pM biotinylated monomeric human TGF-β RII
antigen with 100 nM competition for 30 min. Phage were ied between rounds of selection by
centrifugation of an overnight culture of phage infected TG1 cells for 30 minutes at 4000 g. 40 ml of
supernatant containing the amplified phage was added to 10ml of PEG/NaCl (20% v/w PEG 8000 +
2.5M NaCl) and incubated on ice for 60 minutes. The samples were centrifuged for 40 minutes at
4000 g to pellet the precipitated phage. The supernatant was discarded and the phage pellet was
resuspended in 1ml 15% v/v glycerol/PBS. The phage sample was transferred to 2 ml Eppendorf
tubes and centrifuged for 10 minutes to remove any remaining bacterial cell debris. Diversified
domain dy Vh genes from the phage selections were PCR amplified using primers
PEP011VHStopNotIR (5’-CCCTGGTCACCGTCTCGAGCTAATAGGCGGCCGCGAATTC-3’ (SEQ ID NO:
231) and Nco1 VH F (5’-TATCGTCCATGGCGGAGGTGCAGCTGTTGGAGTCTGG-3’ (SEQ ID NO: 232) ,
digested with NcoI and NotI restriction cleases and ligated into the pC10 vector also ed
with NcoI and NotI. pC10 is a plasmid vector based on pUC119 vector, with expression under the
control an enhanced LacZ promoter designed for e sion of dAbs. Expression of dAbs into
the supernatant is enabled by fusion of the dAb gene to the pelB signal peptide at the N-terminal
end. The ligation products were transformed into chemically competent E. coli HB2151 cells and
plated on nutrient agar plates supplemented with 100 µg/ml of icillin. Individual clones were
picked and expressed in ght express auto-induction medium (high-level protein expression
system, Novagen), supplemented with 100 µg/ml carbenicillin in 96-well plates and grown with
shaking at 250 rpm for either 66 hours at 30°C or 24 hours at 37°C. Expression plates were then
centrifuged at 3500g for 15 minutes and the supernatants filtered using 0.45µ filter plates
(Millipore). atants containing the domain antibodies were screened on the E™ B4000
against human TGF-β RII and human TGF-β RII/Fc to determine the off-rate (Kd) (data not shown).
Biotinylated antigens were captured on an SA chip in accordance with the manufacturer’s
instructions, analysis was carried out at 25°C using HBS-EP buffer. Samples were run on BIACORE™
at a flow rate of 30 µl / min. Regeneration of the chip was achieved using glycine at low pH. The
data (not shown) were ed for off-rate by fitting the dissociation phase to the 1:1 dissociation
model inherent to the software, supernatants were also analyzed for protein A binding to estimate
levels of expression. Domain antibodies with off rates improved over parent were selected for
further study. Improved clones were expressed in overnight express autoinduction medium
(ONEX™, Novagen) supplemented with 100 ug/ml carbenicillin and antifoam (Sigma) at 30°C for 48
to 72 hours with shaking at 250 rpm. The cultures were centrifuged (4,200 rpm for 40 minutes) and
the supernatants were incubated with LINE™-protein A beads (Amersham Biosciences, GE
HEALTHCARE™, UK. g capacity: 5 mg of dAb per ml of , at 4°C or at room ature
for at least one hour. The beads were packed into a chromatography column and washed with PBS,
followed by 10 mM Cl pH 7.4 (Sigma, UK). Bound dAbs were eluted with 0.1 M glycine-HCl pH
2.0 and neutralized with 1M Tris-HCL pH 8.0. The OD at 280 nm of the dAbs was measured and
protein concentrations were determined using extinction coefficients ated from the amino acid
compositions of the dAbs. Affinity matured domain antibodies were tested on the BIACORE™ T200
(data for two preferred dAbs are shown in Example 9) and in the SBE-bla HEK 293T Cell Sensor
assay (data for two preferred dAbs are shown in Example 11) to determine their affinity and
potency. Biophysical properties including thermal stability and on state were determined using
Differential Scanning Colourimetry (DSC) and size exclusion tography with multi-angle-
LASER-light scattering (SEC-MALS) (data for two preferred dAbs are shown in Example 10).
The amino acid and nucleic acid sequences of affinity d DOM23h20 and DOM23h
50 anti-human TGFRII dAbs
DOM23h25 nucleic acid sequence (SEQ ID NO: 233)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
ATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTG
TCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGTTTTATGACTACTGGGGTCAGGGAACCCTGGTCACC
GTCTCGAGC
DOM23h25 amino acid sequence (SEQ ID NO: 234)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSS
DOM23h123 nucleic acid sequence (SEQ ID NO: 235)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCT
GGTCACCGTCTCGAGC
DOM23h123 amino acid sequence (SEQ ID NO: 236)
EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSS
DOM23h35 nucleic acid sequence (SEQ ID NO: 237)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTC
TCCTGTGCAGCCTCCGGATTCACCTTTGGGACCGATCAGATGTGGTGGGTCCGCCAGGCT
CCAGGGAAGGGTCTAGAGTTTGTCTCACGCATTGATTCCCCCGGTGGGCGGACATACTAC
TCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTAT
CTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCAG
GGGGTGTCGGGGAAGTACGTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTC
TCGAGC
DOM23h35 amino acid ce (SEQ ID NO: 238)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGTDQMWWVRQAPGKGLEFVSRIDSPGGRTYYANSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKRQPAGVSGKYVDYWGQGTLVTVSS
DOM23h129 nucleic acid sequence (SEQ ID NO: 239)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTC
TCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCT
CCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTAC
GCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTAT
CTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCG
CCCAATCAGAGGTATGTTGCCCGGGGCCGCTTGGACTACTGGGGTCAGGGAACCCTGGTC
ACCGTCTCGAGC
DOM23h129 amino acid sequence (SEQ ID NO: 240)
EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQAPNQRYVARGRLDYWGQGTLVTVSS
The CDRs as defined by Kabat of these anti-human TGFRII dAb affinity leads are shown in Table 13
Table 13. CDR Sequences of affinity matured anti-human TGFβRII dAbs
CDR1 (Kabat 26-35) CDR2 (Kabat 50-65) CDR3 (Kabat 95-102)
DOM23h- GSTFTEYRMW (SEQ AIEPIGHRTYYADSVKG QAPGEKWARRWDLDY
ID NO: 241) (SEQ ID NO: 242) (SEQ ID NO: 243)
271-123
DOM23h- GSTFTEYRMW (SEQ AIEPIGHRTYYADSVKG QAPNQRYVARGRLDY
ID NO: 244) (SEQ ID NO: 245) (SEQ ID NO: 246)
271-129
DOM23h- GFTFGTEQMW (SEQ RIDSPGGRTYYADSVKG RRPTGVSGTFYDY (SEQ
ID NO: 247) (SEQ ID NO: 248) ID NO: 249)
439-25
DOM23h- DQMW (SEQ RIDSPGGRTYYANSVKG SGKYVDY (SEQ
ID NO: 250) (SEQ ID NO: 251) ID NO: 252)
439-35
Further cation of the DOM23h25 and DOM23h123 nucleotide sequence
The D61N and K64R mutations as described in example 7 were introduced into the DOM23h25,
DOM23h123 and 129 affinity matured dAbs either in combination or tely.
Introduction of a V48I mutation in the DOM23h25 and DOM23h123 dAbs was also tested
to determine whether it could confer improvements in potency. Spontaneous mutation at kabat
position 48 was observed in a number of improved dAbs from the -439 lineage following
both test tion and CDR-directed affinity maturation. An Alanine at the C-terminus of the Vh
region, immediately after kabat residue 113 was also added to 25, DOM23h123
and DOM23h129 and all variants of these dAbs with the afore-mentioned mutations. Mutations
were introduced into the DOM23h25 (SEQ ID NO: 234), DOM23h123 (SEQ ID NO: 236)
and DOM23h129 (SEQ ID NO: 240) nes by overlap extension using the polymerase
chain reaction (PCR) essentially as described in Example 7. Specific mutations in the nucleotide
sequence were introduced by incorporating nucleotide changes into the overlapping oligonucleotide
primers, the insertion of the Alanine at the end of the Vh region was achieved by using a 3’
oligonucleotide designed to orate the Alanine e after kabat position 113.
The following oligonucleotides were used to introduce the mutations (mutated residues underlined):
439 48I SDM F (SEQ ID NO: 253)
’-GGGTCTAGAGTTTATTTCACGTATTGATTCGCC-3’
439 61N SDM F (SEQ ID NO: 254)
’-GGGAGGACATACTACGCAAACTCCGTGAAGGGCCGG-3’
439 64R SDM F (SEQ ID NO: 255)
’-CGCAGACTCCGTGCGTGGCCGGTTCACC-3’
439 61N 64R SDM F (SEQ ID NO: 256)
’-GGGAGGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACC-3’
271 61N SDM F (SEQ ID NO: 257)
’-GGACATACTACGCAAACTCCGTGAAGGGCCGG-3’
271 64R SDM F (SEQ ID NO: 258)
’-CGCAGACTCCGTGCGTGGCCGGTTCACC-3’
271 61N 64R SDM F (SEQ ID NO: 259)
5’-GGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACC-3’
567 +A rev (Flanks the 3’ end of the dAb Vh gene)(SEQ ID NO: 260)
’-CCCTGGTCACCGTCTCGAGCGCGTAATAAGCGGCCGCAGATTA-3’
21-23 Fwd (Flanks the 5’ end of the dAb Vh gene)(SEQ ID NO: 261)
’-ATAAGGCCATGGCGGAGGTGCAGCTGTTGGAGTCTG-3’
To determine the effect of the mutations the dAbs were expressed in TB ONEX™ supplemented with
100 ug/ml carbenicillin and antifoam (Sigma) at 30°C for 72 hours with shaking at 250 rpm. The
cultures were centrifuged (4,200 rpm for 40 minutes) and dAbs ty ed using
STREAMLINE™-protein A beads (Amersham Biosciences, GE HEALTHCARE™, UK) as before. ty
and potency of the domain dy variants were determined on the BIACORE™ T200 (data for
preferred dAbs is shown in Example 9) and in the a HEK 293T Cell Sensor assay (data for
preferred dAbs is shown in Example 11). .
The amino acid and nucleic acid sequences of DOM23h25 (SEQ ID NO: 234) and DOM23h
123 (SEQ ID NO: 236) anti-human TGFRII dAbs modified to include the C-terminal Alanine and
D61N, K64R or V48I mutations are given below:
DOM23h40 (DOM23h25 + C-terminal Alanine) Nucleic acid sequence (SEQ ID NO: 262)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTG
TCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGTTTTATGACTACTGGGGTCAGGGAACCCTGGTCACC
GTCTCGAGCGCG
40 (DOM23h25 + C-terminal Alanine) Amino acid ce (SEQ ID NO: 263)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSA
DOM23h41 (DOM23h25 + C-terminal Alanine + 48I) Nucleic acid sequence (SEQ ID NO:
264)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTA
TTTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGTTTTATGACTACTGGGGTCAGGGAACCCTGGTCACC
GTCTCGAGCGCG
DOM23h41 h25 + C-terminal Alanine + 48I) Amino acid sequence (SEQ ID NO:
265)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFISRIDSPGGRTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSA
DOM23h42 (DOM23h25 + C-terminal Alanine + 61N) Nucleic acid sequence (SEQ ID NO:
266)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTG
TCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGTTTTATGACTACTGGGGTCAGGGAACCCTGGTCACC
GTCTCGAGCGCG
DOM23h42 (DOM23h25 + C-terminal Alanine + 61N) Amino acid sequence (SEQ ID NO:
267)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYANSVKGRFTISRD
YLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSA
DOM23h43 (DOM23h25 + C-terminal Alanine + 64R) Nucleic acid sequence (SEQ ID NO:
268)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTG
TCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAGACTCCGTGCGTGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
AACGGCGACCCACGGGGGTGTCCGGGACGTTTTATGACTACTGGGGTCAGGGAACCCTGGTCACC
GTCTCGAGCGCG
DOM23h43 (DOM23h25 + C-terminal Alanine + 64R) Amino acid sequence (SEQ ID NO:
269)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYADSVRGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSA
DOM23h44 (DOM23h25 + C-terminal Alanine + 61N64R) c acid sequence (SEQ ID
NO: 270)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTG
TCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGTTTTATGACTACTGGGGTCAGGGAACCCTGGTCACC
GTCTCGAGCGCG
DOM23h44 (DOM23h25 + C-terminal Alanine + 61N64R) Amino acid sequence (SEQ ID
NO: 271)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYANSVRGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSA
130 (DOM23h123 + C-terminal e) Nucleic acid sequence (SEQ ID NO: 272)
CAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCT
GGTCACCGTCTCGAGCGCG
DOM23h130 (DOM23h123 + C-terminal Alanine) Amino acid sequence (SEQ ID NO: 273)
SGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSA
DOM23h131 (DOM23h123 + C-terminal Alanine + 61N) Nucleic acid sequence (SEQ ID
NO: 274)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCC
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCT
GGTCACCGTCTCGAGCGCG
131 (DOM23h123 + C-terminal Alanine + 61N) Amino acid sequence (SEQ ID
NO: 275)
EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYANSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSA
DOM23h132 (DOM23h123 + C-terminal Alanine + 64R) Nucleic acid sequence (SEQ ID
NO: 276)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGCGTGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCT
GGTCACCGTCTCGAGCGCG
DOM23h132 (DOM23h123 + C-terminal Alanine + 64R) Amino acid sequence (SEQ ID
NO: 277)
SGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVRGRFTISRD
YLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSA
DOM23h133 (DOM23h123+ C-terminal Alanine + ) Nucleic acid sequence (SEQ ID
NO: 278)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCT
GGTCACCGTCTCGAGC GCG
DOM23h133 (DOM23h123+ C-terminal Alanine + 61N64R) Amino acid sequence (SEQ ID
NO: 279)
EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYANSVRGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSA
DOM23h134 (DOM23h123+ C-terminal Alanine + 48I) Nucleic acid sequence (SEQ ID
NO: 280)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGA
TTTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
AACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCT
GGTCACCGTCTCGAGCGCG
DOM23h134 (DOM23h123+ C-terminal Alanine + 48I) Amino acid sequence (SEQ ID NO:
281)
EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWISAIEPIGHRTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSA
DOM23h135 (DOM23h129 + C-terminal e) Nucleic acid sequence (SEQ ID NO: 282)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGGCGCCCAATCAGAGGTATGTTGCCCGGGGCCGCTTGGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGCGCG
DOM23h135 h129 + C-terminal Alanine) Amino acid sequence (SEQ ID NO: 283)
SGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQAPNQRYVARGRLDYWGQGTLVTVSSA
DOM23h136 (DOM23h129 + C-terminal Alanine + 61N) Nucleic acid sequence (SEQ ID
NO: 284)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
GTGCGAAACAGGCGCCCAATCAGAGGTATGTTGCCCGGGGCCGCTTGGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGCGCG
DOM23h136 (DOM23h129 + C-terminal Alanine + 61N) Amino acid sequence (SEQ ID
NO: 285)
EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYANSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQAPNQRYVARGRLDYWGQGTLVTVSSA
137 (DOM23h129 + C-terminal Alanine + 61N64R) Nucleic acid sequence (SEQ
ID NO: 286)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGG
TCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACT
AACAGGCGCCCAATCAGAGGTATGTTGCCCGGGGCCGCTTGGACTACTGGGGTCAGGGAACCCTG
GTCACCGTCTCGAGCGCG
DOM23h137 (DOM23h129 + inal Alanine + 61N64R) Amino acid sequence (SEQ ID
NO: 287)
EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYANSVRGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKQAPNQRYVARGRLDYWGQGTLVTVSSA
DOM23h47 h42 + 48I) Nucleic acid sequence (SEQ ID NO: 288)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTA
TTTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGTTTTATGACTACTGGGGTCAGGGAACCCTGGTCACC
GTCTCGAGCGCG
DOM23h47 (DOM23h42 + 48I) Amino acid ce (SEQ ID NO: 289)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFISRIDSPGGRTYYANSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSA
DOM23h48 (DOM23h44 + 48I) Nucleic acid sequence (SEQ ID NO: 290)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC
CTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTA
TTTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACCATCTCCC
GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT
GTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGTTTTATGACTACTGGGGTCAGGGAACCCTGGTCACC
AGCGCG
DOM23h48 (DOM23h44 + 48I) Amino acid sequence (SEQ ID NO: 291)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFISRIDSPGGRTYYANSVRGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSA
Example 9. Biacore kinetic analysis of affinity d domain antibodies
uman IgG was immobilised on a Biacore CM4 chip by primary amine coupling according to the
manufacturer’s instructions. Human TGF-β RII/Fc, cynomolgus TGF-β RII/Fc or human Fc fragment
were captured on this surface. Domain antibodies were passed over the two captured receptors at 3
concentrations of 100, 10 and 1 nM (DOM23h-439 dAbs) or 100, 25 and 6.25 nM (DOM23h-271
dAbs). Only the 100 nM concentration of each dab was passed over human Fc fragment to confirm
specificity of binding to the extracellular TGF-β RII domain. An injection of buffer over the captured
n surface was used for double referencing. The captured surface was regenerated, after each
domain dy injection using 3M magnesium chloride solution; the regeneration removed the
captured antigen but did not significantly affect the ability of the surface to capture antigen in a
subsequent cycle. All runs were carried out at 25°C using HBS-EP buffer as running buffer. Data
were ted using the BIACORE™ T200 and fitted to the 1:1 binding model inherent to the
software. Table 14 shows the binding kinetics of the dAbs tested. The DOM23h-271 dAbs and the
DOM23h-439 lineages were run in separate experiments.
Table 14
Human TGFBRII Cyno TGFBRII
sample Ka (M-1.s-1) Kd (s-1) KD (M) Ka -1) Kd (s-1) KD (M)
DOM23h123 06 2.93E-04 1.02E-10 3.08E+06 3.20E-04 1.04E-10
DOM23h129 5.55E+06 4.11E-04 7.41E-11 6.00E+06 4.27E-04 7.12E-11
DOM23h130 2.51E+06 3.09E-04 1.23E-10 2.61E+06 3.24E-04 1.24E-10
DOM23h131 6.26E+06 04 2.17E-11 6.81E+06 1.44E-04 2.12E-11
DOM23h132 1.22E+07 2.22E-04 1.82E-11 1.29E+07 2.38E-04 1.85E-11
DOM23h133 06 7.70E-05 9.09E-12 06 8.71E-05 9.85E-12
DOM23h25 7.77E+06 6.34E-04 11 8.99E+06 2.73E-03 3.04E-10
DOM23h35 2.26E+07 2.22E-04 9.83E-12 2.32E+07 6.75E-04 2.91E-11
DOM23h40 1.34E+07 1.39E-03 1.04E-10 9.34E+06 3.42E-03 3.66E-10
DOM23h41 1.81E+07 1.87E-04 1.04E-11 07 5.22E-04 3.33E-11
42 4.09E+07 1.31E-04 3.20E-12 3.72E+07 4.10E-04 1.10E-11
DOM23h43 8.83E+06 3.73E-04 4.23E-11 06 1.20E-03 1.49E-10
DOM23h44 2.39E+07 6.91E-05 2.89E-12 2.17E+07 1.47E-04 6.77E-12
Example 10. sical Evaluation of affinity matured dAbs
The thermal stability of the dAbs was ined using ential Scanning Calorimeter (DSC).
dAbs were dialysed overnight into PBS buffer and adjusted at a final concentration of 1mg/ml.
Dialysis buffer was used as a reference for all samples. DSC was performed using capillary cell
microcalorimeter VP-DSC (GE care / Microcal), at a heating rate of 180˚C/hour. A typical scan
range was from 20-90˚C for both the reference buffer and the protein sample. After each reference
buffer and sample pair, the capillary cell was cleaned with a solution of 5% Decon (Fisher-Scientific)
in water followed by PBS. Resulting data traces were analyzed using Origin 7.0 software. The DSC
trace obtained from the reference buffer was subtracted from the sample trace. The precise molar
concentration of the sample was entered into the data analysis routine to yield values for g
temperature (Tm), enthalpy (∆H) and Van’t Hoff enthalpy (∆Hv) values. Data were fitted to a non
state model. Best fit and dependencie values were obtained with either 1 or 2 transition events. Onset
unfolding temperature was also determined by integrating from zero each sample thermogram.
This value was then determined as the temperature at which 4 percentage of the sample was
unfolded.
Table 14A
Protein name Apparent Tm (C) On-set
temperature (C)
DOM23h21 60.66 52.5
56.34 49
DOM23h30 57.17 50
DOM23h32 55.01 49
DOM23h33 58.93 51
DOM23h34 56.78 50
DOM23h42 61.90 55
DOM23h43 66.81 58.6
DOM23h44 63.38 56
DOM23h123 54.82 50.6
DOM23h124 58.16 52
DOM23h125 58.26 55
Analysis of solution state by size ion chromatography with multi-angle-LASER-light scattering
(SEC-MALS)
To determine r dAbs are monomeric or form higher order oligomers in solution, they were
analyzed by SEC-MALLS (Size Exclusion Chromatography with Multi-Angle-LASER-Light-Scattering).
Agilent 1100 series HPLC system with an autosampler and a UV detector (controlled by Empower
software) was connected to Wyatt Mini Dawn Treos (Laser Light Scattering (LS) or) and Wyatt
Optilab rEX DRI (Differential Refractive Index (RI) detector). The detectors are ted in the
following order -UV-LS-RI. Both RI and LS instruments operate at a wavelength of 658nm; the UV
signal is monitored at 280nm and 220nm. Domain antibodies (50 microliters injection at a
concentration of 1mg/mL in PBS) were ted according to their hydrodynamic properties by size
exclusion chromatography using a TSK2000 column. The mobile phase was 0.2M NaCl, 0.1M NaPO4,
% n-propanol. The intensity of the scattered light while protein passed through the detector was
measured as a on of angle. This measurement taken er with the protein concentration
determined using the RI detector d calculation of the molar mass using appropriate ons
(integral part of the analysis software Astra v.5.3.4.14). The solution state as percentage monomer
is shown in Table 15.
Table 15
Protein nam Percentage monomer
DOM23h21 100%
DOM23h25 100%
DOM23h30 100%
DOM23h32 100%
DOM23h33 98.2%
DOM23h34 97.7%
DOM23h42 83.8%
DOM23h43 83%
DOM23h44 64.7%
DOM23h123 100%
DOM23h124 97.4%
Example 11. TGFβ-RII inhibition by affinity matured dAbs in the SBE-bla HEK 293T Cell
Sensor assay
The assay was carried out exactly as ed in example 4, assay h2.
The assay was performed multiple times to obtain an average and a range of values which
are summarised in Table 16. The arithmetic mean IC50 was calculated using log IC50s, and the
range was calculated by adding and subtracting the log standard deviation from the mean IC50, and
then transforming back to IC50. The assay QC parameters were met; the robust Z factors were
greater than 0.4 and the TGF-β EC80 was within 6 fold of the concentration added to the assay. The
s are shown in Table 16.
Table 16 Cell Functional assay data for human specific clones plus VH Dummy dAb.
IC50 nM
IC50
range
Mean (+/- log SD) n
DOM23h123 18.3 9.8 - 34.1 11
DOM23h129 22.7 16.6 - 31.1 4
DOM23h130 37.0 15.1 - 90.7 4
DOM23h131 6.3 4.5 - 8.9 4
DOM23h132 21.0 16.1 - 27.5 4
DOM23h133 2.4 1.5 - 3.8 4
DOM23h25 4.0 1.1 - 14.7 17
DOM23h35 0.5 0.4 - 0.7 3
DOM23h37 0.7 0.2 - 2.2 4
DOM23h40 14 10.8 - 18.8 6
41 1.7 1.3 - 2.3 4
42 0.7 0.2 - 2.7 8
DOM23h43 1.0 0.7 - 1.4 4
DOM23h44 1.3 0.5 - 3.3 6
VHDummy2 > 25119 25119 13
Sequence Concordance Table
SEQ ID NO DOM number Description
1 DOM23h-802 amino acid sequence – naive clone
2 DOM23h-803 amino acid sequence – naive clone
3 DOM23h-813 amino acid sequence – naive clone
4 DOM23h-815 amino acid sequence – naive clone
-828 amino acid sequence – naive clone
6 DOM23h-830 amino acid ce – naive clone
7 -831 amino acid sequence – naive clone
8 DOM23h-840 amino acid ce – naive clone
9 DOM23h-842 amino acid ce – naive clone
DOM23h-843 amino acid sequence – naive clone
11 DOM23h-850 amino acid sequence – naive clone
12 DOM23h-854 amino acid sequence – naive clone
13 DOM23h-855 amino acid sequence – naive clone
14 -865 amino acid sequence – naive clone
DOM23h-866 amino acid sequence – naive clone
16 DOM23h-874 amino acid sequence – naive clone
17 DOM23h-883 amino acid sequence – naive clone
18 DOM23h-903 amino acid sequence – naive clone
19 DOM23m-4 amino acid sequence – naive clone
DOM23m-29 amino acid sequence – naive clone
21 -32 amino acid ce – naive clone
22 DOM23m-62 amino acid sequence – naive clone
23 DOM23m-71 amino acid sequence – naive clone
24 DOM23m-72 amino acid sequence – naive clone
DOM23m-81 amino acid sequence – naive clone
26 DOM23m-99 amino acid sequence – naive clone
27 DOM23m-101 amino acid sequence – naive clone
28 DOM23m-352 amino acid sequence – naive clone
29 DOM23h21 amino acid sequence – affinity matured
DOM23h22 amino acid sequence – affinity matured
31 DOM23h27 amino acid sequence – affinity matured
32 DOM23h101 amino acid sequence – affinity matured
33 DOM23h102 amino acid sequence – affinity matured
34 DOM23h105 amino acid ce – affinity matured
DOM23h106 amino acid sequence – affinity matured
36 DOM23h114 amino acid sequence – affinity matured
37 DOM23h39 amino acid sequence – affinity matured plus D61R K64D
mutation
38 DOM23h40 amino acid sequence – affinity matured plus D61R K64F
mutation
39 -802 nucleic acid sequence – naive clone
40 DOM23h-803 nucleic acid sequence – naive clone
41 DOM23h-813 nucleic acid sequence – naive clone
42 DOM23h-815 nucleic acid sequence – naive clone
43 DOM23h-828 c acid sequence – naive clone
44 DOM23h-830 nucleic acid sequence – naive clone
45 DOM23h-831 nucleic acid sequence – naive clone
46 -840 nucleic acid sequence – naive clone
47 DOM23h-842 nucleic acid ce – naive clone
48 -843 nucleic acid sequence – naive clone
49 DOM23h-850 nucleic acid sequence – naive clone
50 DOM23h-854 nucleic acid sequence – naive clone
51 DOM23h-855 nucleic acid ce – naive clone
52 DOM23h-865 nucleic acid sequence – naive clone
53 DOM23h-866 nucleic acid sequence – naive clone
54 DOM23h-874 nucleic acid sequence – naive clone
55 DOM23h-883 nucleic acid sequence – naive clone
56 DOM23h-903 nucleic acid sequence – naive clone
57 DOM23m-4 nucleic acid sequence – naive clone
58 DOM23m-29 nucleic acid ce – naive clone
59 DOM23m-32 nucleic acid sequence – naive clone
60 DOM23m-62 nucleic acid sequence – naive clone
61 DOM23m-71 nucleic acid sequence – naive clone
62 DOM23m-72 c acid sequence – naive clone
63 DOM23m-81 nucleic acid sequence – naive clone
64 DOM23m-99 nucleic acid sequence – naive clone
65 DOM23m-101 nucleic acid sequence – naive clone
66 DOM23m-352 nucleic acid sequence – naive clone
67 DOM23h21 nucleic acid sequence – affinity d
68 DOM23h22 nucleic acid sequence – affinity matured
69 DOM23h27 nucleic acid sequence – affinity matured
70 DOM23h101 nucleic acid sequence – affinity matured
71 DOM23h102 nucleic acid sequence – affinity matured
72 DOM23h105 nucleic acid sequence – ty d
73 106 nucleic acid sequence – affinity d
74 DOM23h114 nucleic acid sequence – affinity matured
75 DOM23h39 nucleic acid sequence – affinity matured plus D61R K64D
mutation
76 DOM23h40 nucleic acid sequence – affinity d plus D61R K64F
mutation
77 DOM23h-802 CDR1
113 .. CDR2
149 .. CDR3
78 DOM23h-803 CDR1
114 .. CDR2
150 .. CDR3
79 DOM23h-813 CDR1
115 .. CDR2
151 .. CDR3
80 DOM23h-815 CDR1
116 .. CDR2
152 .. CDR3
81 DOM23h-828 CDR1
117 .. CDR2
153 .. CDR3
82 -830 CDR1
118 .. CDR2
154 .. CDR3
83 DOM23h-831 CDR1
119 .. CDR2
155 .. CDR3
84 DOM23h-840 CDR1
120 .. CDR2
156 .. CDR3
85 DOM23h-842 CDR1
121 .. CDR2
157 .. CDR3
86 DOM23h-843 CDR1
122 .. CDR2
158 .. CDR3
87 DOM23h-850 CDR1
123 .. CDR2
159 .. CDR3
88 DOM23h-854 CDR1
124 .. CDR2
160 .. CDR3
89 DOM23h-855 CDR1
125 .. CDR2
161 .. CDR3
90 DOM23h-865 CDR1
126 .. CDR2
162 .. CDR3
91 DOM23h-866 CDR1
127 .. CDR2
163 .. CDR3
92 DOM23h-874 CDR1
128 .. CDR2
164 .. CDR3
93 DOM23h-883 CDR1
129 .. CDR2
165 .. CDR3
94 DOM23h-903 CDR1
130 .. CDR2
166 .. CDR3
95 DOM23m-4 CDR1
131 .. CDR2
167 .. CDR3
96 DOM23m-29 CDR1
132 .. CDR2
168 .. CDR3
97 DOM23m-32 CDR1
133 .. CDR2
169 .. CDR3
98 DOM23m-62 CDR1
134 .. CDR2
170 .. CDR3
99 DOM23m-71 CDR1
135 .. CDR2
171 .. CDR3
100 DOM23m-72 CDR1
136 .. CDR2
172 .. CDR3
101 DOM23m-81 CDR1
137 .. CDR2
173 .. CDR3
102 DOM23m-99 CDR1
138 .. CDR2
174 .. CDR3
103 -101 CDR1
139 .. CDR2
175 .. CDR3
104 DOM23m-352 CDR1
140 .. CDR2
176 .. CDR3
105 DOM23h21 CDR1
141 .. CDR2
177 .. CDR3
106 DOM23h22 CDR1
142 .. CDR2
178 .. CDR3
107 DOM23h27 CDR1
143 .. CDR2
179 .. CDR3
108 DOM23h101 CDR1
144 .. CDR2
180 .. CDR3
109 DOM23h102 CDR1
145 .. CDR2
181 .. CDR3
110 DOM23h105 CDR1
146 .. CDR2
182 .. CDR3
111 DOM23h106 CDR1
147 .. CDR2
183 .. CDR3
112 DOM23h114 CDR1
148 .. CDR2
184 .. CDR3
185 DOM008 primer
186 DOM009 primer
187 DOM172 primer
188 DOM173 primer
189 271-7R1deg CDR1 primer
190 271-7R2deg CDR2 primer
191 271-7R3deg CDR3 primer
192 PE008 primer
193 271-6164 R primer
194 271-6164 deg-F primer
195 AS1309 primer
196 271-6164 NR-F primer
197 DOM57 primer
198 DOM6 primer
199 DOM23h-271 amino acid sequence – naive clone
200 DOM23h-271 nucleic acid sequence – naive clone
201 DOM23h7 amino acid sequence – naive clone
202 DOM23h7 c acid sequence – naive clone
203 DOM23h21 nucleic acid sequence – test matured clone
204 DOM23h21 amino acid sequence – test matured clone
205 DOM23h13 nucleic acid ce – test matured clone
206 DOM23h13 amino acid sequence – test matured clone
207 DOM23h20 nucleic acid sequence – test matured clone
208 DOM23h20 amino acid sequence – test matured clone
209 PEPF Primer
210 PelB NcoVh Primer
211 PEP011 Primer
212 DOM50 c acid sequence – rected affinity matured clone
213 DOM50 amino acid sequence – CDR-directed affinity matured clone
214 DOM50 amino acid sequence – CDR-directed affinity matured clone
(*duplicate of 213 above*)
215 PEP044 Primer
216 23h20 CDRH1 Primer
217 23h20 CDRH2 Primer
218 23h20 CDRH3 Primer
219 23h13 CDRH1 Primer
220 23h13 CDRH2 Primer
221 23h13 CDRH3 Primer
222 23h21 CDRH1 Primer
223 23h21 CDRH2 Primer
224 23h21 CDRH3 Primer
225 H143 R Primer
226 H2p143F Primer
227 H2p243 F Primer
228 H3p143 F Primer
229 H3p243 F Primer
230 71-43 F Primer
231 PEP011VHStopNotIR Primer
232 Nco1 VH F Primer
233 25 nucleic acid sequence – CDR-directed affinity matured clone
234 DOM23h25 amino acid sequence – CDR-directed affinity matured clone
235 DOM23h123 nucleic acid sequence – CDR-directed affinity matured clone
236 123 amino acid sequence – rected affinity matured clone
237 DOM23h35 c acid sequence – CDR-directed affinity matured clone
238 DOM23h35 amino acid sequence – CDR-directed affinity d clone
239 DOM23h129 nucleic acid sequence – CDR-directed affinity d clone
240 DOM23h129 amino acid ce – CDR-directed affinity matured clone
241 DOM23h123 CDR1
242 DOM23h123 CDR2
243 DOM23h123 CDR3
244 DOM23h129 CDR1
245 DOM23h129 CDR2
246 DOM23h129 CDR3
247 DOM23h25 CDR1
248 DOM23h25 CDR2
249 DOM23h25 CDR3
250 DOM23h35 CDR1
251 DOM23h35 CDR2
252 DOM23h35 CDR3
253 439 48I SDM F Primer
254 439 61N SDM F Primer
255 439 64R SDM F Primer
256 439 61N 64R SDM F Primer
257 271 61N SDM F Primer
258 271 64R SDM F Primer
259 271 61N 64R SDM F Primer
260 567 +A rev Primer
261 21-23 Fwd Primer
262 DOM23h40 nucleic acid sequence – CDR-directed ty matured clone
263 40 amino acid sequence – CDR-directed affinity matured clone
264 DOM23h41 nucleic acid sequence – CDR-directed affinity matured clone
265 DOM23h41 amino acid sequence – CDR-directed affinity matured clone
266 DOM23h42 nucleic acid sequence – CDR-directed affinity d clone
267 DOM23h42 amino acid sequence – CDR-directed affinity matured clone
268 DOM23h43 nucleic acid sequence – CDR-directed affinity matured clone
269 DOM23h43 amino acid sequence – CDR-directed affinity matured clone
270 DOM23h44 nucleic acid sequence – CDR-directed affinity matured clone
271 DOM23h44 amino acid sequence – CDR-directed affinity matured clone
272 DOM23h130 nucleic acid sequence – CDR-directed affinity matured clone
273 DOM23h130 amino acid ce – CDR-directed affinity matured clone
274 DOM23h131 nucleic acid sequence – CDR-directed affinity matured clone
275 DOM23h131 amino acid sequence – CDR-directed ty d clone
276 DOM23h132 nucleic acid sequence – CDR-directed affinity matured clone
277 DOM23h132 amino acid sequence – CDR-directed affinity matured clone
278 DOM23h133 c acid sequence – rected affinity matured clone
279 DOM23h133 amino acid sequence – CDR-directed affinity matured clone
280 134 c acid sequence – CDR-directed affinity matured clone
281 DOM23h134 amino acid sequence – CDR-directed affinity matured clone
282 DOM23h135 nucleic acid sequence – CDR-directed affinity matured clone
283 135 amino acid sequence – CDR-directed affinity matured clone
284 DOM23h136 nucleic acid sequence – CDR-directed affinity matured clone
285 DOM23h136 amino acid sequence – CDR-directed affinity matured clone
286 DOM23h137 nucleic acid sequence – CDR-directed affinity matured clone
287 DOM23h137 amino acid sequence – CDR-directed affinity matured clone
288 DOM23h47 nucleic acid sequence - DOM23h42 + 48I
289 DOM23h47 amino acid sequence - DOM23h42 + 48I
290 48 nucleic acid sequence - DOM23h44 + 48I
291 DOM23h48 amino acid sequence - DOM23h44 + 48I
Claims (31)
1. An anti-TGFbetaRII immunoglobulin single variable domain comprising an amino acid sequence as set forth in SEQ ID NO:267 having up to 5 amino acid substitutions, deletions or additions, in any combination. 5
2. An anti-TGFbetaRII immunoglobulin single variable domain as d in claim 1, wherein the said amino acid substitutions, ons or additions are not within CDR3.
3. An anti-TGFbetaRII globulin single variable domain as claimed in claim 1 or claim 2, wherein the said amino acid substitutions, ons or additions are not within any of the CDRs.
4. An anti-TGFbetaRII immunoglobulin single variable domain as claimed in claim 1 consisting of 10 an amino acid ce as set forth in SEQ ID NO:267.
5. An isolated ptide comprising an anti-TGFbetaRII immunoglobulin single variable domain as claimed in any one of the preceding claims, wherein said isolated polypeptide binds to TGFbetaRII.
6. An anti-TGFbetaRII immunoglobulin single variable domain as claimed in claim 1, 2 or 3, or a 15 polypeptide as claimed in claim 5, further comprising at least one of the following amino acids selected from the group: R at position 39, I at on 48, D at position 53, N at position 61, R at position 61, K at position 61, R at position 64, F at position 64, D at on 64, E at position 64, M at on 64, Y at position 64, H at position 102, or S at position 103 of the immunoglobulin single variable domain, said positions being according to the Kabat numbering 20 convention.
7. An anti-TGFbetaRII immunoglobulin single variable domain or a polypeptide as claimed in claim 6, comprising an R or a K at position 61 according to the Kabat numbering convention.
8. An anti-TGFbetaRII immunoglobulin single variable domain as claimed in claim 1, 2 or 3, or a ptide as claimed in claim 5, further comprising one of the following amino acid 25 combinations selected from the group: N at position 61 and R at position 64; R at position 61 and E at position 64; R at position 61 and M at position 64; R at position 61 and F at position 64; R at position 61 and Y at position 64; and R at position 61 and D at position 64 of the immunoglobulin single variable domain, said positions being according to the Kabat numbering convention. 30
9. An anti-TGFbetaRII immunoglobulin single variable domain or a polypeptide as claimed in claim 6, 7 or 8, comprising an isoleucine residue at position 48 according to the Kabat numbering tion.
10. An anti-TGFbetaRII immunoglobulin single le domain or polypeptide as claimed in any one of claims 1-9, wherein said anti-TGFbetaRII immunoglobulin single variable domain or 35 polypeptide binds to human TGFbetaRII.
11. An anti-TGFbetaRII immunoglobulin single variable domain or polypeptide as claimed in claim 10, wherein said anti-TGFbetaRII immunoglobulin single variable domain or polypeptide also binds to mouse TGFbetaRII and/or cyno TGFbetaRII.
12. An immunoglobulin single variable domain or ptide as claimed in any one of claims 1-11, 5 wherein said immunoglobulin single le domain or ptide neutralises TGFbeta ty.
13. An immunoglobulin single variable domain or polypeptide as claimed in any one of claims 1-12, wherein said globulin single variable domain or polypeptide inhibits binding of TGFbeta to TGFbetaRII.
14. An isolated nucleic acid ng an anti-TGFbetaRII immunoglobulin single le domain or 10 polypeptide as claimed in any one of claims 1-13.
15. An isolated nucleic acid molecule as claimed in claim 14, comprising at least one nucleic acid molecule of SEQ ID NO:266.
16. A vector comprising a nucleic acid molecule as claimed in claim 14 or claim 15.
17. A host cell comprising a nucleic acid or a vector as claimed in any one of claims 14-16, wherein 15 the host cell is not a human cell within a human.
18. A method of producing a polypeptide comprising an anti-TGFbetaRII immunoglobulin single variable domain as claimed in any one of claims 1-13, the method comprising maintaining a host cell as claimed in claim 17 under ions suitable for expression of said nucleic acid or vector, whereby a polypeptide comprising an immunoglobulin single le is produced. 20
19. An GFbetaRII immunoglobulin single variable domain or polypeptide as claimed in any one of claims 1-13 for use as a ment.
20. A ceutical composition sing an anti-TGFbetaRII immunoglobulin single variable domain or polypeptide as claimed in any one of claims 1-13.
21. An anti-TGFbetaRII immunoglobulin single variable domain or polypeptide as claimed in claim 25 19 or pharmaceutical composition as claimed in claim 20 for treatment of a disease associated with TGFbeta signalling selected from the group of: tissue fibrosis, such as pulmonary fibrosis, ing idiopathic pulmonary fibrosis; liver fibrosis, including cirrhosis and chronic hepatitis; rheumatoid arthritis; ocular disorders; fibrosis of the skin, including keloid of skin, and Dupuytren’s Contracture; kidney fibrosis such as nephritis and nephrosclerosis, wound healing; 30 and a vascular condition, such as restenosis.
22. An anti-TGFbetaRII immunoglobulin single variable domain, ptide or pharmaceutical composition as claimed in claim 19, for use in treating tissue fibrosis or for use in wound healing and/or scarring reduction.
23. An anti-TGFbetaRII immunoglobulin single variable domain as claimed in claim 19 or 21, 35 wherein the disease is keloid disease or Dupuytren’s Contracture.
24. Use of an anti-TGFbetaRII single le domain or polypeptide as claimed in any one of claims 1-13 in the cture of a medicament for the treatment of a disease associated with TGFbeta signalling selected from the group of: tissue fibrosis, such as pulmonary fibrosis, including idiopathic pulmonary fibrosis; liver fibrosis, including sis and chronic tis; rheumatoid arthritis; ocular disorders; fibrosis of the skin, including keloid of skin, and Dupuytren’s Contracture; kidney is such as tis and nephrosclerosis, wound healing; and a vascular condition, such as restenosis. 5
25. A use as claimed in claim 24, for use in treating tissue fibrosis or for use in wound healing and/or scarring reduction.
26. A use as claimed in claim 24 or 25, wherein the disease is keloid disease or ren’s Contracture.
27. A kit comprising an anti-TGFbetaRII single variable domain or polypeptide as claimed in any 10 one of claims 1-13 and a device for applying said single variable domain or polypeptide to the skin.
28. A kit as claimed in claim 27, wherein the device is an intradermal delivery device.
29. An anti-TGFbetaRII immunoglobulin single variable domain as claimed in claim 1 substantially as herein described with reference to any example thereof. 15
30. An isolated peptide as claimed in claimed in claim 5 substantially as herein described with reference to any example thereof.
31. A pharmaceutical ition as claimed in claim 20 substantially as herein described with reference to any example thereof.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161430235P | 2011-01-06 | 2011-01-06 | |
US61/430,235 | 2011-01-06 | ||
PCT/EP2012/050061 WO2012093125A1 (en) | 2011-01-06 | 2012-01-04 | Ligands that bind tgf-beta receptor ii |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ612839A NZ612839A (en) | 2014-09-26 |
NZ612839B2 true NZ612839B2 (en) | 2015-01-06 |
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