U.S. patent application number 13/585192 was filed with the patent office on 2013-02-14 for nucleic acid molecules encoding anti-inflammatory domain antibodies.
This patent application is currently assigned to CEPHALON AUSTRALIA PTY LTD.. The applicant listed for this patent is Anthony G. Doyle, Philip A. Jennings, Jennifer A. Lee, Ian M. Tomlinson, Benjamin P. Woolven. Invention is credited to Anthony G. Doyle, Philip A. Jennings, Jennifer A. Lee, Ian M. Tomlinson, Benjamin P. Woolven.
Application Number | 20130040383 13/585192 |
Document ID | / |
Family ID | 38188157 |
Filed Date | 2013-02-14 |
United States Patent
Application |
20130040383 |
Kind Code |
A1 |
Woolven; Benjamin P. ; et
al. |
February 14, 2013 |
Nucleic acid molecules encoding anti-inflammatory domain
antibodies
Abstract
The present invention provides a recombinant domain antibody
(dAb) which binds to human TNF-.alpha., the dAb comprising an
immunoglobulin heavy or light chain variable domain, comprising at
least one complementarity determining region (CDR) having a
sequence derived from a New World primate.
Inventors: |
Woolven; Benjamin P.;
(Cambridgeshire, GB) ; Tomlinson; Ian M.;
(Cambridgeshire, GB) ; Lee; Jennifer A.;
(Cambridgeshire, GB) ; Doyle; Anthony G.;
(Drummoyne, AU) ; Jennings; Philip A.; (Warrawee,
AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Woolven; Benjamin P.
Tomlinson; Ian M.
Lee; Jennifer A.
Doyle; Anthony G.
Jennings; Philip A. |
Cambridgeshire
Cambridgeshire
Cambridgeshire
Drummoyne
Warrawee |
|
GB
GB
GB
AU
AU |
|
|
Assignee: |
CEPHALON AUSTRALIA PTY LTD.
Macquarie Park
AU
|
Family ID: |
38188157 |
Appl. No.: |
13/585192 |
Filed: |
August 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13008342 |
Jan 18, 2011 |
8263076 |
|
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13585192 |
|
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11659009 |
Jan 30, 2007 |
7981414 |
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PCT/AU2006/001940 |
Dec 20, 2006 |
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13008342 |
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60817272 |
Jun 28, 2006 |
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Current U.S.
Class: |
435/320.1 ;
536/23.53 |
Current CPC
Class: |
A61P 35/04 20180101;
A61P 1/00 20180101; C07K 16/241 20130101; C07K 2317/569 20130101;
A61P 1/16 20180101; A61P 19/00 20180101; C07K 2317/24 20130101;
A61P 21/00 20180101; A61P 9/10 20180101; A61P 1/04 20180101; A61P
9/00 20180101; A61P 11/00 20180101; A61P 17/00 20180101; A61P 19/02
20180101; A61P 25/00 20180101; A61P 31/00 20180101; A61P 37/06
20180101; C07K 2317/565 20130101; A61P 31/04 20180101; A61P 17/02
20180101; A61P 19/10 20180101; A61P 29/00 20180101; A61P 3/10
20180101; A61P 37/00 20180101; A61P 35/00 20180101 |
Class at
Publication: |
435/320.1 ;
536/23.53 |
International
Class: |
C12N 15/13 20060101
C12N015/13; C12N 15/63 20060101 C12N015/63 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 2005 |
AU |
2005907124 |
Claims
1. (canceled)
2. An isolated nucleic acid molecule encoding a domain antibody
(dAb) which binds to human TNF-.alpha., wherein the dAb comprises
an amino acid sequence selected from the group consisting of:
TABLE-US-00009 (SEQ ID NO: 8)
DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR; (SEQ
ID NO: 9) DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR; (SEQ
ID NO: 10) DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR; (SEQ
ID NO: 50) DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKPPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR; (SEQ
ID NO: 51) DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR; and
(SEQ ID NO: 52) DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR.
3. The isolated nucleic acid molecule of claim 2, wherein the dAb
comprises the amino acid sequence: TABLE-US-00010 (SEQ ID NO: 10)
DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR.
4. An expression vector comprising the nucleic acid molecule of
claim 2.
5. An expression vector comprising the nucleic acid molecule of
claim 3.
6. An isolated nucleic acid molecule encoding a domain antibody
(dAb) which binds to human TNF-.alpha., wherein the dAb consists of
an amino acid sequence selected from the group consisting of:
TABLE-US-00011 (SEQ ID NO: 8)
DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR; (SEQ
ID NO: 9) DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR; (SEQ
ID NO: 10) DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR; (SEQ
ID NO: 50) DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKPPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR; (SEQ
ID NO: 51) DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR; and
(SEQ ID NO: 52) DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR.
7. The isolated nucleic acid molecule of claim 6, wherein the dAb
consists of the amino acid sequence: TABLE-US-00012 (SEQ ID NO: 10)
DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR.
8. An expression vector comprising the nucleic acid molecule of
claim 6.
9. An expression vector comprising the nucleic acid molecule of
claim 7.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/008,342, filed on Jan. 18, 2011 and issued as U.S. Pat. No.
8,263,076 on Sep. 11, 2012, which is a continuation of U.S.
application Ser. No. 11/659,009, filed Jan. 30, 2007, and issued as
U.S. Pat. No. 7,981,414 on Jul. 19, 2011, which is a National Phase
filing of International Application PCT/AU2006/001940, filed Dec.
20, 2006, which claims the benefit of U.S. provisional patent
application Ser. No. 60/817,272, filed Jun. 28, 2006 and Australian
application AU 2005907124, filed Dec. 20, 2005, the disclosures of
which are incorporated herein by reference in their entirety.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes a Sequence Listing submitted
electronically as a text file named CAP552C20110531SEQLSTTEXT.txt,
created on May 31, 2011, with a size of 41,262 bytes. The Sequence
Listing is incorporated by reference herein.
FIELD OF THE INVENTION
[0003] The invention relates to recombinant domain antibodies
(dAbs) useful for human therapy. More particularly, the present
invention relates to a domain antibody (dAb) which binds to human
TNF-.alpha. and its use in the treatment of disorders characterised
by human TNF-.alpha. activity.
BACKGROUND OF THE INVENTION
[0004] Tumor necrosis factor alpha (TNF-.alpha.) is a cytokine
produced by numerous cell types, including monocytes and
macrophages, that has been implicated in mediating shock and the
pathophysiology of a variety of human diseases and disorders
including sepsis, infections, autoimmune diseases, transplant
rejection and graft-versus-host disease.
[0005] In an effort to counter the harmful effects mediated by
human TNF-.alpha., antibodies that bind to and neutralise human
TNF-.alpha. have been sought as a means to inhibit TNF-.alpha.
activity. Some of the earliest antibodies directed against human
TNF-.alpha. were mouse monoclonal antibodies secreted from
hybridoma cell lines prepared from lymphocytes harvested from mice
immunized with human TNF-.alpha.. Although such antibodies were
effective in binding to and neutralising human TNF-.alpha., their
use in in vivo therapy has been limited by problems associated with
the administration of mouse antibodies to humans, in particular,
elicitation of an unwanted immune response against the mouse
antibody in a human, referred to as human anti-mouse antibody
(HAMA) reactions.
[0006] In an attempt to overcome these problems, murine anti-human
TNF-.alpha. antibodies have been genetically engineered to be more
human-like. For example, human/mouse chimeric antibodies have been
created in which antibody variable region sequences from the mouse
genome are combined with antibody constant region sequences from
the human genome. The chimeric antibodies exhibit the binding
characteristics of the parental mouse antibody, and the effector
functions associated with the human constant region. Although these
chimeric antibodies have been used in human therapy, they still
retain some murine sequences and therefore still may elicit
anti-chimeric antibody reactions in human recipients, particularly
when administered for prolonged periods thus limiting their
therapeutic application.
[0007] Human monoclonal antibodies against human TNF-.alpha. have
been developed using human hybridoma techniques. This approach,
however, suffers from ethical, clinical and immunological
limitations on immunization of human subjects.
[0008] It has been postulated that non-human primate antibodies
will be tolerated in humans because they are structurally similar
to human antibodies (Ehrlich P H et al., Human and primate
monoclonal antibodies for in vivo therapy. Clin Chem. 34:9 pg
1681-1688 (1988)). Furthermore, because human antibodies are
non-immunogenic in Rhesus monkeys (Ehrich P H et al., Rhesus monkey
responses to multiple injections of human monoclonal antibodies.
Hybridoma 1987; 6:151-60), it is likely that the converse is also
applicable and primate antibodies will be non-immunogenic in
humans.
[0009] Evolutionarily distant primates, such as New World primates,
are not only sufficiently different from humans to allow antibodies
against human antigens to be generated, but are sufficiently
similar to humans to have antibodies similar to human antibodies so
that the host does not generate an anti-antibody immune response
when such primate-derived antibodies are introduced into a human.
New World primates (infraorder--Platyrrhini) comprises at least 53
species commonly divided into two families, the Callithricidae and
Cebidae. The Callithricidae consist of marmosets and tamarins. The
Cebidae includes the squirrel monkey, titi monkey, spider monkey,
woolly monkey, capuchin, night or owl monkey and the howler
monkey.
[0010] Previous studies have characterised the expressed
immunoglobulin heavy chain repertoire of the Callithrix jacchus
marmoset (von Budingen H--C et al., Characterization of the
expressed immunoglobulin IGHV repertoire in the New World marmoset
Callithrix jacchus. Immunogenetics 2001; 53:557-563). Six IGHV
subgroups were identified which showed a high degree of sequence
similarity to their human IGHV counterparts. The framework regions
were more conserved when compared to the complementarity
determining regions (CDRs). The degree of similarity between C.
jacchus and human IGHV sequences was less than between Old World
primates and humans.
Domain Antibodies
[0011] Domain antibodies (dAb) are the smallest functioning binding
units of antibodies and correspond to the variable regions of
either the heavy (V.sub.H) or light (V.sub.L) chains of antibodies.
Domain antibodies have a molecular weight of approximately 13 kDa,
or less than one tenth the size of a full antibody.
[0012] Immunoglobulin light chains are referred to as either kappa
or lambda light chains and the heavy chains as gamma, mu, delta,
alpha or epsilon. The variable region gives the antibody its
specificity. Within each variable region are regions of
hypervariability, otherwise known as complementarity determining
regions (CDRs) which are flanked by more conserved regions referred
to as framework regions. Within each variable region are three CDRs
and four framework regions.
[0013] In contrast to conventional antibodies, domain antibodies
are well expressed in bacterial, yeast and mammalian systems. Their
small size allows for higher molar quantities per gram of product,
thus providing a significant increase in potency per dose. In
addition, domain antibodies can be used as a building block to
create therapeutic products such as multiple targeting dAbs in
which a construct containing two or more variable domains bind to
two or more therapeutic targets, or dAbs targeted for pulmonary or
oral administration.
SUMMARY OF THE INVENTION
[0014] In a first aspect, the present invention provides a
recombinant domain antibody (dAb) which binds to human TNF-.alpha.,
the dAb comprising an immunoglobulin heavy or light chain variable
domain, wherein said variable domain comprises at least one
complementarity determining region (CDR) having a sequence derived
from a New World primate wherein the CDR is selected from the group
the group consisting of YAATKLQS (SEQ ID No:1), YEASSLQS (SEQ ID
No:2), YEASKLQS (SEQ ID No:3), YSASNLET (SEQ ID No:4).
[0015] In a second aspect, the invention provides a pharmaceutical
composition comprising an effective amount of the dAb according to
the first aspect of the invention, together with a pharmaceutically
acceptable carrier or diluent.
[0016] In a third aspect, the present invention provides for the
use of a dAb according to the first aspect of the invention in a
diagnostic application for detecting human TNF-.alpha..
[0017] In a fourth aspect, the invention provides a method for
treating a disorder characterised by human TNF-.alpha. activity in
a human subject, comprising administering to the subject a
pharmaceutical composition according to the second aspect of the
invention.
[0018] In a fifth aspect the invention provides a nucleic acid
sequence encoding the dAb of the first aspect of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows the amino acid (SEQ ID No:6) and nucleotide
sequence (SEQ ID No:5) of the acceptor dAb.
[0020] FIGS. 2A-2E show the nucleotide and amino acid sequences of
eleven (11) marmoset and six (6) Owl monkey V.kappa. gene
segments.
[0021] FIG. 3 shows the acceptor dAb amino acid and nucleotide
sequence (both strands). The restriction digest sites for Kpn I and
San DI which excises a region including the CDR2 is indicated in
the figure. CDR2 residues removed are indicated in underlined.
[0022] FIGS. 4A-4D show sequence alignments showing
oligonucleotides used during cloning and final sequence
confirmation of the nucleotide (FIGS. 4A-4C) and amino acid (FIG.
4D) sequences shown in FIGS. 2A-2E.
[0023] FIG. 5 demonstrates the ability of CDR2-grafted dAbs to
inhibit the binding of TNF to recombinant TNF receptor. The dAbs
tested were as follows: Owl Monkey 1 (CDR=YAATKLQS; SEQ ID No:1),
Owl Monkey 2 (CDR=YEASSLQS; SEQ ID No:2), Marmoset 1 (CDR=YEASKLQS;
SEQ ID No:3), Mainioset 2 (CDR=YSASNLET; SEQ ID No:4) and Acceptor
dAb (CDR=YSASELQS; SEQ ID No:49).
[0024] FIG. 6 demonstrates the improved ability of Compounds 100
and 123 to neutralise the cytotoxic activity of TNF on mouse L929
fibroblasts relative to acceptor dAb (Compound 145).
DETAILED DESCRIPTION OF THE INVENTION
[0025] In a first aspect, the present invention provides a
recombinant domain antibody (dAb) which binds to human TNF-.alpha.,
the dAb comprising an immunoglobulin heavy or light chain variable
domain, wherein said variable domain comprises at least one
complementarity determining region (CDR) having a sequence derived
from a New World primate wherein the CDR is selected from the group
the group consisting of YAATKLQS (SEQ ID No:1), YEASSLQS (SEQ ID
No:2), YEASKLQS (SEQ ID No:3), YSASNLET (SEQ ID No:4).
[0026] Preferably, the CDR is CDR2.
[0027] In a preferred embodiment the dAb has a sequence selected
from:
TABLE-US-00001 [Compound 145; SEQ ID No: 7]
DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
[Compound 123; SEQ ID No: 8]
DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
[Compound 100; SEQ ID No: 9]
DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
[Compound 196; SEQ ID No: 10]
DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
[Compound 134; SEQ ID No: 50]
DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKPPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
[Compound 137; SEQ ID No: 51]
DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
[Compound 121; SEQ ID No: 52]
DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
[0028] In a further aspect the invention provides a nucleic acid
sequence encoding the dAb of the first aspect of the invention.
[0029] The term "binds to" as used herein, is intended to refer to
the binding of an antigen by an immunoglobulin variable region with
a dissociation constant (K.sub.d) of 1 .mu.M or lower as measured
by surface plasmon resonance analysis using, for example a
BIAcore.TM. surface plasmon resonance system and BIAcore.TM.
kinetic evaluation software (eg. version 2.1). The affinity or
dissociation constant (K.sub.d) for a specific binding interaction
is preferably about 500 nM or lower, more preferably about 300 nM
or lower and preferably at least 300 nM to 50 pM, 200 nM to 50 pM,
and more preferably at least 100 nM to 50 pM, 75 nM to 50 pM, 10 nM
to 50 pM.
[0030] The term "variable domain" as used herein is meant a folded
polypeptide domain which comprises sequences characteristic of
immunoglobulin heavy or light chain variable domains and which
specifically binds an antigen. A domain antibody or dAb is
equivalent to a single variable domain polypeptide.
[0031] It will be appreciated by persons skilled in the art that
the remainder of the variable domain sequence may be derived from
either a human, New World primate or Old World primate variable
domain sequence which, because of their evolutionary association
with humans, share a high degree of homology with the human
sequence. Thus, for example, a CDR selected from the sequences
above may be grafted into the human or primate variable region
sequence to replace the wild-type CDR.
[0032] Accordingly, the invention is further based on a method for
amplification of New World primate immunoglobulin variable domain
genes, for example by polymerase chain reaction (PCR) from nucleic
acid extracted from New World primate lymphocytes using primers
specific for heavy and light chain variable domain gene families.
For example, information regarding the boundaries of the variable
domains of heavy and light chain genes (V.sub.H and V.sub.L
respectively) can be used to design PCR primers that amplify the
variable domain from a cloned heavy or light chain coding sequence
encoding an antibody known to bind a given antigen. The amplified
variable domain is then inserted either alone or as a fusion with
another polypeptide sequence into a suitable expression vector. The
expressed variable domain is then screened for high affinity
binding to the desired antigen.
[0033] The repertoire of V.sub.H and V.sub.L domains can be a
naturally occurring repertoire of immunoglobulin sequences or a
synthetic repertoire. A naturally occurring repertoire is one
prepared, for example, from immunoglobulin expressing cells
harvested from one or more primates. Such repertoires can be naive
ie. prepared from newborn immunoglobulin expressing cells, or
rearranged ie. prepared from, for example, adult primate B cells.
If desired, clones identified from a natural repertoire, or any
repertoire that bind the target antigen are then subject to
mutagenesis and further screening in order to produce and select
variants with improved binding characteristics.
[0034] Synthetic repertoires of single immunoglobulin variable
domains are prepared by artificially introducing diversity into a
cloned variable domain.
[0035] A repertoire of V.sub.H and V.sub.L domains can be screened
for desired binding specificity and functional behaviour by, for
example phage display. Methods for the construction of
bacteriophage display libraries and lambda phage expression
libraries are well known in the art. The phage display technique
has been described extensively in the art and examples of methods
and compounds for generating and screening such libraries and
affinity maturing the products of them can be found in, for
example, Barbas et al. (1991) PNAS 88:7978-7982; Clarkson et al.
(1991) Nature 352:624:628; Dower et al. PCT. 91/17271, U.S. Pat.
No. 5,427,908, U.S. Pat. No. 5,580,717 and EP 527,839; Fuchs et al.
(1991) Bio/Technology 9:1370-1372; Garrad et al. (1991)
Bio/Technology 9:1373:1377; Garrard et al. PCT WO 92/09690; Gram et
al. (1992) PNAS 89:3576-3580; Griffiths et al. (1993) EMBO J
12:725:734; Griffiths et al. U.S. Pat. No. 5,885,793 and EP
589,877; Hawkins et al. (1992) J Mol Biol 226:889-896; Hay et al.
(1992) Hum Antibod Hybridomas 3:81-85; Hoogenboom et al. (1991) Nuc
Acid Res 19:4133-4137; Huse et al. (1989) Science 246:1275-1281;
Knappik et al. (2000) J Mol Biol 296:57-86; Knappik et al. PCT WO
97/08320; Ladner et al. U.S. Pat. No. 5,223,409, U.S. Pat. No.
5,403,484, U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,837,500 and EP
436,597; McCafferty et al. (1990) Nature 348:552-554; McCafferty et
al. PCT. WO 92/01047, U.S. Pat. No. 5,969,108 and EP 589,877;
Salfeld et al. PCT WO 97/29131, U.S. Provisional Application No.
60/126,603; and Winter et al. PCT WO 92/20791 and EP 368,684.
[0036] Recombinant libraries expressing the repertoire of V.sub.H
and V.sub.L domains can be expressed on the surface of
microorganisms eg. Yeast or bacteria (see PCT publications
WO99/36569 and 98/49286).
[0037] The Selective Lymphocyte Antibody Method or SLAM as it is
referred to in the state of the art, is another means of generating
high affinity antibodies rapidly. Unlike phage display approaches
all antibodies are fully divalent. In order to generate New World
primate antibodies, New World primates are immunised with a human
antigen eg. a TNF-.alpha. polypeptide. Following immunisation cells
are removed and selectively proliferated in individual micro wells.
Supernatants are removed from wells and tested for both binding and
function. Gene sequences can be recovered for subsequent
manipulations eg. humanisation, Fab fragment, scFv or dAb
generation. Thus another example is the derivation of the antibody
or antibody species of the invention by SLAM and its derivatives
(Babcock, J. S. et al 1996, Proc. Natl. Acad. Sci, USA 93;
7843-7848, U.S. Pat. No. 5,627,052 and PCT publication WO92/02551).
Adaptations of SLAM, such as the use of alternatives to testing
supernatants such as panning, also lie within the scope of this
invention.
[0038] In one expression system the recombinant peptide/protein
library is displayed on ribosomes (for examples see Roberts, R W
and Szostak, J. W. 1997. Proc. Natl. Acad. Sci. USA.
94:12297-123202 and PCT Publication No. WO98/31700). Thus another
example involves the generation and in vitro transcription of a DNA
library (eg of antibodies or derivatives preferably prepared from
immunised cells, but not so limited), translation of the library
such that the protein and "immunised" mRNAs stay on the ribosome,
affinity selection (eg by binding to RSP), mRNA isolation, reverse
translation and subsequent amplification (eg by polymerase chain
reaction or related technology). Additional rounds of selection and
amplification can be coupled as necessary to affinity maturation
through introduction of somatic mutation in this system or by other
methods of affinity maturation as known in the state of the
art.
[0039] Another example sees the application of emulsion
compartmentalisation technology to the generation of the domain
antibodies of the invention. In emulsion compartmentalisation, in
vitro and optical sorting methods are combined with
co-compartmentalisation of translated protein and its nucleotide
coding sequence in aqueous phase within an oil droplet in an
emulsion (see PCT publications no's WO99026711 and WO0040712). The
main elements for the generation and selection of antibodies are
essentially similar to the in vitro method of ribosome display.
[0040] The CDR sequences may be obtained from several sources, for
example, databases e.g. The National Centre for Biotechnology
Information protein and nucleotide databases, The Kabat Database of
Sequences of Proteins of Immunological Interest. Alternatively, the
CDR regions can be predicted from the V.sub.H and V.sub.L domain
repertoire (see for example Kabat E A and Wu T T. Attempts to
locate complementarity determining residues in the variable
positions of light and heavy chains. Ann. NY Acad. Sci. 190:382-93
(1971)). The CDR sequence may be a genomic DNA or a cDNA.
[0041] There are a number of ways in which a replacement CDR may be
grafted into a variable domain sequence and such methods will be
familiar to those skilled in the art. The preferred method of the
present invention involves replacement of the CDR2 in the variable
region domain via primer directed mutagenesis. This method consists
of annealing a synthetic oligonucleotide encoding a desired
mutations to a target region where it serves as a primer for
initiation of DNA synthesis in vitro, extending the oligonucleotide
by a DNA polymerase to generate a double-stranded DNA that carries
the desired mutations, and ligating and cloning the sequence into
an appropriate expression vector.
[0042] Preferably, the domain antibody according to the invention
has low immunogenicity in humans.
[0043] By reference to the term "low immunogenicity" it is meant
that the domain antibody does not raise an antibody response in a
human of sufficient magnitude to reduce the effectiveness of
continued administration of the antibody for a sufficient time to
achieve therapeutic efficacy.
[0044] Preferably, the variable region sequence into which the CDR
is grafted is the "dAb acceptor sequence" (designated Compound 128)
provided in FIG. 1.
[0045] The dAb acceptor sequence consists of the amino acid
sequence set forth in SEQ ID No:6:
TABLE-US-00002 (SEQ ID No: 6)
DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASELQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR.
[0046] This sequence is encoded by the nucleotide sequence set
forth in SEQ ID No:5:
TABLE-US-00003 GAC ATC CAG ATG ACC CAG TCT CCA TCC TCT CTG TCT GCA
TCT GTA GGA GAC CGT GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT GAT
AGT TAT TTA CAT TGG TAC CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG
ATC TAT AGT GCA TCC GAG TTG CAA AGT GGG GTC CCA TCA CGT TTC AGT GGC
AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT GAA
GAT TTT GCT ACG TAC TAC TGT CAA CAG GTT GTG TGG CGT CCT TTT ACG TTC
GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG
[0047] In one preferred embodiment of the present invention, a
marmoset CDR sequence YSASNLET (SEQ ID No:4) is grafted into the
dAb acceptor sequence so as to replace the CDR2 sequence (YSASELQS;
SEQ ID No:49) of the dAb acceptor sequence to produce the following
dAb (designated Compound 145):
Compound 145
TABLE-US-00004 [0048] (SEQ ID No: 7)
DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
[0049] Thus, in one preferred embodiment, the dAb which binds to
human TNF-.alpha. comprises the amino acid sequence of SEQ ID
No:7.
[0050] It is within the scope of the present invention, that the
dAb sequence may be further subject to affinity maturation in order
to improve its antigen binding characteristics. This may
necessitate the modification of certain amino acid residues within
CDR1 and CDR3.
[0051] For example, the marmoset CDR-grafted dAb set forth in SEQ
ID No:7 was affinity matured as set out in the Materials and
Methods and tested for TNF-binding. In a further preferred
embodiment, the dAb which binds to human TNF-.alpha. comprises the
amino acid sequence of SEQ ID No:8 or SEQ ID No:9. These have been
designated Compound 123 and Compound 100 respectively and their
sequences are shown below:
Compound 123
TABLE-US-00005 [0052] (SEQ ID No: 8)
DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
Compound 100 (SEQ ID No: 9)
DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
[0053] In a particularly preferred embodiment, the dAb which binds
to human TNF-.alpha. comprises the amino acid sequence of SEQ ID
No:10. This has ben designated Compound 196 and the sequence is
provided below:
Compound 196
TABLE-US-00006 [0054] (SEQ ID No: 10)
DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYS
ASNLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQ GTKVEIKR
[0055] The dAb according to the invention may further comprise an
immunoglobulin constant region (Fc region) connected thereto. The
constant region sequence may be derived from human or primate
sequences. The primate sequence may be a New World primate or an
Old World primate sequence. Suitable Old World primates include
chimpanzee, or other hominid ape eg. gorilla or orang utan, which
because of their close phylogenetic proximity to humans, share a
high degree of homology with the human constant region
sequence.
[0056] The dAb (with or without the constant region connected
thereto) can be derivatised or linked to another functional
molecule. For example, the dAb can be functionally linked by
chemical coupling, genetic fusion, noncovalent association or
otherwise, to one or more other molecular entities, such as another
antibody, a detectable agent, a cytotoxic agent, a pharmaceutical
agent, and/or a protein or peptide that can mediate association of
the antibody with another molecule (such as a streptavidin core
region or a polyhistidine tag).
[0057] Useful detectable agents with which the dAb may be
derivatised include fluorescecnt compounds. Exemplary fluorescent
detectable agents include fluorescein, fluorescein isothiocyanate,
rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride,
phycoerythrin and the like. The dAb may also be derivatised with
detectable enzymes such as alkaline phosphatase, horseradish
peroxidase, glucose oxidase and the like. When a dAb is derivatized
with a detectable enzyme, it is detected by adding additional
reagents that the enzyme uses to produce a detectable reaction
product. A dAb may also be derivatised with biotin, and detected
through indirect measurement of avidin or streptavidin binding.
[0058] The present invention also extends to PEGylated dAbs (with
or without the constant region connected thereto) which provide
increased half-life and resistance to degradation without a loss in
activity (eg. binding affinity) relative to non-PEGylated antibody
polypeptides.
[0059] The dAb can be coupled, using methods known in the art, to
polymer molecules (preferably PEG) useful for achieving the
increased half-life and degradation resistance properties. Polymer
moieties which can be utilised in the invention can be synthetic or
naturally occurring and include, but or not limited to straight or
branched chain polyalkylene, polyalkenylene or polyoxyalkylene
polymers, or a branched or unbranched polysaccharide such as a
homo- or heteropolysaccharide. Preferred examples of synthetic
polymers which can be used in the invention include straight or
branched chain poly(ethylene glycol) (PEG), polypropylene glycol),
or poly(vinyl alcohol) and derivatives or substituted forms
thereof. Particularly preferred substituted polymers for linkage to
dAbs include substituted PEG, including methoxy(polyethylene
glycol). Naturally occurring polymer moieties which can be used in
addition to or in place of PEG include lactose, amylose, dextran,
or glycogen, as well as derivatives thereof which would be
recognised by persons skilled in the art.
[0060] Derivatized forms of polymer molecules include, for example,
derivatives which have additional moieties or reactive groups
present therein to permit interaction with amino acid residues of
the domain antibody polypeptides described herein. Such derivatives
include N-hydroxylsuccinimide (NHS) active esters, succinimidyl
propionate polymers, and sulfhydryl-selective reactive agents such
as maleimide, vinyl sulfone, and thiol. PEG polymers useful in the
invention can be linear molecules, or can be branched wherein
multiple PEG moieties are present in a single polymer.
[0061] The reactive group (e.g., MAL, NHS, SPA, VS, or Thiol) may
be attached directly to the PEG polymer or may be attached to PEG
via a linker molecule.
[0062] The size of polymers useful in the invention can be in the
range of between 500 Da to 60 kDa, for example, between 1000 Da and
60 kDa, 10 kDa and 60 kDa, 20 kDa and 60 kDa, 30 kDa and 60 kDa, 40
kDa and 60 kDa, and up to between 50 kDa and 60 kDa. The polymers
used in the invention, particularly PEG, can be straight chain
polymers or may possess a branched conformation.
[0063] The polymer (PEG) molecules useful in the invention can be
attached to a domain antibody using methods which are well known in
the art. The first step in the attachment of PEG or other polymer
moieties to an antibody polypeptide monomer or multimer of the
invention is the substitution of the hydroxyl end-groups of the PEG
polymer by electrophile-containing functional groups. Particularly,
PEG polymers are attached to either cysteine or lysine residues
present in the domain antibody. The cysteine and lysine residues
can be naturally occurring, or can be engineered into the antibody
polypeptide molecule. For example, cysteine residues can be
recombinantly engineered at the C-terminus of a dAb polypeptide, or
residues at specific solvent accessible locations in a dAb or other
antibody polypeptide can be substituted with cysteine or
lysine.
[0064] The dAb according to the invention may be linked to one or
more molecules which can increase its half-life in vivo. These
molecules may be linked to the dAb via a linker so that they do not
interfere/sterically hinder the antigen binding site.
Alternatively, they may be linked to the constant region.
Typically, such molecules are polypeptides which occur naturally in
vivo and which resist degradation or removal by endogenous
mechanisms. Molecules which increase half life may be selected from
the following:
(a) proteins from the extracellular matrix, eg. collagen, laminin,
integrin and fibronectin; (b) proteins found in blood, eg. fibrin
.alpha.-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen B,
serum amyloid protein A, heptaglobin, protein, ubiquitin,
uteroglobulin, B-2 microglobulin, plasminogen, lysozyme, cystatin
C, alpha-1-antitrypsin and pancreatic kypsin inhibitor; (c) immune
serum proteins, eg. IgE, IgG, IgM; (d) transport proteins, eg.
retinol binding protein, .alpha.-1 microglobulin; (e) defensins,
eg. beta-defensin 1, Neutrophil defensins 1, 2 and 3; (f) proteins
found at the blood brain barrier or in neural tissues, eg.
melanocortin receptor, myelin, ascorbate transporter; (g)
transferrin receptor specific ligand-neuropharmaceutical agent
fusion proteins (see U.S. Pat. No. 5,977,307); brain capillary
endothelial cell receptor, transferrin, transferrin receptor,
insulin, insulin-like growth factor 1 (IGF 1) receptor,
insulin-like growth factor 2 (IGF 2) receptor, insulin receptor;
(h) proteins localised to the kidney, eg. polycystin, type IV
collagen, organic anion transporter K1, Heymann's antigen; (i)
proteins localised to the liver, eg. alcohol dehydrogenase, G250;
(j) blood coagulation factor X; (k) .alpha.-1 antitrypsin;
(l) HNF 1.alpha.;
[0065] (m) proteins localised to the lung, eg. secretory component
(binds IgA); (n) proteins localised to the Heart, eg. HSP 27; (o)
proteins localised to the skin, eg, keratin; (p) bone specific
proteins, such as bone morphogenic proteins (BMPs) eg. BMP-2, -4,
-5, -6, -7 (also referred to as osteogenic protein (OP-1) and -8
(OP-2); (q) tumour specific proteins, eg. human trophoblast
antigen, herceptin receptor, oestrogen receptor, cathepsins eg
cathepsin B (found in liver and spleen); (r) disease-specific
proteins, eg. antigens expressed only on activated T-cells:
including LAG-3 (lymphocyte activation gene); osteoprotegerin
ligand (OPGL) see Nature 402, 304-309, 1999; OX40 (a member of the
TNF receptor family, expressed on activated T cells and the only
costimulatory T cell molecule known to be specifically up-regulated
in human T cell leukaemia virus type-I (HTLV-I)-producing
cells--see J. Immunol. 2000 Jul. 1; 16561):263-70; metalloproteases
(associated with arthritis/cancers), including CG6512 Drosophila,
human paraplegin, human FtsH, human AFG3L2, murine ftsH; angiogenic
growth factors, including acidic fibroblast growth factor (FGF-1),
basic fibroblast growth factor (FGF-2), vascular endothelial growth
factor/vascular permeability factor (VEGF/VPF), transforming growth
factor-.alpha. (TGF-.alpha.), tumor necrosis factor-alpha
(TNF-.alpha.), angiogenin, interleukin-3 (IL-3), interleukin-8
(IL-8), platelet derived endothelial growth factor (PD-ECGF),
placental growth factor (PlGF), midkine platelet-derived growth
factor-BB (PDGF), fractalkine; (s) stress proteins (heat shock
proteins); (t) proteins involved in Fc transport; and (u)
antibodies, fragments or derivatives directed against endogenous
proteins e.g. serum albumin.
[0066] In a further embodiment of the present invention, the dAb
according to the first aspect may be multimerised, as for example,
hetero- or homodimers, hetero- or homotrimers, hetero- or
homotetramers, or higher order hetero- or homomultimers.
Multimerisation can increase the strength of antigen binding,
wherein the strength of binding is related to the sum of the
binding affinities of the multiple binding sites.
[0067] Thus, the invention provides a domain antibody according to
the first aspect, wherein the domain antibody is linked to at least
one further domain antibody. Each dAb may bind to the same or
different antigens.
[0068] The dAb multimers may further comprise one or more dAbs
which are linked and wherein each dAb binds to a different antigen,
multi-specific ligands including so-called "dual-specific ligands".
For example, the dual specific ligands may comprise a pair of
V.sub.H domains or a pair of V.sub.L domains. Such dual-specific
ligands are described in WO 2004/003019 (PCT/GB2003/002804) in the
name of Domantis Ltd.
[0069] In a second aspect, the invention provides a pharmaceutical
composition comprising an effective amount of the dAb according to
the first aspect of the invention, together with a pharmaceutically
acceptable carrier or diluent.
[0070] A "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
antifungal agents, isotonic and absorption delaying agents, and the
like which are physiologically compatible. Examples of
pharmaceutically acceptable carriers include one or more of water,
saline, phosphate buffered saline, dextrose, glycerol, ethanol, and
the like as well as combinations thereof. In many cases it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Pharmaceutically acceptable substances such as wetting
or minor amounts of auxiliary substances such as wetting or
emulsifying agents, preservatives or buffers.
[0071] The composition may be in a variety of forms, including
liquid, semi-solid and solid dosage forms, such as liquid solutions
(eg injectable and infusible solutions), dispersions or
suspensions, tablets, pills, powders, liposomes and suppositories.
Preferably, the composition is in the form of an injectable
solution for immunization. The administration may be intravenous,
subcutaneous, intraperitoneal, intramuscular, transdermal,
intrathecal, and intra-arterial.
[0072] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
compositions can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (ie. dAb) into the required
amount in an appropriate solvent with one or a combination of
ingredients listed above, followed by filtered sterilisation.
[0073] The composition may also be formulated as a sterile powder
for the preparation of sterile injectable solutions. The proper
fluidity of a solution can be maintained by for example, use of a
coating such as lecithin and/or surfactants.
[0074] In certain embodiments, the active compound may be prepared
with a carrier that will protect the compound against rapid
release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Compatible polymers may be used such as ethylene vinyl
acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters and polylactic acid.
[0075] The composition may also be formulated for oral
administration. In this embodiment, the dAb may be enclosed in a
hard or soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet.
[0076] The composition may also be formulated for rectal
administration.
[0077] Supplementary active compounds can also be incorporated into
the composition. The domain antibody may be co-formulated with
and/or co-administered with one or more additional therapeutic
agents eg. anti-inflammatory compounds, soluble TNF-.alpha.
receptor or a chemical agent that inhibits human TNF-.alpha.
production, or antibodies that bind other targets such as cytokines
or cell surface molecules. Alternatively, it may be co-administered
with a soluble immunochemical reagent such as protein A, C, G or
L.
[0078] An effective amount may include a therapeutically effective
amount or prophylactically effective amount of the dAb of the
invention. A therapeutically effective amount refers to an amount
effective at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A prophylactically effective amount
refers to an amount effective, at dosages and for periods of time
necessary, to achieve the desired prophylactic result.
[0079] In a preferred embodiment the composition is administered to
mammals, preferably humans or primates.
[0080] In a third aspect, the present invention provides for the
use of a dAb according to the first aspect of the invention in a
diagnostic application for detecting human TNF-.alpha..
[0081] For example, the anti-human TNF-.alpha. dAb according to the
invention can be used to detect human TNF-.alpha. for example in a
biological sample, such as serum or plasma using a conventional
immunoassay, such as an enzyme linked immunosorbent assay (ELISA),
a radioimmunoassay (RIA) or tissue immunohistochemistry. The
anti-human TNF-.alpha. dAb according to the invention can be
assayed in biological fluids by a competition immunoassay using
recombinant human TNF-.alpha. standards labelled with a detectable
substance and an unlabelled anti-human TNF-.alpha. antibody.
[0082] The anti-human TNF-.alpha. dAb according to the invention
may also be used to detect TNF-.alpha.from species other than
humans eg. chimpanzee, marmoset, rhesus, mouse, pig.
[0083] The anti-human TNF-.alpha. dAb according to the invention
may also be used in cell culture applications where it is desired
to inhibit TNF-.alpha. activity.
[0084] In a fourth aspect, the invention provides a method for
treating a disorder characterised by human TNF-.alpha. activity in
a human subject, comprising administering to the subject a
pharmaceutical composition according to the second aspect of the
invention.
[0085] A disorder characterised by human TNF-.alpha. activity is
intended to include diseases and other disorders in which the
presence of TNF-.alpha. in a subject suffering from the disorder
has been shown to be or is suspected of being either responsible
for the pathophysiology of the disorder or a factor which
contributes to a worsening of the disorder. Preferably, the
disorder characterised by human TNF-.alpha. activity is selected
from the group consisting of inflammation, inflammatory diseases,
sepsis, including septic shock, endotoxic shock, gram negative
sepsis and toxic shock syndrome; autoimmune disease, including
rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and
gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes,
autoimmune uveitis and nephrotic syndrome; infectious disease,
including fever and myalgias due to infection and cachexia
secondary to infection; graft versus host disease; tumour growth or
metastasis; pulmonary disorders including adult respiratory
distress syndrome, shock lung, chronic pulmonary inflammatory
disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis;
inflammatory bowel disorders including Crohn's disease and
ulcerative colitis; cardiac disorders; inflammatory bone disorders,
hepatitis, coagulation disturbances, burns, reperfusion injury,
keloid formation and scar tissue formation.
[0086] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0087] All publications mentioned in this specification are herein
incorporated by reference. Any discussion of documents, acts,
materials, devices, articles or the like which has been included in
the present specification is solely for the purpose of providing a
context for the present invention. It is not to be taken as an
admission that any or all of these matters form part of the prior
art base or were common general knowledge in the field relevant to
the present invention as it existed in Australia or elsewhere
before the priority date of each claim of this application.
[0088] In order that the nature of the present invention may be
more clearly understood, preferred forms thereof will now be
described with reference to the following non-limiting
examples.
Example 1
Materials and Methods
Isolation of New World Primate VL Genes
[0089] Marmoset (genus Callithrix, species unknown) and Owl monkey
(Aotus trivirgatus) genomic DNA were obtained from the European
Collection of Cell Cultures (ECACC), catalogue numbers 85011419 and
90110510 respectively. Marmoset DNA was derived from cell line
B95-8 while Owl monkey DNA came from cell line OMK 637-69.
[0090] Degenerate primers based on human V.kappa. leader sequences
and recombination signal sequences (RSS) were derived from Walter
and Tomlinson, Antibody Engineering: A Practical Approach (1996).
The primers used for amplification of germline V.kappa. DNA were as
follows:
TABLE-US-00007 Primer VK1BL (SEQ ID No: 11) AATCKCAGGTKCCAGATG
Primer VK1BL35a (SQ ID No: 12) GTTYRGGTKKGTAACACT Primer VK1BL35b
(SEQ ID No: 13) ATGMCTTGTWACACTGTG
[0091] Genomic PCR (30 cycles) was performed using Taq polymerase
with either primer pair VK1BL.times.VK1BL35a or
VK1BL.times.VK1BL35b. There was overlap between the sequences
cloned and the two primer sets used.
[0092] PCR products were cloned into Invitrogen's TOPO TA cloning
kit (Cat No K4500-01) and sequenced with M13 forward and pUC
reverse primers. Sequence was confirmed in forward and reverse
directions. In order to further confirm key sequences were not
subject to PCR errors, the PCR and cloning process was repeated
twice for marmoset sequences. Nucleotide (SEQ ID Nos:14-24 and SEQ
ID Nos:36-41) and amino acid (SEQ ID Nos:25-35 and SEQ ID
Nos:42-47) are given in FIG. 2. Marmoset sequences 1, 2 and 3 were
confirmed. Sequences 4, 5, 6, 7 and 8 were seen only in the initial
PCR. Sequences 9, 10 and 11 were seen only in the repeat (ie
second) PCR and cloning.
Oligo Synthesis and Cloning into Acceptor Sequence
[0093] Four CDR sequences, namely YAATKLQS (SEQ ID No:1) from Owl
monkey sequence 1 (SEQ ID No:42), YEASSLQS (SEQ ID No:2) from Owl
monkey sequence 2 (SEQ ID No:43), YEASKLQS (SEQ ID No:3) from
Marmoset sequence 1 (SEQ ID No:25), and YSASNLET (SEQ ID No:4) from
Marmoset sequence 2 (SEQ ID No:26), were chosen from the amino acid
sequences shown in FIG. 2 as indicated. Owl Monkey sequence 5,
YYASSLQS (SEQ ID No:48) was found to be identical to GI6176295 an
Aotus nancymaae (Ma's night monkey) cDNA sequence, all other
sequences were unique.
[0094] An acceptor variable region (anti-TNF domain antibody)
sequence in the expression vector (Domantis proprietary vector) was
digested (25 .mu.g) sequentially with KpnI and SanDI which excises
the majority of FR2 as well as CDR2 as indicated on the restriction
digest map. The vector was then gel purified to remove the excised
wild-type FR2 and CDR2 sequence.
[0095] Oligo annealing was performed by incubating oligo pairs (500
pmol of each as shown in FIGS. 4A and 4B) at 95.degree. C. for 5
minutes followed by 65.degree. C. for 5 minutes and then allowed to
reach room temperature slowly on a hot block. Overlaps were then
filled in during a Klenow reaction in the presence of dNTPs.
Affinity Maturation
[0096] The marmoset CDR-grafted dAb Compound 145 (SEQ ID No:7) was
affinity matured by constructing 14 separate libraries, each a
diversification of the sequence of SEQ ID No:7 at a single amino
acid residue. The selected residues are shown shaded below.
TABLE-US-00008 ##STR00001##
[0097] The selection was based upon residues in CDR1 and CDR3 that
are known to be diversified in the mature human Ig repertoire, and
framework residues that have been observed to produce functional
proteins after mutagenesis in related dAbs. For each of the
selected residues, complimentary forward and reverse PCR primer
pairs were designed with NKK degeneracy, and two initial PCR
reactions were performed each with a single mutagenic primer and
flanking primer. After clean-up, the two PCR products were annealed
and then amplified using flanking primers alone (splicing by
overlap extension of PCR; Lowman H. L. & Clackson T. (eds),
Phage Display: A practical approach, Oxford University Press,
Oxford, UK). Clones were initially screened by ELIZA using
solid-phase TNF, and positive clones were sequenced. dAb protein
was purified from the best clones and evaluated for potency in
receptor binding assays and L929 cytotoxicity assays. Compounds 100
(SEQ ID No:9) and 123 (SEQ ID No:8) were found to have improved
TNF-neutralization relative to the parent dAb, Compound 145 (SEQ ID
No:7).
[0098] Combination of the affinity-enhancing substitutions of
Compounds 100 (SEQ ID No:9) and 123 (SEQ ID No:8), yielded an
anti-TNF dAb with further improved potency in the L929 cytotoxicity
assay (Compound 196; SEQ ID No:10).
Results
Potency of Anti-TNF dAb Clones in Receptor Binding Assay (RBA) and
Cytotoxocity Assay
[0099] The ability of the anti-TNF dAbs to inhibit TNF binding to
its receptor and to neutralize TNF-mediated cytotoxicity of L929
cells was conducted as follows:
Receptor Binding Assay
[0100] dAbs diversified in the 14 selected positions were tested
for the ability to inhibit the binding of TNF to recombinant TNF
receptor 1 (p55). Briefly, Maxisorp plates were incubated overnight
with 30 mg/ml anti-human Fc mouse monoclonal antibody (Zymed, San
Francisco, USA). The wells were washed with phosphate buffered
saline (PBS) containing 0.05% Tween-20 and then blocked with 1% BSA
in PBS before being incubated with 100 ng/ml TNF receptor 1 Fc
fusion protein (R&D Systems, Minneapolis, USA). Each dAb was
mixed with TNF which was added to the washed wells at a final
concentration of 10 ng/ml. TNF binding was detected with 0.2 mg/ml
biotinylated anti-TNF antibody (HyCult biotechnology, Uben,
Netherlands) followed by 1 in 500 dilution of horse radish
peroxidase labelled streptavidin (Amersham Biosciences, UK) and
then incubation with TMB substrate (KPL, Gaithersburg, USA). The
reaction was stopped by the addition of HCl and the absorbance was
read at 450 nm. Anti-TNF dAb activity lead to a decrease in TNF
binding and therefore a decrease in absorbance compared with the
TNF only control (FIG. 5).
L929 Cytotoxicity Assay
[0101] Anti-TNF dAbs identified by the minilibrary diversification
approach, including Compounds 100 (SEQ ID No:9) and 123 (SEQ ID
No:8), were also tested for the ability to neutralise the cytotoxic
activity of TNF on mouse L929 fibroblasts (Evans, T. (2000)
Molecular Biotechnology 15, 243-248). Briefly, L929 cells plated in
microtitre plates were incubated overnight with anti-TNF dAb, 100
pg/ml TNF and 1 mg/ml actinomycin D (Sigma, Poole, UK). Cell
viability was measured by reading absorbance at 490 nm following an
incubation with
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carbboxymethoxyphenyl)-2-(4-sulfopheny-
l)-2H-tetrazolium (Promega, Madison, USA). Anti-TNF dAb activity
lead to a decrease in TNF cytotoxicity and therefore an increase in
absorbance compared with the TNF only control. The results, in
comparison with the parent dAb Compound 145 (SEQ ID No:7) are
presented in FIG. 6.
[0102] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
Sequence CWU 1
1
7717PRTAotus trivirgatus 1Ala Ala Thr Lys Leu Gln Ser 1 5
27PRTAotus trivirgatus 2Glu Ala Ser Ser Leu Gln Ser 1 5
37PRTCallithrix 3Glu Ala Ser Lys Leu Gln Ser 1 5 47PRTCallithrix
4Ser Ala Ser Asn Leu Glu Thr 1 5 5324DNAArtificial
SequenceSynthesized construct 5gacatccaga tgacccagtc tccatcctct
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
agttatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatagt gcatccgagt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 3246108PRTArtificial
SequenceSynthesized construct 6Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Asp Ser Tyr 20 25 30 Leu His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala
Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg Pro Phe
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
7108PRTArtificial SequenceSynthesized construct 7Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Ser Tyr 20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val
Val Trp Arg Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 8108PRTArtificial SequenceSynthesized construct
8Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Asp Ser
Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Leu Glu Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Val Val Trp Arg Pro Phe 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 9108PRTArtificial
SequenceSynthesized construct 9Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Asp Ser Tyr 20 25 30 Leu His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala
Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Leu Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg Pro Phe
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
10108PRTArtificial SequenceSynthesized construct 10Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Asp Ser Tyr 20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Leu Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val
Val Trp Arg Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 1118DNAArtificial SequenceSynthesized construct
11aatckcaggt kccagatg 181218DNAArtificial SequenceSynthesized
construct 12gttyrggtkk gtaacact 181318DNAArtificial
SequenceSynthesized construct 13atgmcttgtw acactgtg
1814267DNACallithrix 14gacatccaga tgacccagtc tccatcttcc ctgactgcat
ctgtaggagg caaagtcacc 60atcacttgcc gggcgagtca ggacattaac aagtggttag
cctggtatca gcagaaacca 120gggacagtcc ctaagcccct gatctatgag
gcatccaaat tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacatat tttactctca ccatcagcag cctgcagcct 240gaagatgctg
caacttatta ctgtcag 26715267DNACallithrix 15gacatccaga tgatccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgct gggcaagtca
gggtattagc cactggttag cctggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctatagt gcatcaaatt tagaaacagg ggtcccatca
180aggttcagtg gaagtggatc caggacagat tttactctca ccatcagcag
cctgcagcct 240gaagatattg caacatatta ctgtcaa 26716267DNACallithrix
16gacatccaga tgacccagac tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc gggcaagtca gggtattagc agctggttag cctggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctatggg gcatcaaatt tggaaacagg
ggtcccatca 180agattcagcg gaagtggatc tgggacagat tttactctca
ccatcagcag tctgcagcct 240gaagatattg caacatatta ctgtcaa
26717267DNACallithrix 17gacatccaga tgatccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgct gggcaagtca gggtattagc cactggttag
cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatagt
gcatcaaatt taggaacagg ggtcccatca 180aggttcagtg gaagtggatc
caggacagat tttactctca ccatcagcag cctgcagcct 240gaagatattg
caacatatta ctgtcaa 26718267DNACallithrix 18gacatccaga tgacccagtc
tccatcttcc ctgactgcat ctgtaggagg caaagtcacc 60atcacttgcc gggcgtgtca
ggacattaac aagtggttag cctggtatca gcagaaacca 120gggacagtcc
ctaagcccct gatctatgag gcatccaaat tgcaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacatat tttactctca ccatcagcag
cctgcagcct 240gaagatgctg caacttatta ctgtcag 26719267DNACallithrix
19gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagttacc
60atcacttgcc gggcgagtca gggcattagt aattatttag cctggtatca gcagaaacca
120gggaaaactc ctaggctcct gatctatgct gcatccagtt tacaaactgg
gattccctct 180cggttcagcg gcagtggatc tgggacagac tacactctca
ccatcagcag cctgcagtct 240gaagatgttg caatttatta ctgtcaa
26720267DNACallithrix 20gacatccaga tgacccagtc tccatcttcc ctgactgcat
ctgtaggagg caaagtcacc 60atcacttgcc gggcgagtca ggacattaac aagtggttag
cctggtatca gcagaaacca 120gggacagtcc ctaagcccct gatctatgag
gcatccaaat tgcaaagtgg ggtcccatca 180aggctcagcg gcagtggatc
tgggacatat ttcactctca ccatcagcag cctgcagcct 240gaagatgctg
caacttatta ctgtcag 26721267DNACallithrix 21gacatccaga tgacccagtc
tccatcttcc ctgactgcat ctgtaggagg caaagtcacc 60atcacttgcc gggcgagtca
ggacattaac aagtggtcag cctggtatca gcagaaacca 120gggacagtcc
ctaagcccct gatctatgag gcatccaaat tgcaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacatat tttactctca ccatcagcag
cctgcagcct 240gaagatgctg caacttatta ctgtcag 26722267DNACallithrix
22gacatccaga tgacccagtc tccatcttcc ctgactgcat ctgtaggagg caaagtcacc
60gtcacttgcc gggcgagtca ggacattaac aagtggttag cctggtatca gcagaaacca
120gggacagtcc ctaagcccct gatctatgag gcatccaaat tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacatat tttactctca
ccatcagcag cctgcagcct 240gaagatgctg caacttatta ctgtcag
26723267DNACallithrix 23gacatccaga tgacccagtc tccatcttcc ctgactgcat
ctgtaggagg caaagtcacc 60atcacttgcc gggcgagtca ggacattaac aagtggttag
cctggtatca gcagaaacca 120gggacagtcc ttaagcccct gatctatgag
gcatccaaat tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacatat tttactctca ccatcagcag cctgcagcct 240gaagatgctg
caacttatta ctgtcag 26724267DNACallithrix 24gacatccaga tgacccagtc
tccatcttcc ctgactgcat ctgtaggagg caaagtcacc 60atcacttgcc gggcgagtca
ggacattaac aagtggttag cctggtatca gcagaaacca 120gggacagtcc
ctaagcccct gatctatgag gcatccaaat tgcaaagtgg ggtcccatta
180aggttcagcg gcagtggatc tgggacatat tttactctca ccatcagcag
cctgcagcct 240gaagatgctg caacttatta ctgtcag 2672589PRTCallithrix
25Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Thr Ala Ser Val Gly 1
5 10 15 Gly Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Lys
Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Thr Val Pro Lys
Pro Leu Ile 35 40 45 Tyr Glu Ala Ser Lys Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Tyr Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln 85 2689PRTCallithrix 26Asp Ile Gln Met Ile Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Trp Ala Ser Gln Gly Ile Ser His Trp 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala
Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln 85 2789PRTCallithrix 27Asp
Ile Gln Met Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys
Gln 85 2889PRTCallithrix 28Asp Ile Gln Met Ile Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Trp
Ala Ser Gln Gly Ile Ser His Trp 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser
Asn Leu Gly Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln 85 2989PRTCallithrix 29Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Thr Ala Ser Val Gly 1 5 10 15
Gly Lys Val Thr Ile Thr Cys Arg Ala Cys Gln Asp Ile Asn Lys Trp 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Thr Val Pro Lys Pro Leu
Ile 35 40 45 Tyr Glu Ala Ser Lys Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
85 3089PRTCallithrix 30Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Thr Pro Arg Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80 Glu
Asp Val Ala Ile Tyr Tyr Cys Gln 85 3189PRTCallithrix 31Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Thr Ala Ser Val Gly 1 5 10 15 Gly
Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Lys Trp 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Thr Val Pro Lys Pro Leu Ile
35 40 45 Tyr Glu Ala Ser Lys Leu Gln Ser Gly Val Pro Ser Arg Leu
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln 85
3289PRTCallithrix 32Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Thr
Ala Ser Val Gly 1 5 10 15 Gly Lys Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Ile Asn Lys Trp 20 25 30 Ser Ala Trp Tyr Gln Gln Lys Pro
Gly Thr Val Pro Lys Pro Leu Ile 35 40 45 Tyr Glu Ala Ser Lys Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp
Ala Ala Thr Tyr Tyr Cys Gln 85 3389PRTCallithrix 33Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Thr Ala Ser Val Gly 1 5 10 15 Gly Lys
Val Thr Val Thr Cys Arg Ala Ser Gln Asp Ile Asn Lys Trp 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Thr Val Pro Lys Pro Leu Ile 35
40 45 Tyr Glu Ala Ser Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln 85
3489PRTCallithrix 34Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Thr
Ala Ser Val Gly 1 5 10 15 Gly Lys Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Ile Asn Lys Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Thr Val Leu Lys Pro Leu Ile 35 40 45 Tyr Glu Ala Ser Lys Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp
Ala Ala Thr Tyr Tyr Cys Gln 85 3589PRTCallithrix 35Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Thr Ala Ser Val Gly 1 5 10 15 Gly Lys
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Lys Trp 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Thr Val Pro Lys Pro Leu Ile 35
40 45 Tyr Glu Ala Ser Lys Leu Gln Ser Gly Val Pro Leu Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln 85
36267DNAAotus trivirgatus 36gacatccaga tgacccagtc tccatccttc
ctgtctgcat ctgcaggaga cagagtcacc 60atcacctgcc aggtgagtca gggaattagc
agtgaattac tctggtatca gcagaaacca 120gggaaagccc ctatgctctt
gatctatgct gcaaccaaat tgcagtcggg aatcccatct 180cggttcagtg
gccatggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gatgattttg ctacttatta ctgtcaa
26737267DNAAotus trivirgatus 37gacatccaga tgacccagtc tgcattctcc
ctgtctgcat ctgtaggaga cagagtcacc 60attacttgcc aggcgagtca gggcattacc
agtgatttag cctggtatca gcaaaagcca 120gggaacgcct ctaagctcct
gatctatgag gcatccagtt tacaaagcga ggtcccatca 180aggttcagcg
gcagtggatc tgggagagat tttactctca ccatcagcag cctgcagcct
240gaagattttg taacttatta ctgtcaa 26738267DNAAotus trivirgatus
38gacatccaga tgacccagac tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc gggcgagtca agacatttac aattatttag cctggtatca gcagaaacca
120gggaaaactc ctaggctctt gatctatgct gcatccagtt tgcaaactgg
gattccctct 180cggttcagtg gcagtggatc tgggacagac tacactctca
ccatcagcag cctgcagcct 240gatgattttg ccacttatta ctgtcaa
26739267DNAAotus trivirgatus 39gacatccaga tgacccagac tccatcctcc
ctgcctgcat ctgtaggaga caaagtcacc 60atcacttgcc gggcaagtca gggtattagc
agctggttag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatccataag gcatcaaatt tggaaacagg ggtcccatca 180aggttcagtg
gaagtggatc tgggacagat tttactctca ccatcagcag cctgcagcct
240gaagatatcg caacatatta ctgtcaa 26740267DNAAotus trivirgatus
40gacatccaga tgacccagtc tccatcttcc ctgactgcat ctgtaggaga caaagtcacc
60atcacttgcc gggcaagtca gggcattagc aataatttag cctggtatca gcagaaacca
120gggaaagccc ctaagcccct gatctattat gcatccagtt tgcaaagcgg
ggtcccatca 180aggttcagcg gcagtggatc tggggcagat tacactctca
ccaccagcag cctgcagcct 240gaagattttg caacttatta ctgtcaa
26741267DNAAotus trivirgatus 41gacaaccaga tgatccagtc tccatcttcc
ctgactgcat ctgtaggaga cagagtcacc 60atcacttgcc gagccagtca gagtattagc
agctggttag cctggtatca gcagaaacca 120gggacagtcc ctaagcctct
gatctatgac gcatccaaat tgctaagtgg ggtcccatca 180aggttcagtg
gctgtggatc tgggacagat tttactctca ccatcagcag cctgcagcct
240gaagattttg caacttatta ctgtcaa 2674289PRTAotus trivirgatus 42Asp
Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Ala Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Gln Val Ser Gln Gly Ile Ser Ser Glu
20 25 30 Leu Leu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Met Leu
Leu Ile 35 40 45 Tyr Ala Ala Thr Lys Leu Gln Ser Gly Ile Pro Ser
Arg Phe Ser Gly 50 55 60 His Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys
Gln 85 4389PRTAotus trivirgatus 43Asp Ile Gln Met Thr Gln Ser Ala
Phe Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Gln Ala Ser Gln Gly Ile Thr Ser Asp 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Asn Ala Ser Lys Leu Leu Ile 35 40 45 Tyr Glu
Ala Ser Ser Leu Gln Ser Glu Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Arg Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80 Glu Asp Phe Val Thr Tyr Tyr Cys Gln 85 4489PRTAotus
trivirgatus 44Asp Ile Gln Met Thr Gln Thr Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Tyr Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Thr Pro Arg Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln
Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Asp Asp Phe
Ala Thr Tyr Tyr Cys Gln 85 4589PRTAotus trivirgatus 45Asp Ile Gln
Met Thr Gln Thr Pro Ser Ser Leu Pro Ala Ser Val Gly 1 5 10 15 Asp
Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 His Lys Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln 85
4689PRTAotus trivirgatus 46Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Thr Ala Ser Val Gly 1 5 10 15 Asp Lys Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Ser Asn Asn 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile 35 40 45 Tyr Tyr Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Ala Asp Tyr Thr Leu Thr Thr Ser Ser Leu Gln Pro65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 85 4789PRTAotus trivirgatus
47Asp Asn Gln Met Ile Gln Ser Pro Ser Ser Leu Thr Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Thr Val Pro Lys
Pro Leu Ile 35 40 45 Tyr Asp Ala Ser Lys Leu Leu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Cys Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln 85 487PRTAotus trivirgatus 48Tyr Ala Ser Ser Leu Gln Ser 1
5 497PRTArtificial SequenceSynthesized construct 49Ser Ala Ser Glu
Leu Gln Ser 1 5 50108PRTArtificial SequenceSynthesized construct
50Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Ser
Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Leu Glu Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Val Val Trp Arg Pro Phe 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 51108PRTArtificial
SequenceSynthesized construct 51Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Asp Ser Tyr 20 25 30 Leu His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala
Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg Pro Phe
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
52108PRTArtificial SequenceSynthesized construct 52Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Ser Tyr 20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Val Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val
Val Trp Arg Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 53324DNAArtificial SequenceSynthesized
construct 53ccgtttgatt tccaccttgg tcccttggcc gaacgtaaaa ggacgccaca
caacctgttg 60acagtagtac gtagcaaaat cttcaggttg cagactgctg atggtgagag
tgaaatctgt 120cccagatcca ctgccactga aacgtgatgg gaccccactt
tgcaactcgg atgcactata 180gatcaggagc ttaggggctt tccctggttt
ctgctggtac caatgtaaat aactatcaat 240gctctgactt gcccggcaag
tgatggtgac acggtctcct acagatgcag acagagagga 300tggagactgg
gtcatctgga tgtc 3245468DNAAotus trivirgatus 54tttacattgg taccagcaga
aaccagggaa agcccctaag ctcctgatct atgctgcaac 60caaattgc
685545DNAAotus trivirgatus 55cctgatctat gctgcaacca aattgcagtc
gggggtccca tcacg 4556324DNAAotus trivirgatus 56gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gagcattgat agttatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatgct gcaaccaaat tgcagtcggg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
3245768DNAAotus trivirgatus 57tttacattgg taccagcaga aaccagggaa
agcccctaag ctcctgatct atgaggcatc 60cagtttac 685845DNAAotus
trivirgatus 58cctgatctat gaggcatcca gtttacaaag cggggtccca tcacg
4559324DNAAotus trivirgatus 59gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gagcattgat
agttatttac attggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgag gcatccagtt tacaaagcgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 3246068DNACallithrix
60tttacattgg taccagcaga aaccagggaa agcccctaag ctcctgatct atgaggcatc
60caaattgc 686145DNACallithrix 61cctgatctat gaggcatcca aattgcaaag
tggggtccca tcacg 4562324DNACallithrix 62gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca
gagcattgat agttatttac attggtacca gcagaaacca 120gggaaagccc
ctaagctcct gatctatgag gcatccaaat tgcaaagtgg ggtcccatca
180cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag gttgtgtggc
gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
3246368DNACallithrix 63tttacattgg taccagcaga aaccagggaa agcccctaag
ctcctgatct atagtgcatc 60aaatttag 686445DNACallithrix 64cctgatctat
agtgcatcaa atttagaaac aggggtccca tcacg 4565324DNACallithrix
65gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gagcattgat agttatttac attggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctatagt gcatcaaatt tagaaacagg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtcaacag
gttgtgtggc gtccttttac gttcggccaa 300gggaccaagg tggaaatcaa acgg
3246623PRTAotus trivirgatusVARIANT1Xaa = Any Amino Acid 66Xaa Leu
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 1 5 10 15
Ile Tyr Ala Ala Thr Lys Leu 20 6715PRTAotus trivirgatusVARIANT1Xaa
= Any Amino Acid 67Xaa Leu Ile Tyr Ala Ala Thr Lys Leu Gln Ser Gly
Val Pro Ser 1 5 10 15 68108PRTAotus trivirgatus 68Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Ser Tyr 20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ala Ala Thr Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val
Val Trp Arg Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 6923PRTAotus trivirgatusVARIANT1Xaa = Any Amino
Acid 69Xaa Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu 1 5 10 15 Ile Tyr Glu Ala Ser Ser Leu 20 7015PRTAotus
trivirgatusVARIANT1Xaa = Any Amino Acid 70Xaa Leu Ile Tyr Glu Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser 1 5 10 15 71108PRTAotus
trivirgatus 71Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Asp Ser Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg Pro Phe 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
7223PRTCallithrixVARIANT1Xaa = Any Amino Acid 72Xaa Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 1 5 10 15 Ile Tyr Glu
Ala Ser Lys Leu 20 7315PRTCallithrixVARIANT1Xaa = Any Amino Acid
73Xaa Leu Ile Tyr Glu Ala Ser Lys Leu Gln Ser Gly Val Pro Ser 1 5
10 15 74108PRTCallithrix 74Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Asp Ser Tyr 20 25 30 Leu His Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser
Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg Pro Phe 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
7524PRTCallithrixVARIANT1Xaa = Any Amino Acid 75Xaa Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu 1 5 10 15 Leu Ile Tyr
Ser Ala Ser Asn Leu 20 7616PRTCallithrixVARIANT1, 16Xaa = Any Amino
Acid 76Xaa Leu Ile Tyr Ser Ala Ser Asn Leu Glu Thr Gly Val Pro Ser
Xaa 1 5 10 15 77108PRTCallithrix 77Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Asp Ser Tyr 20 25 30 Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg
Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
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