U.S. patent application number 12/307578 was filed with the patent office on 2010-02-25 for novel multivalent immunoglobulins.
This patent application is currently assigned to f-star Biotechnologische Forschungs-und Entwicklungsges.m.b.H.. Invention is credited to Gottfried Himmler, Florian Ruker, Gordana Wozniak-Knopp.
Application Number | 20100048877 12/307578 |
Document ID | / |
Family ID | 38720179 |
Filed Date | 2010-02-25 |
United States Patent
Application |
20100048877 |
Kind Code |
A1 |
Ruker; Florian ; et
al. |
February 25, 2010 |
NOVEL MULTIVALENT IMMUNOGLOBULINS
Abstract
The present invention provides a multivalent immunoglobulin or
part thereof binding specifically to at least two cell surface
molecules of a single cell with at least one modification in at
least one structural loop region of said immunoglobulin determining
binding to an epitope of said cell surface molecules wherein the
unmodified immunoglobulin does not significantly bind to said
epitope, its use and methods for producing it.
Inventors: |
Ruker; Florian; (Wien,
AT) ; Himmler; Gottfried; (Gross-Enzersdorf, AT)
; Wozniak-Knopp; Gordana; (Wien, AT) |
Correspondence
Address: |
SHELDON MAK ROSE & ANDERSON PC
100 Corson Street, Third Floor
PASADENA
CA
91103-3842
US
|
Assignee: |
f-star Biotechnologische
Forschungs-und Entwicklungsges.m.b.H.
Wien
AT
|
Family ID: |
38720179 |
Appl. No.: |
12/307578 |
Filed: |
June 26, 2007 |
PCT Filed: |
June 26, 2007 |
PCT NO: |
PCT/AT07/00313 |
371 Date: |
September 14, 2009 |
Current U.S.
Class: |
530/387.3 ;
435/7.1; 536/23.53 |
Current CPC
Class: |
C07K 16/28 20130101;
C07K 16/005 20130101; C07K 16/468 20130101; C07K 16/2887 20130101;
C07K 2317/52 20130101; A61P 37/04 20180101 |
Class at
Publication: |
530/387.3 ;
536/23.53; 435/7.1 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C07H 21/04 20060101 C07H021/04; G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 5, 2006 |
AT |
A 1147/2006 |
Claims
1. A multivalent immunoglobulin or part thereof binding
specifically to at least two cell surface molecules of a single
cell with at least one modification in at least one structural loop
region of said immunoglobulin determining binding to an epitope of
said cell surface molecules wherein the unmodified immunoglobulin
does not significantly bind to said epitope.
2. Immunoglobulin according to claim 1 wherein the modified
structural loop region is within the constant domain of said
immunoglobulin, preferably within CH1, CH2, CH3, CH4, Igk-C, Igl-C,
or a part thereof.
3. Immunoglobulin according to claim 2, characterised in that said
modified structural loop region comprises at least 6 amino acid
modifications.
4. Immunoglobulin according to claim 2, characterised in that the
modified structural loop region is within a constant domain
selected from the group consisting of a CH1, a CH2, a CH3 and a CH4
domain of human or murine origin and comprises at least one
modification within amino acids 7 to 21, amino acids 25 to 39,
amino acids 41 to 81, amino acids 83 to 85, amino acids 89 to 103,
or amino acids 106 to 117.
5. Immunoglobulin according to claim 2, characterised in that the
immunoglobulin comprises Igk-C or Igl-C modified structural loop
regions that are of human origin and comprise at least one
modification within amino acids 8 to 18, amino acids 27 to 35,
amino acids 42 to 78, amino acids 83 to 85, amino acids 92 to 100,
amino acids 108 to 117, or amino acids 123 to 126.
6. Immunoglobulin according to claim 2, characterised in that the
modified structural loop regions of Igk-C or Igl-C are of murine
origin and comprise at least one modification within amino acids 8
to 20, amino acids 26 to 36, amino acids 43 to 79, amino acids 83
to 85, amino acids 90 to 101, amino acids 108 to 116, or amino
acids 122 to 125.
7. Immunoglobulin according to claim 1, characterised in that the
immunoglobulin comprises a constant domain of camelid origin.
8. Immunoglobulin according to claim 7, characterised in that the
immunoglobulin comprises at least one modified constant domain
selected from the group consisting of a CH1, CH2 and CH3
domain.
9. Immunoglobulin according to claim 8, characterised in that the
modified constant domain comprises at least one modification within
amino acids 8 to 20, amino acids 24 to 39, amino acids 42 to 78,
amino acids 82 to 85, amino acids 91 to 103, or amino acids 108 to
117.
10. Immunoglobulin according to claim 1, characterised in that the
immunoglobulin comprises a variable domain selected from the group
consisting of VH, Vkappa, Vlambda, VHH, and combinations
thereof.
11. Immunoglobulin according to claim 1, characterised in that the
immunoglobulin comprises a modified structural loop region of a VH,
a Vkappa, a Vlambda or a VHH domain comprising at least one
modification within amino acids 7 to 21, amino acids 25 to 39,
amino acids 41 to 81, amino acids 83 to 85, amino acids 89 to 103,
or amino acids 106 to 117, where the numbering of the amino acid
position of the domains is that of the IMGT.
12. Immunoglobulin according to claim 1, characterised in that the
immunoglobulin comprises a modified structural loop region of a VH,
Vkappa, or Vlambda domain of human origin and comprises at least
one modification within amino acids 8 to 20, amino acids 44 to 50,
amino acids 67 to 76 and amino acids 89 to 101, most preferably
amino acid positions 12 to 17, amino acid positions 45 to 50, amino
acid positions 69 to 75, and amino acid positions 93 to 98, where
the numbering of the amino acid position of the domains is that of
the IMGT.
13. Immunoglobulin according to claim 1, characterised in that the
immunoglobulin comprises a modified structural loop region of a VH
domain of murine origin and comprises at least one modification
within amino acids 6 to 20, amino acids 44 to 52, amino acids 67 to
76, and amino acids 92 to 101, where the numbering of the amino
acid position of the domains is that of the IMGT.
14. Immunoglobulin according to claim 1, characterised in that the
immunoglobulin comprises a modified structural loop region of a VHH
domain of camelid origin and comprises at least one modification
within amino acids 7 to 18, amino acids 43 to 55, amino acids 68 to
75, and amino acids 91 to 101, where the numbering of the amino
acid position of the domains is that of the IMGT.
15. Immunoglobulin according to claim 1, characterized in that the
immunoglobulin is further combined with one or more additional
modified immunoglobulins or with unmodified immunoglobulins, or
parts thereof, to obtain a combination immunoglobulin.
16. Immunoglobulin according to claim 1, characterised in that the
modification is a deletion, a substitution, an insertion or a
combination thereof.
17. Nucleic acid encoding an immunoglobulin according to claim 1 or
part thereof.
18. Method for engineering a multivalent immunoglobulin according
to claim 1, comprising the steps of: (a) providing a nucleic acid
encoding an immunoglobulin comprising at least one structural loop
region, (b) modifying at least one nucleotide residue of said
structural loop region, (c) transferring said modified nucleic acid
in an expression system, (d) expressing said multivalent
immunoglobulin, (e) contacting the expressed multivalent
immunoglobulin with an epitope, and (f) determining whether said
multivalent immunoglobulin binds to said epitope.
19. (canceled)
Description
[0001] The present invention provides a multivalent immunoglobulin
or part thereof binding specifically to at least two cell surface
molecules of a single cell, with at least one modification in at
least one structural loop region of said immunoglobulin determining
binding to an epitope of said cell surface molecules wherein the
unmodified immunoglobulin does not significantly bind to said
epitope.
[0002] Monoclonal antibodies have found use in many therapeutic,
diagnostic and analytical applications.
[0003] The basic antibody structure will be explained here using as
example an intact IgG1 immunoglobulin. Two identical heavy (H) and
two identical light (L) chains combine to form the Y-shaped
antibody molecule. The heavy chains each have four domains. The
amino terminal variable domains (VH) are at the tips of the Y.
These are followed by three constant domains: CH1, CH2, and the
carboxy-terminal CH3, at the base of the Y's stem. A short stretch,
the switch, connects the heavy chain variable and constant regions.
The hinge connects CH2 and CH3 (the Fc fragment) to the remainder
of the antibody (the Fab fragments). One Fc and two identical Fab
fragments can be produced by proteolytic cleavage of the hinge in
an intact antibody molecule. The light chains are constructed of
two domains, variable (VL) and constant (CL), separated by a
switch.
[0004] Disulfide bonds in the hinge region connect the two heavy
chains. The light chains are coupled to the heavy chains by
additional disulfide bonds. Asn-linked carbohydrate moieties are
attached at different positions in constant domains depending on
the class of immunoglobulin. For IgG1 two disulfide bonds in the
hinge region, between Cys235 and Cys238 pairs, unite the two heavy
chains. The light chains are coupled to the heavy chains by two
additional disulfide bonds, between Cys229s in the CH1 domains and
Cys214s in the CL domains. Carbohydrate moieties are attached to
Asn306 of each CH2, generating a pronounced bulge in the stem of
the Y.
[0005] These features have profound functional consequences. The
variable regions of both the heavy and light chains (VH) and (VL)
lie at the "tips" of the Y, where they are positioned to react with
antigen. This tip of the molecule is the side on which the
N-terminus of the amino acid sequence is located. The stem of the Y
projects in a way to efficiently mediate effector functions such as
the activation of complement and interaction with Fc receptors, or
ADCC and ADCP. Its CH2 and CH3 domains bulge to facilitate
interaction with effector proteins. The C-terminus of the amino
acid sequence is located on the opposite side of the tip, which can
be termed "bottom" of the Y.
[0006] Two types of light chain, termed lambda (.lamda.) and kappa
(.kappa.), are found in antibodies. A given immunoglobulin either
has .kappa. chains or .lamda. chains, never one of each. No
functional difference has been found between antibodies having
.lamda. or .kappa. light chains.
[0007] Each domain in an antibody molecule has a similar structure
of two beta sheets packed tightly against each other in a
compressed antiparallel beta barrel. This conserved structure is
termed the immunoglobulin fold. The immunoglobulin fold of constant
domains contains a 3-stranded sheet packed against a 4-stranded
sheet. The fold is stabilized by hydrogen bonding between the beta
strands of each sheet, by hydrophobic bonding between residues of
opposite sheets in the interior, and by a disulfide bond between
the sheets. The 3-stranded sheet comprises strands C, F, and G, and
the 4-stranded sheet has strands A, B, E, and D. The letters A
through G denote the sequential positions of the beta strands along
the amino acid sequence of the immunoglobulin fold.
[0008] The fold of variable domains has 9 beta strands arranged in
two sheets of 4 and 5 strands. The 5-stranded sheet is structurally
homologous to the 3-stranded sheet of constant domains, but
contains the extra strands C' and C''. The remainder of the strands
(A, B, C, D, E, F, G) have the same topology and similar structure
as their counterparts in constant domain immunoglobulin folds. A
disulfide bond links strands B and F in opposite sheets, as in
constant domains.
[0009] The variable domains of both light and heavy immunoglobulin
chains contain three hypervariable loops, or
complementarity-determining regions (CDRs). The three CDRs of a V
domain (CDR1, CDR2, CDR3) cluster at one end of the beta barrel.
The CDRs are loops that connect beta strands B-C, C'-C'', and F-G
of the immunoglobulin fold. The residues in the CDRs vary from one
immunoglobulin molecule to the next, imparting antigen specificity
to each antibody.
[0010] The VL and VH domains at the tips of antibody molecules are
closely packed such that the 6 CDRs (3 on each domain) cooperate in
constructing a surface (or cavity) for antigen-specific binding.
The natural antigen binding site of an antibody thus is composed of
the loops which connect strands B-C, C'-C'', and F-G of the light
chain variable domain and strands B-C, C'-C'', and F-G of the heavy
chain variable domain.
[0011] The loops which are not CDR-loops in a native
immunoglobulin, or not part of the antigen-binding pocket as
determined by the CDR loops, do not have antigen binding or epitope
binding specificity, but contribute to the correct folding of the
entire immunoglobulin molecule and/or its effector or other
functions and are therefore called structural loops for the purpose
of this invention. Prior art documents show that the
immunoglobulin-like scaffold has been employed so far for the
purpose of manipulating the existing antigen binding site, thereby
introducing novel binding properties. So far, however, only the CDR
regions have been engineered for antigen binding, in other words,
in the case of the immunoglobulin fold, only the natural antigen
binding site has been modified in order to change its binding
affinity or specificity. A vast body of literature exists which
describes different formats of such manipulated immunoglobulins,
frequently expressed in the form of single-chain Fv fragments
(scFv) or Fab fragments, either displayed on the surface of phage
particles or solubly expressed in various prokaryotic or eukaryotic
expression systems.
[0012] PCT/EP2006/050059 describes a method of engineering an
immunoglobulin which comprises a modification in a structural loop
region to obtain a new antigen binding sites. This method is
broadly applicable to immunoglobulins and may be used to produce a
series of immunoglobulins targeting a variety of antigens.
Multivalent binders of cell-surface targets are not explicitly
described.
[0013] US2005/266000A1 describes polypeptides comprising a variant
heavy chain variable framework domain (VFR). A VFR is part of the
antigen binding pocket or groove that may contact antigen. VFRs are
part of the CDR loop region and located at a variable domain at the
side of the CDR loops to support the antigen binding via the CDR
loop region. Framework loops other than VFR have not been mutated
for the purpose of engineering an antigen binding site.
[0014] Cell surface proteins associated with human cancers can be
effective targets for monoclonal therapy. Antibodies can elicit
antitumor responses by modulating cellular activation or through
recruitment of the immune system.
[0015] Some mAbs exert part of their effect by cross-linking of the
target, which may cluster the targets and result in activation,
inhibition, or amplification of cell signalling, finally ending in
cell arrest and/or apoptosis to the cellular target.
[0016] It has been demonstrated that some MAbs (anti-CD19, -CD20,
-CD21, and -CD22) that have little or no inherent anti-growth
activity on lymphoma cell lines can be converted into potent
antitumor agents by using them as tetravalent homodimers. These
activities might be enhanced in vivo by the recruitment of effector
cells and/or complement.
[0017] Another strategy used for therapeutic mAbs is to couple a
cytotoxic drug to the mAb. Such an immunotoxin may bind to the cell
surface target followed by internalization, releasing the drug to
kill the cell. Clustering of the target as a prerequisite to
internalization may be necessary.
[0018] To enhance the potency of mAbs that exert their effect
through the clustering of target molecules, various multivalent Ab
formats have been designed. Covalently linked full-length IgGs that
form tetravalent Abs and naturally occurring IgM and IgG Abs
mimicking polymeric IgM and IgA via the use of their secretory
tailpiece have been devised. Another tetravalent format was
designed by adding Fab at the C terminus of each H chain of a
full-length IgG.
[0019] To improve tumor penetration, smaller constructs using
single-chain Fv (scFv).sub.2 fragments (each Fv consisting of
variable light and variable heavy domains connected by peptide
linkers) have been joined together to form multivalent complexes.
Such constructs may have relatively short half-lives (compared with
those of full-length mAbs), consequently this has been addressed by
joining these scFv multimers to IgG Fc fragments. With scfv and
similar formats it is difficult to control formation of the exact
multimerization degree, i.e., diners, trimers, tetramers, and
larger complexes may form in varying ratios depending on the basic
construct and expression method.
[0020] Any of the known formats to produce multivalent
immunoglobulins have certain disadvantages, be it immunogenicity,
in vivo-half life or production issues.
[0021] It is the object of the present invention to provide a
modular system which allows designing a cell targeting multivalent
immunoglobulin according to the respective need, to solve prior art
problems.
BRIEF DESCRIPTION OF THE INVENTION
[0022] The present invention provides immunoglobulin domains which
bind to cell surface proteins via modified structural loops to
provide additional binding to a cell surface molecule thus enabling
crosslinking of cell-surface receptors.
[0023] According to the present invention a multivalent
immunoglobulin or binding part thereof is provided that
specifically binds to at least two cell surface molecules of a
single cell with at least one modification in at least one
structural loop region of said immunoglobulin determining binding
to an epitope of said cell surface molecules, including structures
of antigenic properties, located on a single cell or available
within a homogenous cell population, wherein the unmodified
immunoglobulin does not significantly bind to said epitope.
[0024] According to the present invention, the inventive
multivalent immunoglobulin can be further combined with one or more
modified immunoglobulins or with unmodified immunoglobulins, or
parts thereof, to obtain a combination immunoglobulin.
[0025] Preferably, the modification of the structural loop domain
within the nucleotide or amino acid sequence is a deletion, a
substitution, an insertion or a combination thereof.
[0026] The present invention also provides a nucleic acid encoding
the inventive immunoglobulin or part thereof and a method for
engineering a multivalent immunoglobulin according to the invention
comprising the steps of: [0027] providing a nucleic acid encoding
an immunoglobulin comprising at least one structural loop region,
[0028] modifying at least one nucleotide residue of said structural
loop region, [0029] transferring said modified nucleic acid in an
expression system, [0030] expressing said multivalent
immunoglobulin, [0031] contacting the expressed multivalent
immunoglobulin with an epitope, and [0032] determining whether said
multivalent immunoglobulin binds to said epitope.
[0033] Further, the use of the multivalent immunoglobulin according
to the invention for the preparation of a medicament for
therapeutic use, for example for tumor cell treatment and pathogen
infected cells is provided.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The modified immunoglobulin domains according to the
invention can be used as such or incorporated into various known
antibody formats such as complete antibodies, Fabs, single chain
Fvs, Fab2, minibodies and the like to provide additional binding
sites for cell surface epitopes or receptors.
[0035] In particular, the present invention relates to a method for
engineering an immunoglobulin binding specifically to epitopes of
antigens. Through the modification in the structural loop region
the immunoglobulin may be engineered to bind to the epitope. In a
preferred embodiment the immunoglobulin is binding specifically to
at least two such epitopes that differ from each other, originating
from or mimicking either the same antigen or different
antigens.
[0036] For example, the method according to the invention refers to
engineering an immunoglobulin binding specifically to at least one
first epitope and comprising at least one modification in at least
one structural loop region of said immunoglobulin and determining
the specific binding of said at least one loop region to at least
one second epitope, wherein
[0037] the unmodified structural loop region (non-CDR region) does
not specifically bind to said at least one second epitope,
comprising the steps of: [0038] providing a nucleic acid encoding
an immunoglobulin binding specifically to at least one first
epitope and comprising at least one structural loop region, [0039]
modifying at least one nucleotide residue of at least one of said
loop regions encoded by said nucleic acid, [0040] transferring said
modified nucleic acid in an expression system, [0041] expressing
said modified immunoglobulin, [0042] contacting the expressed
modified immunoglobulin with said at least one second epitope, and
[0043] determining whether said modified immunoglobulin binds
specifically to the second epitope.
[0044] The method according to the invention preferably refers to
at least one modification in at least one structural loop region of
said immunoglobulin and determining the specific binding of said at
least one loop region to at least one molecule selected from the
group consisting of cell surface antigens, wherein the
immunoglobulin containing an unmodified structural loop region does
not specifically bind to said at least one molecule.
[0045] The term "immunoglobulin" as used herein is including
immunoglobulins or parts or fragments or derivatives of
immunoglobulins. Thus, it includes an "immunoglobulin domain
peptide" to be modified according to the present invention (as used
herein the terms immunoglobulin and antibody are interchangeable)
as well as immunoglobulin domains or parts thereof that contain a
structural loop, or a structural loop of such domains, such as a
minidomain. The immunoglobulins can be used as isolated peptides or
as combination molecules with other peptides. In some cases it is
preferable to use a defined modified structural loop or a
structural loop region, or parts thereof, as isolated molecules for
binding or combination purposes. The "immunoglobulin domain" as
defined herein contains such immunoglobulin domain peptides or
polypeptides that may have specific binding characteristics upon
modifying and engineering. The peptides are homologous to
immunoglobulin domain sequences, and are preferably at least 5
amino acids long, more preferably at least 10 or even at least 50
or 100 amino acids long, and constitute at least partially a
structural loop or the structural loop region, or the non-CDR loop
region of the domain. Preferably the peptides exclude those
insertions that are considered non-functional amino acids, hybrid
or chimeric CDR-regions or CDR-like regions and/or canonical
structures of CDR regions. The binding characteristics relate to
specific epitope binding, affinity and avidity.
[0046] A derivative of an immunoglobulin according to the invention
is any combination of one or more immunoglobulins of the invention
and or a fusion protein in which any domain or minidomain of the
immunoglobulin of the invention may be fused at any position of one
or more other proteins (such as other immunoglobulins, ligands,
scaffold proteins, enzymes, toxins and the like). A derivative of
the immunoglobulin of the invention may also be obtained by
recombination techniques or binding to other substances by various
chemical techniques such as covalent coupling, electrostatic
interaction, di-sulphide bonding etc.
[0047] The other substances bound to the immunoglobulins may be
lipids, carbohydrates, nucleic acids, organic and anorganic
molecules or any combination thereof (e.g. PEG, prodrugs or drugs).
A derivative is also an immunoglobulin with the same amino acid
sequence but made completely or partly from non-natural or
chemically modified amino acids.
[0048] The engineered molecules according to the present invention
will be useful as stand-alone proteins as well as fusion proteins
or derivatives, most typically fused in such a way as to be part of
larger antibody structures or complete antibody molecules, or parts
thereof such as Fab fragments, Fc fragments, Fv fragments and
others. It will be possible to use the engineered proteins to
produce molecules which are bispecific, trispecific, and maybe even
carry more specificities at the same time, and it will be possible
at the same time to control and preselect the valency of binding at
the same time according to the requirements of the planned use of
such molecules.
[0049] Another aspect of the present invention relates to an
immunoglobulin with at least one loop region, characterised in that
said at least one loop region comprises at least one amino acid
modification forming at least one modified loop region, wherein
said at least one modified loop region binds specifically to at
least one epitope of an antigen
[0050] It is preferred to molecularly combine at least one modified
antibody domain, which is binding to the specific partner via the
non-variable sequences or a structural loop) with at least one
other binding molecule which can be an antibody, antibody fragment,
a soluble receptor, a ligand or another modified antibody
domain.
[0051] The molecule that functions as a part of a binding pair that
is specifically recognized by the immunoglobulin according to the
invention is preferably selected from the group consisting of
proteinaceous molecules, nucleic acids and carbohydrates.
[0052] The loop regions of the modified immunoglobulins may
specifically bind to any kind of binding molecules or structures,
in particular to antigens, proteinaceous molecules, proteins,
peptides, polypeptides, nucleic acids, glycans, carbohydrates,
lipids, small organic molecules, anorganic molecules, or
combinations or fusions thereof. Of course, the modified
immunoglobulins may comprise at least two loops or loop regions
whereby each of the loops or loop regions may specifically bind to
different molecules or epitopes.
[0053] According to the present invention, binding regions to
antigens or antigen binding sites of all kinds of cell surface
antigens, may be introduced into a structural loop of a given
antibody structure.
[0054] The term "antigen" according to the present invention shall
mean molecules or structures known to interact or capable of
interacting with the CDR-loop region of immunoglobulins. Structural
loop regions of the prior art referring to native antibodies, do
not interact with antigens but rather contribute to the overall
structure and/or to the binding to effector molecules. Only upon
engineering according to the invention structural loops may form
antigen binding pockets without involvement of CDR loops or the CDR
region.
[0055] The term "cell surface antigens" according to the present
invention shall include all antigens on capable of being recognised
by an antibody structure on the surface of a cell, and fragments of
such molecules. Preferred "cell surface antigens" are those
antigens, which have already been proven to be or which are capable
of being immunologically or therapeutically relevant, especially
those, for which a preclinical or clinical efficacy has been
tested. Those cell surface molecules are specifically relevant for
the purpose of the present invention, which mediate cell killing
activity. Upon binding of the immunoglobulin according to the
invention to at least two of those cell surface molecules the
immune system provides for cytolysis or cell death, thus a potent
means for attacking human cells may be provided.
[0056] Preferably the antigen is selected from cell surface
antigens, including receptors, in particular from the group
consisting of erbB receptor tyrosine kinases (such as EGFR, HER2,
HER3 and HER4, but not limited to these), molecules of the
TNF-receptor superfamily, such as Apo-1 receptor, TNFR1, TNFR2,
nerve growth factor receptor NGFR, CD40, T-cell surface molecules,
T-cell receptors, T-cell antigen OX40, TACI-receptor, BCMA, Apo-3,
DR4, DR5, DR6, decoy receptors, such as DcR1, DcR2, CAR1, HVEM,
GITR, ZTNFR-5, NTR-1, TNFL1 but not limited to these molecules,
B-cell surface antigens, such as CD10, CD19, CD20, CD21, CD22,
antigens or markers of solid tumors or hematologic cancer cells,
cells of lymphoma or leukaemia, other blood cells including blood
platelets, but not limited to these molecules.
[0057] According to a further preferred embodiment the antigen or
the molecule binding to the modified structural loop region is
selected from the group consisting of tumor associated antigens, in
particular EpCAM, tumor-associated glycoprotein-72 (TAG-72),
tumor-associated antigen CA 125, Prostate specific membrane antigen
(PSMA), High molecular weight melanoma-associated antigen
(HMW-MAA), tumor-associated antigen expressing Lewis Y related
carbohydrate, Carcinoembryonic antigen (CEA), CEACAM5, HMFG PEM,
mucin MUC1, MUC18 and cytokeratin tumor-associated antigen,
bacterial antigens, viral antigens, allergens, allergy related
molecules IgE, cKIT and Fc-epsilon-receptorI, IRp60, IL-5 receptor,
CCR3, red blood cell receptor (CR1), human serum albumin, mouse
serum albumin, rat serum albumin, neonatal Fc-gamma-receptor FcRn,
Fc-gamma-receptors Fc-gamma RI, Fc-gamma-RII, Fc-gamma RIII,
Fc-alpha-receptors, Fc-epsilon-receptors, fluorescein, lysozyme,
toll-like receptor 9, erythropoietin, CD2, CD3, CD3E, CD4, CD11,
CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29,
CD30, CD32, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54,
CD56, CD64, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5,
IL-6, IL-6R, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, LIF, OSM,
interferon alpha, interferon beta, interferon gamma; TNF-alpha,
TNFbeta2, TNFalpha, TNFalphabeta, TNF-R1, TNF-RII, FasL, CD27L,
CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1,
OX40L, TRAIL Receptor-1, A1 Adenosine Receptor, Lymphotoxin Beta
Receptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, integrin beta1,
integrin beta2, integrin alpha4/beta7, integrin alpha2, integrin
alpha3, integrin alpha4, integrin alpha5, integrin alpha6, integrin
alphav, alphaVbeta3 integrin, FGFR-3, Keratinocyte Growth Factor,
GM-CSF, M-CSF, RANKL, VLA-1, VLA-4, L-selectin, anti-Id,
E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2,
VNRintegrin, TGFbeta1, TGFbeta2, eotaxin1, BLyS (B-lymphocyte
Stimulator), complement C5, IgE, IgA, IgD, IgM, IgG, factor VII,
CBL, NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4
(ErbB4), Tissue Factor, VEGF, VEGFR, endothelin receptor, VLA-4,
carbohydrates such as blood group antigens and related
carbohydrates, Galili-Glycosylation, Gastrin, Gastrin receptors,
tumor associated carbohydrates, Hapten NP-cap or NIP-cap, T cell
receptor alpha/beta, E-selectin, P-glycoprotein, MRP3, MRP5,
glutathione-S-transferase pi (multi drug resistance proteins),
alpha-granule membrane protein (GMP) 140, digoxin, placental
alkaline phosphatase (PLAP) and testicular PLAP-like alkaline
phosphatase, transferrin receptor, Heparanase I, human cardiac
myosin, Glycoprotein IIb/IIIa (GPIIb/IIIa), human cytomegalovirus
(HCMV) gH envelope glycoprotein, HIV gp120, HCMV, respiratory
syncital virus RSV F, RSVF Fgp, VNRintegrin, Hep B gp120, CMV,
gpIIbIIIa, HIV IIIB gp120 V3 loop, respiratory syncytial virus
(RSV) Fgp, Herpes simplex virus (HSV) gD glycoprotein, HSV gB
glycoprotein, HCMV gB envelope glycoprotein, Clostridium
perfringens toxin and fragments thereof.
[0058] Substructures of antigens are generally referred to as
"epitopes" (e.g. B-cell epitopes, T-cell epitopes), as long as they
are immunologically relevant, i.e. are also recognisable by natural
or monoclonal antibodies. The term "epitope" according to the
present invention shall mean a molecular structure which may
completely make up a specific binding partner or be part of a
specific binding partner to the binding domain or the
immunoglobulin of the present invention.
[0059] Chemically, an epitope may either be composed of a
carbohydrate, a peptide, a fatty acid, an anorganic substance or
derivatives thereof and any combinations thereof. If an epitope is
a peptide or polypeptide, there will usually be at least 3 amino
acids, preferably 8 to 50 amino acids, and more preferably between
about 10-20 amino acids included in the peptide. There is no
critical upper limit to the length of the peptide, which could
comprise nearly the full length of the polypeptide sequence.
Epitopes can be either be linear or conformational epitopes. A
linear epitope is comprised of a single segment of a primary
sequence of a polypeptide chain. Linear epitopes can be contiguous
or overlapping. Conformational epitopes are comprised of amino
acids brought together by folding of the polypeptide to form a
tertiary structure and the amino acids are not necessarily adjacent
to one another in the linear sequence.
[0060] Specifically, epitopes are at least part of diagnostically
relevant molecules, i.e. the absence or presence of an epitope in a
sample is qualitatively or quantitatively correlated to either a
disease or to the health status or to a process status in
manufacturing or to environmental and food status. Epitopes may
also be at least part of therapeutically relevant molecules, i.e.
molecules which can be targeted by the specific binding domain
which changes the course of the disease.
[0061] Preferably, the new antigen binding sites in the structural
loops are introduced by substitution, deletion and/or insertion of
one or more elements in the sequence of the immunoglobulin, in
particular of the nucleotide sequence.
[0062] According to another preferred embodiment of the present
invention the modification of at least one nucleotide results in a
substitution, deletion and/or insertion of the amino acid sequence
of the immunoglobulin encoded by said nucleic acid.
[0063] The modification of the at least one loop region may result
in a substitution, deletion and/or insertion of 1 or more
nucleotides or amino acids, preferably a point mutation, or even
the exchange of whole loops, more preferred the change of at least
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, up to 30 amino
acids. Thereby the modified sequence comprises amino acids not
included in the conserved regions of the structural loops, the
newly introduced amino acids being naturally occurring, but foreign
to the site of modification, or substitutes of naturally occurring
amino acids. When the foreign amino acid is selected from a
specific group of amino acids, such as amino acids with specific
polarity, or hydrophobicity, a library enriched in the specific
group of amino acids at the randomized positions can be obtained
according to the invention. Such libraries are also called
"focused" libraries.
[0064] The randomly modified nucleic acid molecule may comprise the
herein identified repeating units, which code for all known
naturally occurring amino acids or a subset thereof. Those
libraries that contain modified sequences wherein a specific subset
of amino acids are used for modification purposes are called
"focused" libraries. The member of such libraries have an increased
probability of an amino acid of such a subset at the modified
position, which is at least two times higher than usual, preferably
at least 3 times or even at least 4 times higher. Such libraries
have also a limited or lower number of library members, so that the
number of actual library members reaches the number of theoretical
library members. In some cases the number of library members of a
focused library is not less than 10.sup.3 times the theoretical
number, preferably not less than 10.sup.2 times, most preferably
not less than 10 times.
[0065] A library according to the invention may be designed as a
dedicated library that contains at least 50% specific formats,
preferably at least 60%, more preferred at least 70%, more
preferred at least 80%, more preferred at least 90%, or those that
mainly consist of specific antibody formats. Specific antibody
formats are preferred, such that the preferred library according to
the invention it is selected from the group consisting of a VH
library, VHH library, Vkappa library, Vlambda library, Fab library,
a CH1/CL library and a CH3 library. Libraries characterized by the
content of composite molecules containing more than one antibody
domains, such as an IgG library or Fc library are specially
preferred. Other preferred libraries are those containing T-cell
receptors, forming T-cell receptor libraries. Further preferred
libraries are epitope libraries, wherein the fusion protein
comprises a molecule with a variant of an epitope, also enabling
the selection of competitive molecules having similar binding
function, but different functionality. Exemplary is a TNFalpha
library, wherein trimers of the TNFalpha fusion protein are
displayed by a single genetic package.
[0066] However, the maximum number of amino acids inserted into a
loop region of an immunoglobulin preferably may not exceed the
number of 30, preferably 25, more preferably 20 amino acids at a
maximum. The substitution and the insertion of the amino acids
occurs preferably randomly or semi-randomly using all possible
amino acids or a selection of preferred amino acids for
randomization purposes, by methods known in the art and as
disclosed in the present patent application.
[0067] The site of modification may be at a specific single
structural loop or a structural loop region. A loop regions usually
is composed of at least two, preferably at least 3 or at least 4
loops that are adjacent to each other, and which may contribute to
the binding of an antigen through forming an antigen binding site
or antigen binding pocket. It is preferred that the one or more
sites of modification are located within the area of 10 amino
acids, more preferably within 20, 30, 40, 50, 60, 70, 80, 90 up to
100 amino acids, in particular within a structural region to form a
surface or pocket where the antigen can sterically access the loop
regions.
[0068] The at least one loop region is preferably mutated or
modified to produce libraries, preferably by random, semi-random
or, in particular, by site-directed random mutagenesis methods, in
particular to delete, exchange or introduce randomly generated
inserts into structural loops. Alternatively preferred is the use
of combinatorial approaches. Any of the known mutagenesis methods
may be employed, among them cassette mutagenesis. These methods may
be used to make amino acid modifications at desired positions of
the immunoglobulin of the present invention. In some cases
positions are chosen randomly, e.g. with either any of the possible
amino acids or a selection of preferred amino acids to randomize
loop sequences, or amino acid changes are made using simplistic
rules. For example all residues may be mutated preferably to
specific amino acids, such as alanine, referred to as amino acid or
alanine scanning. Such methods may be coupled with more
sophisticated engineering approaches that employ selection methods
to screen higher levels of sequence diversity.
[0069] A preferred method according to the invention refers to a
randomly modified nucleic acid molecule coding for an
immunoglobulin, immunoglobulin domain or a part thereof which
comprises at least one nucleotide repeating unit within a
structural loop coding region having the sequence 5'-NNS-3',
5'-NNN-3', 5'-NNB-3' or 5'-NNK-3'. In some embodiments the modified
nucleic acid comprises nucleotide codons selected from the group of
TMT, WMT, BMT, RMC, RMG, MRT, SRC, KMT, RST, YMT, MKC, RSA, RRC,
NNK, NNN, NNS or any combination thereof (the coding is according
to IUPAC).
[0070] The modification of the nucleic acid molecule may be
performed by introducing synthetic oligonucleotides into a larger
segment of nucleic acid or by de novo synthesis of a complete
nucleic acid molecule. Synthesis of nucleic acid may be performed
with tri-nucleotide building blocks which would reduce the number
of nonsense sequence combinations if a subset of amino acids is to
be encoded (e.g. Yanez et al. Nucleic Acids Res. (2004) 32:e158;
Virnekas et al. Nucleic Acids Res. (1994) 22:5600-5607).
[0071] The randomly modified nucleic acid molecule may comprise the
above identified repeating units, which code for all known
naturally occurring amino acids.
[0072] As is well-known in the art, there are a variety of
selection technologies that may be used for the identification and
isolation of proteins with certain binding characteristics and
affinities, including, for example, display technologies such as
phage display, ribosome display, cell surface display, and the
like, as described below. Methods for production and screening of
antibody variants are well-known in the art. General methods for
antibody molecular biology, expression, purification, and screening
are described in Antibody Engineering, edited by Duebel &
Kontermann, Springer-Verlag, Heidelberg, 2001; and Hayhurst &
Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard &
Georgiou, 2000, Annu Rev Biomed Eng 2:339-76.
[0073] A "structural loop" or "non-CDR-loop" according to the
present invention is to be understood in the following manner:
immunoglobulins are made of domains with a so called immunoglobulin
fold. In essence, antiparallel beta sheets are connected by loops
to form a compressed antiparallel beta barrel. In the variable
region, some of the loops of the domains contribute essentially to
the specificity of the antibody, i.e. the binding to an antigen by
the natural binding site of an antibody. These loops are called
CDR-loops. The CDR loops are located within the CDR loop region,
which may in some cases also the variable framework region (called
"VFR") adjacent to the CDR loops. It is known that VFRs may
contribute to the antigen binding pocket of an antibody, which
generally is mainly determined by the CDR loops. Thus, those VFRs
are considered as part of the CDR loop region, and would not be
appropriately used for the purpose of the invention. Contrary to
those VFRs within the CDR loop region or located proximal to the
CDR loops, other VFRs of variable domains would be particularly
suitable to be used according to the invention. Those are the
structural loops of the VFRs located opposite to the CDR loop
region, or at the C-terminal side of a variable immunoglobulin
domain.
[0074] All other loops of antibody domains are rather contributing
to the structure of the molecule and/or the effector function.
These loops are defined herein as "structural loops" or
non-CDR-loops, which would also exclude any VFRs within the CDR
loop region.
[0075] The nucleic acid molecules encoding the modified
immunoglobulins (and always included throughout the whole
specification below: immunoglobulin fragments or derivatives) may
be cloned into host cells, expressed and assayed for their binding
specificities. These practices are carried out using well-known
procedures, and a variety of methods that may find use in the
present invention are described in Molecular Cloning--A Laboratory
Manual, 3.sup.rd Ed. (Maniatis, Cold Spring Harbor Laboratory
Press, New York, 2001), and Current Protocols in Molecular Biology
(John Wiley & Sons). The nucleic acids that encode the modified
immunoglobulins of the present invention may be incorporated into
an expression vector in order to express said immunoglobulins.
Expression vectors typically comprise an immunoglobulin operably
linked that is placed in a functional relationship, with control or
regulatory sequences, selectable markers, any fusion partners,
and/or additional elements. The modified immunoglobulins of the
present invention may be produced by culturing a host cell
transformed with nucleic acid, preferably an expression vector,
containing nucleic acid encoding the modified immunoglobulins,
under the appropriate conditions to induce or cause expression of
the modified immunoglobulins. The methods of introducing exogenous
nucleic acid molecules into a host are well known in the art, and
will vary with the host used. Of course, also acellular or cell
free expression systems for the expression of modified
immunoglobulins may be employed.
[0076] The term "expression system" refers to nucleic acid
molecules containing a desired coding sequence and control
sequences in operable linkage, so that hosts transformed or
transfected with these sequences are capable of producing the
encoded proteins. In order to effect transformation, the expression
system may be included on a vector; however, the relevant DNA may
than also be integrated into the host chromosome.
[0077] According to a preferred embodiment of the present invention
the expression system comprises a vector. Any expression vector
known in the art may be used for this purpose as appropriate.
[0078] The modified immunoglobulin is preferably expressed in a
host, preferably in a bacterial, a yeast, a plant cell, in an
animal cell or in a plant or animal.
[0079] A wide variety of appropriate host cells may be used to
express the modified immunoglobulin, including but not limited to
mammalian cells (animal cells) or and plant cells), bacteria (e.g.
Bacillus subtilis, Escherichia coli), insect cells, and yeast (e.g.
Pichia pastoris, Saccharomyces cerevisiae). For example, a variety
of cell lines that may find use in the present invention are
described in the ATCC cell line catalog, available from the
American Type Culture Collection. Furthermore, also plants and
animals may be used as hosts for the expression of the
immunoglobulin according to the present invention. The expression
as well as the transfection vectors or cassettes may be selected
according to the host used.
[0080] Of course also acellular or cell free protein expression
systems may be used. In vitro transcription/translation protein
expression platforms, that produce sufficient amounts of protein
offer many advantages of a cell-free protein expression,
eliminating the need for laborious up- and down-stream steps (e.g.
host cell transformation, culturing, or lysis) typically associated
with cell-based expression systems.
[0081] In a preferred embodiment of the present invention, the
modified immunoglobulins are purified or isolated after expression.
Modified immunoglobulins may be isolated or purified in a variety
of ways known to those skilled in the art. Standard purification
methods include chromatographic techniques, including affinity
chromatography, ion exchange or hydrophobix chromatography,
electrophoretic, immunological, precipitation, dialysis,
filtration, concentration, and chromatofocusing techniques.
Purification is often enabled by a particular fusion partner. For
example, antibodies may be purified using glutathione resin if a
GST fusion is employed, Ni+2 affinity chromatography if a His-tag
is employed or immobilized anti-flag antibody if a flag-tag is
used. For general guidance in suitable purification techniques, see
Antibody Purification: Principles and Practice, 3.sup.rd Ed.,
Scopes, Springer-Verlag, NY, 1994. Of course, it is also possible
to express the modified immunoglobulins according to the present
invention on the surface of a host, in particular on the surface of
a bacterial, insect or yeast cell or on the surface of phages or
viruses.
[0082] Modified immunoglobulins may be screened using a variety of
methods, including but not limited to those that use in vitro
assays, in vivo and cell-based assays, and selection technologies.
Automation and high-throughput screening technologies may be
utilized in the screening procedures. Screening may employ the use
of a fusion partner or label, for example an enzyme, an immune
label, isotopic label, or small molecule label such as a
fluorescent or calorimetric dye or a luminogenic molecule.
[0083] In a preferred embodiment, the functional and/or biophysical
properties of the immunoglobulins are screened in an in vitro
assay. In a preferred embodiment, the antibody is screened for
functionality, for example its ability to catalyze a reaction or
its binding affinity to its target.
[0084] Assays may employ a variety of detection methods including
but not limited to chromogenic, fluorescent, luminescent, or
isotopic labels.
[0085] As is known in the art, a subset of screening methods are
those that select for favorable members of a library. The methods
are herein referred to as "selection methods", and these methods
find use in the present invention for screening modified
immunoglobulins. When immunoglobulins libraries are screened using
a selection method, only those members of a library that are
favorable, that is which meet some selection criteria, are
propagated, isolated, and/or observed. As will be appreciated,
because only the most fit variants are observed, such methods
enable the screening of libraries that are larger than those
screenable by methods that assay the fitness of library members
individually. Selection is enabled by any method, technique, or
fusion partner that links, covalently or noncovalently, the
phenotype of immunoglobulins with its genotype, that is the
function of a antibody with the nucleic acid that encodes it. For
example the use of phage display as a selection method is enabled
by the fusion of library members to the gene III protein. In this
way, selection or isolation of modified immunoglobulins that meet
some criteria, for example binding affinity to the immunoglobulin's
target, also selects for or isolates the nucleic acid that encodes
it. Once isolated, the gene or genes encoding modified
immunoglobulins may then be amplified. This process of isolation
and amplification, referred to as panning, may be repeated,
allowing favorable antibody variants in the library to be enriched.
Nucleic acid sequencing of the attached nucleic acid ultimately
allows for gene identification.
[0086] A variety of selection methods are known in the art that may
find use in the present invention for screening immunoglobulin
libraries. These include but are not limited to phage display
(Phage display of peptides and antibodies: a laboratory manual, Kay
et al., 1996, Academic Press, San Diego, Calif., 1996; Low-man et
al., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science
228:1315-1317) and its derivatives such as selective phage
infection (Malmborg et al., 1997, J Mol Biol 273:544-551),
selectively infective phage (Krebber et al., 1997, J Mol Biol
268:619-630), and delayed infectivity panning (Benhar et al., 2000,
J Mol Biol 301:893-904), cell surface display (Witrrup, 2001, Curr
Opin Biotechnol, 12:395-399) such as display on bacteria (Georgiou
et al., 1997, Nat Biotechnol 15:29-34; Georgiou et al., 1993,
Trends Biotechnol 11:6-10; Lee et al., 2000, Nat Biotechnol
18:645-648; Jun et al., 1998, Nat Biotechnol 16:576-80), yeast
(Boder & Wittrup, 2000, Methods Enzymol 328:430-44; Boder &
Wittrup, 1997, Nat Biotechnol 15:553-557), and mammalian cells
(Whitehorn et al., 1995, Bio/technology 13:1215-1219), as well as
in vitro display technologies (Amstutz et al., 2001, Curr Opin
Biotechnol 12:400-405) such as polysome display (Mattheakis et al.,
1994, Proc Natl Acad Sci USA 91:9022-9026), ribosome display (Hanes
et al., 1997, Proc Natl Acad Sci USA 94:4937-4942), mRNA display
(Roberts & Szostak, 1997, Proc Natl Acad Sci USA
94:12297-12302; Nemoto et al., 1997, FEBS Lett 414:405-408), and
ribosome-inactivation display system (Zhou et al., 2002, J Am Chem
Soc 124, 538-543).
[0087] Other selection methods that may find use in the present
invention include methods that do not rely on display, such as in
vivo methods including but not limited to periplasmic expression
and cytometric screening (Chen et al., 2001, Nat Biotechnol
19:537-542), the antibody fragment complementation assay (Johnsson
& Varshavsky, 1994, Proc Natl Acad Sci USA 91:10340-10344;
Pelletier et al., 1998, Proc Natl Acad Sci USA 95:12141-12146), and
the yeast two hybrid screen (Fields & Song, 1989, Nature
340:245-246) used in selection mode (Visintin et al., 1999, Proc
Natl Acad Sci USA 96:11723-11728). In an alternate embodiment,
selection is enabled by a fusion partner that binds to a specific
sequence on the expression vector, thus linking covalently or
noncovalently the fusion partner and associated Fc variant library
member with the nucleic acid that encodes them.
[0088] In an alternative embodiment, in vivo selection can occur if
expression of the antibody imparts some growth, reproduction, or
survival advantage to the cell.
[0089] A subset of selection methods referred to as "directed
evolution" methods are those that include the mating or breeding of
favourable sequences during selection, sometimes with the
incorporation of new mutations. As will be appreciated by those
skilled in the art, directed evolution methods can facilitate
identification of the most favourable sequences in a library, and
can increase the diversity of sequences that are screened. A
variety of directed evolution methods are known in the art that may
find use in the present invention for screening antibody variants,
including but not limited to DNA shuffling (PCT WO 00/42561 A3; PCT
WO 01/70947 A3), exon shuffling (U.S. Pat. No. 6,365,377; Kolkman
& Stemmer, 2001, Nat Biotechnol 19:423-428), family shuffling
(Crameri et al., 1998, Nature 391:288-291; U.S. Pat. No.
6,376,246), RACHITT.TM. (Coco et al., 2001, Nat Bio-technol
19:354-359; PCT WO 02/06469), STEP and random priming of in vitro
recombination (Zhao et al., 1998, Nat Biotechnol 16:258-261; Shao
et al., 1998, Nucleic Acids Res 26:681-683), exonuclease mediated
gene assembly (U.S. Pat. No. 6,352,842; U.S. Pat. No. 6,361,974),
Gene Site Saturation Mutagenesis.TM. (U.S. Pat. No. 6,358,709),
Gene Reassembly.TM. (U.S. Pat. No. 6,358,709), SCRATCHY (Lutz et
al., 2001, Proc Natl Acad Sci USA 98:11248-11253), DNA
fragmentation methods (Kikuchi et al., Gene 236:159-167),
single-stranded DNA shuffling (Kikuchi et al., 2000, Gene
243:133-137), and AMEsystem.TM. directed evolution antibody
engineering technology (Applied Molecular Evolution) (U.S. Pat. No.
5,824,514; U.S. Pat. No. 5,817,483; U.S. Pat. No. 5,814,476; U.S.
Pat. No. 5,763,192; U.S. Pat. No. 5,723,323).
[0090] According to a preferred embodiment of the present invention
the specific binding of the modified immunoglobulin to the molecule
is determined by a binding assay selected from the group consisting
of immunological assays, preferably enzyme linked immunosorbent
assays (ELISA), surface plasmon resonance assays, saturation
transfer difference nuclear magnetic resonance spectroscopy,
transfer NOE (trNOE) nuclear magnetic resonance spectroscopy,
competitive assays, tissue binding assays, live cell binding assays
and cellular extract assays.
[0091] Binding assays can be carried out using a variety of methods
known in the art, including but not limited to FRET (Fluorescence
Resonance Energy Transfer) and BRET (Bioluminescence Resonance
Energy Transfer)-based assays, AlphaScreen.TM. (Amplified
Luminescent Proximity Homogeneous Assay), Scintillation Proximity
Assay, ELISA (Enzyme-Linked Immunosorbent Assay), SPR (Surface
Plasmon Resonance, also known as BIACORE.TM.), isothermal titration
calorimetry, differential scanning calorimetry, gel
electrophoresis, and chromatography including gel filtration. These
and other methods may take advantage of some fusion partner or
label.
[0092] The modified immunoglobulin is preferably conjugated to a
label or reporter molecule, selected from the group consisting of
organic molecules, enzyme labels, radioactive labels, colored
labels, fluorescent labels, chromogenic labels, luminescent labels,
haptens, digoxigenin, biotin, metal complexes, metals, colloidal
gold and mixtures thereof. Modified immunoglobulins conjugated to
labels or reporter molecules may be used, for instance, in
diagnostic methods.
[0093] The modified immunoglobulin may be conjugated to other
molecules which allow the simple detection of said conjugate in,
for instance, binding assays (e.g. ELISA) and binding studies.
[0094] In a preferred embodiment, antibody variants are screened
using one or more cell-based or in vivo assays. For such assays,
purified or unpurified modified immunoglobulins are typically added
exogenously such that cells are exposed to individual
immunoglobulins or pools of immunoglobulins belonging to a library.
These assays are typically, but not always, based on the function
of the immunoglobulin; that is, the ability of the antibody to bind
to its target and mediate some biochemical event, for example
effector function, ligand/receptor binding inhibition, apoptosis,
and the like. Such assays often involve monitoring the response of
cells to the antibody, for example cell survival, cell death,
change in cellular morphology, or transcriptional activation such
as cellular expression of a natural gene or reporter gene. For
example, such assays may measure the ability of antibody variants
to elicit ADCC, ADCP, or CDC. For some assays additional cells or
components, that is in addition to the target cells, may need to be
added, for example serum complement, or effector cells such as
peripheral blood monocytes (PBMCs), NK cells, macrophages, and the
like. Such additional cells may be from any organism, preferably
humans, mice, rat, rabbit, and monkey. Immunoglobulins may cause
apoptosis of certain cell lines expressing the target, or they may
mediate attack on target cells by immune cells which have been
added to the assay. Methods for monitoring cell death or viability
are known in the art, and include the use of dyes, immunochemical,
cytochemical, and radioactive reagents. For example, caspase
staining assays may enable apoptosis to be measured, and uptake or
release of radioactive substrates or fluorescent dyes such as
alamar blue may enable cell growth or activation to be
monitored.
[0095] In a preferred embodiment, the DELFIART EuTDA-based
cytotoxicity assay (Perkin Elmer, MA) may be used. Alternatively,
dead or damaged target cells may be monitored by measuring the
release of one or more natural intracellular components, for
example lactate dehydrogenase. Transcriptional activation may also
serve as a method for assaying function in cell-based assays. In
this case, response may be monitored by assaying for natural genes
or immunoglobulins which may be upregulated, for example the
release of certain interleukins may be measured, or alternatively
readout may be via a reporter construct. Cell-based assays may also
involve the measure of morphological changes of cells as a response
to the presence of modified immunoglobulins. Cell types for such
assays may be prokaryotic or eukaryotic, and a variety of cell
lines that are known in the art may be employed. Alternatively,
cell-based screens are performed using cells that have been
transformed or transfected with nucleic acids encoding the
variants. That is, antibody variants are not added exogenously to
the cells. For example, in one embodiment, the cell-based screen
utilizes cell surface display. A fusion partner can be employed
that enables display of modified immunoglobulins on the surface of
cells (Witrrup, 2001, Curr Opin Biotechnol, 12:395-399).
[0096] In a preferred embodiment, the immunogenicity of the
modified immunoglobulins may be determined experimentally using one
or more cell-based assays. In a preferred embodiment, ex vivo
T-cell activation assays are used to experimentally quantitate
immunogenicity. In this method, antigen presenting cells and naive
T cells from matched donors are challenged with a peptide or whole
antibody of interest one or more times. Then, T cell activation can
be detected using a number of methods, for example by monitoring
production of cytokines or measuring uptake of tritiated thymidine.
In the most preferred embodiment, interferon gamma production is
monitored using Elispot assays (Schmittel et. al., 2000, J.
Immunol. Meth., 24: 17-24).
[0097] The biological properties of the modified immunoglobulins of
the present invention may be characterized ex vivo in cell, tissue,
and whole organism experiments. As is known in the art, drugs are
often tested in vivo in animals, including but not limited to mice,
rats, rabbits, dogs, cats, pigs, and monkeys, in order to measure a
drug's efficacy for treatment against a disease or disease model,
or to measure a drug's pharmacokinetics, pharmacodynamics,
toxicity, and other properties. The animals may be referred to as
disease models. Therapeutics are often tested in mice, including
but not limited to nude mice, SCID mice, xenograft mice, and
transgenic mice (including knockins and knockouts). Such
experimentation may provide meaningful data for determination of
the potential of the antibody to be used as a therapeutic with the
appropriate half-life, effector function, apoptotic activity,
cytotoxic or cytolytic activity. Any organism, preferably mammals,
may be used for testing. For example because of their genetic
similarity to humans, primates, monkeys can be suitable therapeutic
models, and thus may be used to test the efficacy, toxicity,
pharmacokinetics, pharmacodynamics, half-life, or other property of
the modified immunoglobulins of the present invention. Tests of the
substances in humans are ultimately required for approval as drugs,
and thus of course these experiments are contemplated. Thus the
modified immunoglobulins of the present invention may be tested in
humans to determine their therapeutic efficacy, toxicity,
immunogenicity, pharmacokinetics, and/or other clinical properties.
Especially those multivalent immunoglobulins according to the
invention that bind to single cell through at least two surface
antigens, preferably binding of at least three structures
cross-linking target cells, would be considered preapoptotic and
exert apoptotic activity upon cell targeting and cross-linking.
Multivalent binding provides a relatively large association of
binding partners, also called cross-linking, which is a
prerequisite for apoptosis.
[0098] The modified immunoglobulins of the present invention may
find use in a wide range of antibody products. In one embodiment
the antibody variant of the present invention is used for therapy
or prophylaxis, e.g. as an active or passive immunotherapy, for
preparative, industrial or analytic use, as a diagnostic, an
industrial compound or a research reagent, preferably a
therapeutic. The modified immunoglobulin or antibody variant may
find use in an antibody composition that is monoclonal or
polyclonal. In a preferred embodiment, the modified immunoglobulins
of the present invention are used to capture or kill target cells
that bear the target antigen, for example cancer cells. In an
alternate embodiment, the modified immunoglobulins of the present
invention are used to block, antagonize, or agonize the target
antigen, for example by antagonizing a cytokine or cytokine
receptor.
[0099] In an alternately preferred embodiment, the modified
immunoglobulins of the present invention are used to block,
antagonize, or agonize growth factors or growth factor receptors
and thereby mediate killing the target cells that bear or need the
target antigen.
[0100] In an alternately preferred embodiment, the modified
immunoglobulins of the present invention are used to block,
antagonize, or agonize enzymes and substrate of enzymes.
[0101] The modified immunoglobulins of the present invention may be
used for various therapeutic purposes, preferably for active or
passive immunotherapy.
[0102] Specifically the immunoglobulin according to the present
invention or obtainable by a method according to the present
invention can be used for the preparation of a vaccine for active
immunization. Hereby the immunoglobulin is either used as an
antigenic drug substance to formulate a vaccine or used for fishing
or capturing antigenic structures ex vivo or in vivo for use in a
vaccine formulation.
[0103] In a preferred embodiment, an antibody comprising the
modified immunoglobulins is ad-ministered to a patient to treat a
specific disorder. A "patient" for the purposes of the present
invention includes both humans and other animals, preferably
mammals and most preferably humans. By "specific disorder" herein
is meant a disorder that may be ameliorated by the administration
of a pharmaceutical composition comprising a modified
immunoglobulin of the present invention.
[0104] In one embodiment, a modified immunoglobulin according to
the present invention is the only therapeutically active agent
administered to a patient. Alternatively, the modified
immunoglobulin according the present invention is administered in
combination with one or more other therapeutic agents, including
but not limited to cytotoxic agents, chemotherapeutic agents,
cytokines, growth inhibitory agents, anti-hormonal agents, kinase
inhibitors, anti-angiogenic agents, cardioprotectants, or other
therapeutic agents. The modified immunoglobulins may be
administered concomitantly with one or more other therapeutic
regimens. For example, an antibody variant of the present invention
may be administered to the patient along with chemotherapy,
radiation therapy, or both chemotherapy and radiation therapy. In
one embodiment, the modified immunoglobulins of the present
invention may be administered in conjunction with one or more
antibodies, which may or may not comprise a antibody variant of the
present invention. In accordance with another embodiment of the
invention, the modified immunoglobulins of the present invention
and one or more other anti-cancer therapies are employed to treat
cancer cells ex vivo. It is contemplated that such ex vivo
treatment may be useful in bone marrow transplantation and
particularly, autologous bone marrow transplantation. It is of
course contemplated that the antibodies of the invention can be
employed in combination with still other therapeutic techniques
such as surgery.
[0105] A variety of other therapeutic agents may find use for
administration with the modified immunoglobulins of the present
invention. In one embodiment, the modified immunoglobulin is
administered with an anti-angiogenic agent, which is a compound
that blocks, or interferes to some degree, the development of blood
vessels. The anti-angiogenic factor may, for instance, be a small
molecule or a protein, for example an antibody, Fc fusion molecule,
or cytokine, that binds to a growth factor or growth factor
receptor involved in promoting angiogenesis. The preferred
anti-angiogenic factor herein is an antibody that binds to Vascular
Endothelial Growth Factor (VEGF). In an alternate embodiment, the
modified immunoglobulin is administered with a therapeutic agent
that induces or enhances adaptive immune response, for example an
antibody that targets CTLA-4. In an alternate embodiment, the
modified immunoglobulin is administered with a tyrosine kinase
inhibitor, which is a molecule that inhibits to some extent
tyrosine kinase activity of a tyrosine kinase. In an alternate
embodiment, the modified immunoglobulins of the pre-sent invention
are administered with a cytokine. By "cytokine" as used herein is
meant a generic term for proteins released by one cell population
that act on another cell as intercellular mediators including
chemokines.
[0106] Pharmaceutical compositions are contemplated wherein
modified immunoglobulins of the present invention and one or more
therapeutically active agents are formulated. Stable formulations
of the antibody variants of the present invention are prepared for
storage by mixing said immunoglobulin having the desired degree of
purity with optional pharmaceutically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed., 1980), in the form of lyophilized
formulations or aqueous solutions. The formulations to be used for
in vivo administration are preferably sterile. This is readily
accomplished by filtration through sterile filtration membranes or
other methods. The modified immunoglobulins and other
therapeutically active agents disclosed herein may also be
formulated as immunoliposomes, and/or entrapped in
microcapsules
[0107] Administration of the pharmaceutical composition comprising
a modified immunoglobulin of the present invention, preferably in
the form of a sterile aqueous solution, may be done in a variety of
ways, including, but not limited to, orally, subcutaneously,
intravenously, intranasally, intraotically, transdermally, mucosal,
topically (e.g., gels, salves, lotions, creams, etc.),
intraperitoneally, intramuscularly, intrapulmonary (e.g., AERx.TM.
inhalable technology commercially available from Aradigm, or
Inhance.TM. pulmonary delivery system commercially available from
Inhale Therapeutics), vaginally, parenterally, rectally, or
intraocularly.
[0108] As used herein, the term "specifically binds" refers to a
binding reaction which is determinative of the cognate ligand of
interest in a heterogeneous population of molecules. Thus, under
designated conditions (e.g. immunoassay conditions in the case of
an immunoglobulin), the specified antibody binds to its particular
"target" and does not bind in a significant amount to other
molecules present in a sample. Comparable to CDRs of antibodies the
modified structural loop regions are antigen-, structure- or
molecule-binding protein moieties and not antigens as such.
[0109] Another aspect of the present invention relates to a method
for manufacturing an immunoglobulin or a pharmaceutical preparation
thereof comprising at least one modification in a structural loop
region of said immunoglobulin and determining the binding of said
immunoglobulin to an epitope of an antigen, wherein the unmodified
immunoglobulin does not significantly bind to said epitope,
comprising the steps of: [0110] providing a nucleic acid encoding
an immunoglobulin comprising at least one loop region, [0111]
modifying at least one nucleotide residue of at least one of said
loop regions, [0112] transferring said modified nucleic acid in an
expression system, [0113] expressing said modified immunoglobulin,
[0114] contacting the expressed modified immunoglobulin with an
epitope, [0115] determining whether said modified immunoglobulin
binds to said epitope, and [0116] providing the modified
immunoglobulin binding to said epitope and optionally finishing it
to a pharmaceutical preparation.
[0117] In a preferred embodiment the immunoglobulin according to
the invention is a bispecific antibody or a bispecific single chain
antibody. Further preferred is that the immunoglobulin comprises a
bispecific domain or a part thereof including a minidomain.
[0118] In particular the present invention relates to a method for
manufacturing a multi-specific immunoglobulin binding specifically
to at least one first molecule or a pharmaceutical preparation
thereof comprising at least one modification in at least one
structural loop region of said immunoglobulin and determining the
specific binding of said at least one loop region to at least one
second molecule, which is an antigen such as selected from the
group consisting of allergens, tumor associated antigens, self
antigens, enzymes, bacterial antigens, fungal antigens, protozooal
antigens and viral antigens, wherein the immunoglobulin containing
an unmodified structural loop region does not specifically bind to
said at least one second molecule, comprising the steps of: [0119]
providing a nucleic acid encoding an immunoglobulin binding
specifically to at least one first molecule comprising at least one
structural loop region, [0120] modifying at least one nucleotide
residue of at least one of said loop regions encoded by said
nucleic acid, [0121] transferring said modified nucleic acid in an
expression system, [0122] expressing said modified immunoglobulin,
[0123] contacting the expressed modified immunoglobulin with said
at least one second molecule, and [0124] determining whether said
modified immunoglobulin binds specifically to the second molecule
and [0125] providing the modified immunoglobulin binding
specifically to said at least one second molecule and optionally
finishing it to a pharmaceutical preparation.
[0126] The engineering of more than one specificity into a member
of a specific binding pair is preferred (Kufer et al. (2004) Trends
in Biotechnology vol. 22 pages 238-244).
[0127] Numerous attempts have been made to produce multi-specific,
e.g. bispecific, monoclonal antibodies or antibody fragments. One
problem in the production of bispecific antibodies made of two
different polypeptide chains (heavy and light chain) is the
necessity to express four different chains (two heavy and two light
chains) in one cell resulting in a number of various combinations
of molecules which have to be separated from the desired bispecific
molecule in the mixture. Due to their similarity the separation of
these molecules is difficult and expensive. A number of techniques
have been employed to minimize the occurrence of such unwanted
pairings (Carter (2001) Journal of Immunological Methods, vol 248,
pages 7-15)
[0128] One solution to the problem is the production of one
poly-peptide chain with two specificities, like e.g. two scFvs
linked to each other or the production of so-called diabodies. Such
molecules have been shown to be far away from the fold of a natural
molecule and are notoriously difficult to produce (Le-Gall et al.
(2004) Protein Engineering, Design & Selection vol 17 pages
357-366).
[0129] Another problem of the current design of bispecific
antibodies is the fact that even if the parent antibodies are
bivalently binding to their respective binding partner (e.g. IgG),
the resulting bispecific antibody is monovalent for each of the
respective binding partner.
[0130] The preferred multi-specific molecules of the present
invention solve these problems: Expression of a bispecific molecule
as one polypeptide chain is possible (a modified Ig domain with two
binding specificities, see example section), which is easier to
accomplish than the expression of two antibody polypeptide chains
(Cabilly et al. Proc. Natl. Acad. Sci. USA 81:3273-3277
(1984)).
[0131] It can also be produced as an antibody like molecule (i.e.
made of two polypeptide chains, either homodimeric or
heterodimeric), due to the fact that the second specificity is
located in the non-variable part of the molecule there is no need
for two different heavy chains or different light chains. Thus,
there is no possibility of wrong pairing of the two chains.
[0132] An antibody of the present invention may consist of a heavy
chain and a light chain, which form together a variable region
binding to a specific binding partner by a first specificity. The
second specificity may be formed by a modified loop of any of the
structural loops of either the heavy chain or the light chain. The
binding site may also be formed by more than one non-CDR loops
which may be structurally neighbored (either on the heavy chain or
on the light chain or on both chains).
[0133] The modified antibody or derivative may be a complete
antibody or an antibody fragment (e.g. Fab, CH1-CH2, CH2-CH3, Fc,
with or without the hinge region).
[0134] It may bind mono- or multivalently to the same or different
binding partners or even with different valency for the different
binding partners, depending on the design.
[0135] As there are a number of various loops available for
selection and design of a specific binding site in the non-CDR
regions of heavy and light chains it is possible to design antibody
derivatives with even more than two specificities without the
problems mentioned above.
[0136] The specific binding domains within one polypeptide chain
may be connected with or without a peptide linker.
[0137] The modified structural loop region of said inventive
immunoglobulin can be within the constant and/or the variable
domain of said immunoglobulin. In case the modified structural loop
is within the constant domain, it is preferably within CH1, CH2,
CH3, CH4, Igk-C, Igl-C, or a part thereof.
[0138] According to a preferred embodiment of the present invention
the immunoglobulin is of human or murine origin.
[0139] Since the modified immunoglobulin may be employed for
various purposes, in particular in pharmaceutical compositions, the
immunoglobulin is preferably of human or murine origin. Of course,
the modified immunoglobulin may also be a humanized or chimeric
immunoglobulin.
[0140] According to another preferred embodiment of the present
invention the human immunoglobulin is selected from the group
consisting of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and
IgM.
[0141] The murine immunoglobulin is preferably selected from the
group consisting of IgA, IgD, IgE, IgG1, IgG2A, IgG2B, IgG2C, IgG3
and IgM.
[0142] The modified immunoglobulin may be derived from one of the
above identified immunoglobulin classes, and structurally changed
thereafter.
[0143] The immunoglobulin comprises preferably a heavy and/or light
chain of the immunoglobulin or a part thereof. Either a
heterodimeric or a homodimeric molecule may be preferably provided
for the purpose of the invention, as well as monomeric
immunoglobulins.
[0144] The modified immunoglobulin may comprise a heavy and/or
light chain, and at least one variable and/or constant domain.
[0145] The immunoglobulin according to the present invention
comprises preferably at least one constant and/or at least one
variable domain of the immunoglobulin or a part thereof including a
minidomain.
[0146] A constant domain is an immunoglobulin fold unit of the
constant part of an immunoglobulin molecule, also referred to as a
domain of the constant region (e.g. CH1, CH2, CH3, CH4, Ck,
Cl).
[0147] A variable domain is an immunoglobulin fold unit of the
variable part of an immunoglobulin, also referred to as a domain of
the variable region (e.g. Vh, Vk, Vl, Vd)
[0148] A preferred immunoglobulin according to the invention
consists of a constant domain selected from the group consisting of
CH1, CH2, CH3, CH4, Igk-C, Igl-C, or a part or combinations
thereof, including a mini-domain, with at least one loop region,
and is characterised in that said at least one loop region
comprises at least one amino acid modification forming at least one
modified loop region, wherein said at least one modified loop
region binds specifically to at least one epitope of an
antigen.
[0149] The modified immunoglobulin according to the present
invention may comprise one or more constant domains (e.g. at least
two, three, four, five, six, ten domains). If more than one do-main
is present in the modified immunoglobulin these domains may be of
the same type or of varying types (e.g. CH1-CH1-CH2, CH3-CH3, Fc
region, (CH2)2-(CH3)2). Of course also the order of the single
domains may be of any kind (e.g. CH1-CH3-CH2, CH4-CH1-CH3-CH2).
[0150] According to another preferred embodiment of the present
invention the modified loop regions of CH1, CH2, CH3 and CH4
comprise amino acids 7 to 21, amino acids 25 to 39, amino acids 41
to 81, amino acids 83 to 85, amino acids 89 to 103 and amino acids
106 to 117.
[0151] According to another preferred embodiment of the present
invention the amino acid residues of positions 15 to 17, 29 to 34,
85.4 to 85.3, 92 to 94, 97 to 98 and/or 108 to 110 of CH3 are
modified.
[0152] The loop regions of Igk-C and Igl-C of human origin comprise
preferably amino acids 8 to 18, amino acids 27 to 35, amino acids
42 to 78, amino acids 83 to 85, amino acids 92 to 100, amino acids
108 to 117 and amino acids 123 to 126.
[0153] The loop regions of Igk-C and Igl-C of murine origin
comprise preferably amino acids 8 to 20, amino acids 26 to 36,
amino acids 43 to 79, amino acids 83 to 85, amino acids 90 to 101,
amino acids 108 to 116 and amino acids 122 to 125.
[0154] According to a specific embodiment the immunoglobulin
according to the invention may contain a modification within the
variable domain, which is selected from the group of VH, Vkappa,
Vlambda, VHH and combinations thereof. More specifically, they
comprise at least one modification within amino acids 7 to 21,
amino acids 25 to 39, amino acids 41 to 81, amino acids 83 to 85,
amino acids 89 to 103 or amino acids 106 to 117, where the
numbering of the amino acid position of the domains is that of the
IMGT.
[0155] Another preferred immunoglobulin according to the invention
consists of a variable domain of a heavy or light chain, or a part
thereof including a minidomain, with at least one loop region,
preferably a structural loop region, and is characterised in that
said at least one loop region comprises at least one amino acid
modification forming at least one modified loop region, wherein
said at least one modified loop region binds specifically to at
least one epitope of an antigen.
[0156] In an alternative embodiment, the immunoglobulin according
to the invention is characterised in that the loop regions of VH or
Vkappa or Vlambda of human origin comprise at least one
modification within amino acids 8 to 20, amino acids 44 to 50,
amino acids 67 to 76 and amino acids 89 to 101, most preferably
amino acid positions 12 to 17, amino acid positions 45 to 50, amino
acid positions 69 to 75 and amino acid positions 93 to 98, where
the numbering of the amino acid position of the domains is that of
the IMGT.
[0157] The structural loop regions of the variable domain of the
immunoglobulin of human origin, as possible selected for
modification purposes according to the invention comprise
preferably amino acids 8 to 20, amino acids 44 to 50, amino acids
67 to 76 and amino acids 89 to 101.
[0158] According to a preferred embodiment of the present invention
the structural loop regions of the variable domain of the
immunoglobulin of murine origin as possible selected for
modification purposes according to the invention comprise amino
acids 6 to 20, amino acids 44 to 52, amino acids 67 to 76 and amino
acids 92 to 101.
[0159] The immunoglobulin according to the invention is preferably
also of camel origin. Camel antibodies comprise only one heavy
chain and have the same antigen affinity as normal antibodies
consisting of light and heavy chains. Consequently camel antibodies
are much smaller than, e.g., human antibodies, which allows them to
penetrate dense tissues to reach the antigen, where larger proteins
cannot. Moreover, the comparative simplicity, high affinity and
specificity and the potential to reach and interact with active
sites, camel's heavy chain antibodies present advantages over
common antibodies in the design, production and application of
clinically valuable compounds.
[0160] The immunoglobulin of camel or camelid origin comprises
preferably at least one constant domain selected from the group
consisting of CH1, CH2 and CH3. According to a preferred embodiment
of the present invention the loop regions of CH1, CH2 and CH3 of
the camel immunoglobulin comprise amino acids 8 to 20, amino acids
24 to 39, amino acids 42 to 78, amino acids 82 to 85, amino acids
91 to 103 and amino acids 108 to 117.
[0161] Even more specified, the immunoglobulin loop regions of VH
of murine origin comprise at least one modification within amino
acids 6 to 20, amino acids 44 to 52, amino acids 67 to 76 and amino
acids 92 to 101, where the numbering of the amino acid position of
the domains is that of the IMGT. The modified loop regions of a VHH
of camelid origin preferably comprise at least one modification
within amino acids 7 to 18, amino acids 43 to 55, amino acids 68 to
75 and amino acids 91 to 101, where the numbering of the amino acid
position of the domains is that of the IMGT.
[0162] The above identified amino acid regions of the respective
immunoglobulins are loop regions specified to be suitable for
modification purposes according to the invention.
[0163] Yet another aspect of the present invention relates to a
method for specifically binding and/or detecting a molecule
comprising the steps of: [0164] (a) contacting a modified
immunoglobulin according to the present invention or a modified
immunoglobulin obtainable by a method according to the present
invention with a test sample suspected to contain said molecule,
and [0165] (b) detecting the potential formation of a specific
immunoglobulin/molecule complex.
[0166] Another aspect of the present invention relates to a method
for specifically isolating a molecule comprising the steps of:
[0167] (a) contacting a modified immunoglobulin according to the
present invention or a modified immunoglobulin obtainable by a
method according to the present invention with a sample containing
said molecule, [0168] (b) separating the specific
immunoglobulin/molecule complex formed, and [0169] (c) optionally
isolating the molecule from said complex.
[0170] The immunoglobulins according to the present invention may
be used to isolate specifically molecules from a sample. If
multi-specific immunoglobulins are used more than one molecules may
be isolated from a sample. It is especially advantageous using
modified immunoglobulins in such methods because it allows, e.g.,
to generate a matrix having a homogeneous surface with defined
amounts of binding partners (i.e. Modified immunoglobulins)
immobilised thereon which able to bind to the molecules to be
isolated. In contrast thereto, if mono-specific binding partners
are used no homogeneous matrix can be generated because the single
binding partners do not bind with the same efficiency to the
matrix.
[0171] Another aspect of the present invention relates to a method
for targeting a compound to a target comprising the steps of:
[0172] (a) contacting a modified immunoglobulin according to the
present invention or a modified immunoglobulin obtainable by a
method according to the present invention capable to specifically
bind to said compound, [0173] (b) delivering the
immunoglobulin/compound complex to the target.
[0174] Modified immunoglobulins according to the present invention
may be used to deliver at least one compound bound to the CDRs
and/or modified loop regions to a target. Such immunoglobulins may
be used to target therapeutic substances to a preferred site of
action in the course of the treatment of a disease.
[0175] Another aspect of the present invention relates to the use
of an immunoglobulin according to the present invention or
obtainable by a method according to the present invention for the
preparation of a protein library of immunoglobulins. Further
libraries according to the invention not just contain a variety of
proteins or fusion proteins, genetic packages, but also precursors
of proteins, nucleic acids, ribosomes, cells, virus, phages, and
other display systems which express information encoding the
proteins and/or the proteins as such.
[0176] Another aspect of the present invention relates to a protein
library comprising an immunoglobulin according to the present
invention or obtainable by the method according to the present
invention.
[0177] Preferred methods for constructing said library can be found
above and in the examples. The library according to the present
invention may be used to identify immunoglobulins binding to a
distinct molecule.
[0178] In particular the present invention relates to the use of a
protein library comprising an immunoglobulin according to the
present invention or obtainable by the method according to the
present invention for the design of immunoglobulin derivatives.
[0179] An existing immunoglobulin can be changed to introduce
antigen binding sites into any domain or minidomain by using a
protein library of the respective domain of at least 10, preferably
100, more preferably 1000, more preferably 10000, more preferably
100000, most preferably more than 1000000 variant domains or
minidomains with at least one modified loop, in particular one or
more structural loops. The number of members of a library can even
be higher, in most cases up to 10e12, with some display systems,
such as ribosomal display the number can even be higher than
that.
[0180] The library is then screened for binding to the specific
antigen. After molecular characterization for the desired
properties the selected domain or minidomain is cloned into the
original immunoglobulin by genetic engineering techniques so that
it replaces the wild type region. Alternatively, only the DNA
coding for the loops or coding for the mutated amino acids may be
exchanged to obtain an immunoglobulin with the additional binding
site for the specific antigen.
[0181] The choice of the site for the mutated, antigen-specific
structural loop is dependent on the structure of the original
immunoglobulin and on the purpose of the additional binding site.
If, for example, the original molecule is a complete immunoglobulin
which needs to have inserted an additional antigen binding site
without disturbance of the effector function, the loops to be
modified would be selected from domains distant from CH2 and CH3
which are the natural binding partners to Fc-effector molecules. If
the original immunoglobulin is a Fab fragment, modification of
loops in constant domains of the light chains or the heavy chains
or the respective variable domains is possible. To generate a
library one may prepare libraries of mutant original molecules
which have mutations in one or more structural loops of one or more
domains. The selection with complete mutated original molecules may
have some advantages as the selection for antigen binding with a
modified structural loop will deliver the sterically advantageous
modifications if tested also for the other properties the mutated
immunoglobulin should show. In particular an Fc library is
preferred, e.g. with binding sites in the C-terminal loop
region.
[0182] The size requirement (i.e. the number of variant proteins)
of a protein library of a mutated domain or a minidomain or a
fusion molecule of a domain is dependent on the task. In general, a
library to generate an antigen binding site de novo needs to be
larger than a library used to further modify an already existing
engineered antigen binding site made of a modified structural loop
(e.g. for enhancing affinity or changing fine specificity to the
antigen).
[0183] The present invention also relates to an immunoglobulin
library or a nucleic acid library comprising a plurality of
immunoglobulins, e.g. a constant or variable domain, a minidomain
and/or at least one structural loop region contained in a
mini-domain, or nucleic acid molecules encoding the same. The
library contains members with different modifications, wherein the
plurality is defined by the modifications in the at least one
structural loop region. The nucleic acid library preferably
includes at least 10 different members with a difference in the
nucleotide sequence to obtain at least one different amino acid
(resulting in one amino acid exchange) and more preferably includes
at least 100, more preferably 1000 or 10000 different members (e.g.
designed by randomization strategies or combinatory techniques).
Even more diversified individual member numbers, such as at least
1000000 or at least 10000000 are also preferred.
[0184] A further aspect of the invention is the combination of two
different immunoglobulins, domains or minidomains selected from at
least two libraries according to the invention in order to generate
multispecific immunoglobulins. These selected specific
immunoglobulins may be combined with each other and with other
molecules, similar to building blocks, to design the optimal
arrangement of the domains or minidomains to get the desired
properties. For example, a molecule based on Fc can be used as
such, with antigen-binding properties, as a carrier for other
binding motifs or as a building block to build an immunoglobulin
with constant or variable domains, or else combined with constant
domains only, such as multimeric Fc molecules, preferably with 2,
3, or 4 antigen binding sites.
[0185] Furthermore, one or more modified immunoglobulins according
to the invention may be introduced at various or all the different
sites of a protein possible without destruction of the structure of
the protein. By such a "domain shuffling" technique new libraries
are created which can again be selected for the desired
properties.
[0186] Preferably, the immunoglobulin according to the present
invention is composed of at least two immunoglobulin domains, or a
part thereof including a minidomain, and each domain contains at
least one antigen binding site.
[0187] Also preferred is an immunoglobulin according to the
invention, which comprises at least one domain of the constant
region and/or at least one domain of the variable region of the
immunoglobulin, or a part thereof including a minidomain. Thus, a
variable domain, which is for example modified in the C-terminal
region, or the variable domain linked to a modified CH1 region, for
instance a modified CH1 minidomain, is one of the preferred
embodiments.
[0188] The preferred library contains immunoglobulins according to
the invention, selected from the group consisting of domains of an
immunoglobulin, minidomains or derivatives thereof.
[0189] A preferred embodiment of the present invention is a binding
molecule for an antigen (antigen binding molecule) comprising at
least one immunoglobulin domain and a structural loop region
modified according to the present invention to bind to the antigen,
wherein said binding molecule does not comprise variable domains of
an antibody. It may comprise other parts useable for antibody
activities (e.g. such as natural or modified effector regions
(sequences); however, it lacks the "natural" binding region of
antibodies, i.e. the variable domains or CDR loops, including VFR
loops within the CDR region, in their naturally occurring position.
These antigen binding molecules according to the present invention
have the advantages described above for the present molecules, yet
without the specific binding activity of antibodies mediated by CDR
loops; however with a newly introduced specific binding activity in
the structural loop region.
[0190] Preferably, these antigen binding molecules according to the
present invention comprise CH1, CH2, CH3, CH4, Igk-C, Igl-C and
combinations thereof; said combinations comprising at least two,
preferably at least four, especially at least six constant domains
and at least one structural loop or loop region modified according
to the present invention. Preferably these structural loop regions
are either connected via structural loop region modified according
to the present invention or the structural loops being naturally
present between such two constant domains. An embodiment of these
antigen binding molecules according to the present invention
consists of the Fc region of an antibody with at least one
modification in a structural loop according to the present
invention. Also for the antigen binding molecules according to the
present invention it is preferred that the new antigen binding
sites in the structural loops are introduced by randomizing
technologies, i.e. by exchanging one or more amino acid residues of
the loop by randomization techniques or by introducing randomly
generated inserts into such structural loops. Alternatively
preferred is the use of combinatorial approaches. Preferably the
antigen binding sites in the modified structural loops are selected
from suitable libraries.
[0191] According to another aspect, the present invention relates
to a modified immunoglobulin having an antigen binding site to
provide a specificity foreign to the unmodified immunoglobulin and
incorporated in one or more structural loops. The term "foreign"
means that the antigen is not recognized by the specific CDR
binding region or other natural or intrinsic binding regions of the
immunoglobulin. A foreign binding partner, but not the natural
binding partner of an immunoglobulin, may thus be bound by the
newly formed antigen binding site of a structural loop. This means
that a natural binding partner, such as a an Fc-receptor or an
effector of the immune system, is not considered to be bound by the
antigen binding site foreign to the unmodified immunoglobulin.
[0192] Preferred immunoglobulins according to the present invention
comprise at least two antigen binding sites, the first site binding
to a first epitope, and the second site binding to a second
epitope.
[0193] According to a preferred embodiment, the present
immunoglobulin comprises at least two loop regions, the first loop
region binding to a first epitope, and the second loop region
binding to a second epitope. Either the at least first or at least
second loop region or both may contain a structural loop. The
immunoglobulins according to the present inventions include the
fragments thereof known in the art to be functional which contain
the essential elements according to the present invention: the
structural loop or loop region modified according to the present
invention.
[0194] The preferred immunoglobulin according to the invention
comprises a domain that has at least 50% homology with the
unmodified domain.
[0195] The term "homology" indicates that polypeptides have the
same or conserved residues at a corresponding position in their
primary, secondary or tertiary structure. The term also extends to
two or more nucleotide sequences encoding the homologous
polypeptides.
[0196] "Homologous immunoglobulin domain" means an immunoglobulin
domain according to the invention having at least about 50% amino
acid sequence identity with regard to a full-length native sequence
immunoglobulin domain sequence or any other fragment of a
full-length immunoglobulin domain sequence as disclosed herein.
Preferably, a homologous immunoglobulin domain will have at least
about 50% amino acid sequence identity, preferably at least about
55% amino acid sequence identity, more preferably at least about
60% amino acid sequence identity, more preferably at least about
65% amino acid sequence identity, more preferably at least about
70% amino acid sequence identity, more preferably at least about
75% amino acid sequence identity, more preferably at least about
80% amino acid sequence identity, more preferably at least about
85% amino acid sequence identity, more preferably at least about
90% amino acid sequence identity, more preferably at least about
95% amino acid sequence identity to a native immunoglobulin domain
sequence, or any other specifically defined fragment of a
full-length immunoglobulin domain sequence as disclosed herein.
[0197] "Percent (%) amino acid sequence identity" with respect to
the immunoglobulin domain sequences identified herein is defined as
the percentage of amino acid residues in a candidate sequence that
are identical with the amino acid residues in the specific
immunoglobulin domain sequence, after aligning the sequence and
introducing gaps, if necessary, to achieve the maximum percent
sequence identity, and not considering any conservative
substitutions as part of the sequence identity. Alignment for
purposes of determining percent amino acid sequence identity can be
achieved in various ways that are within the skill in the art, for
instance, using publicly available computer software such as BLAST,
BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the
art can determine appropriate parameters for measuring alignment,
including any algorithms needed to achieve maximal alignment over
the full length of the sequences being compared.
[0198] Percent (%) amino acid sequence identity values may be
obtained as de-scribed below by using the WU-BLAST-2 computer
program (Altschul et al., Methods in Enzymology 266:460-480
(1996)). Most of the WU-BLAST-2 search parameters are set to the
default values. Those not set to default values, i.e., the
adjustable parameters, are set with the following values: overlap
span=1, overlap fraction=0.125, word threshold (T)=11, and scoring
matrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid
sequence identity value is determined by dividing (a) the number of
matching identical amino acid residues between the amino acid
sequence of the immunoglobulin domain of interest having a sequence
derived from the native immunoglobulin domain and the comparison
amino acid sequence of interest (i.e., the sequence against which
the immunoglobulin domain of interest is being compared which may
be the unmodified immunoglobulin domain) as determined by
WU-BLAST-2 by (b) the total number of amino acid residues of the
non-randomized parts of the immunoglobulin do-main of interest. For
example, in the statement "a polypeptide comprising an amino acid
sequence A which has or having at least 80% amino acid sequence
identity to the amino acid sequence B", the amino acid sequence A
is the comparison amino acid sequence of interest and the amino
acid sequence B is the amino acid sequence of the immunoglobulin
domain of interest.
[0199] Another aspect of the present invention relates to a kit of
binding partners containing
[0200] (a) a modified immunoglobulin having an antigen binding site
foreign to the immunoglobulin incorporated in one or more
structural loops, and
[0201] (b) a binding molecule containing an epitope of said
antigen.
[0202] Such a binding molecule of this kit according to the present
invention may be used as a capturing agent for identifying the
binding specificity of the modified immunoglobulin according to the
present invention. By using the binding molecule of this kit
according to the pre-sent invention, the potency of the modified
immunoglobulins according to the present invention may be
determined.
[0203] Potency as defined here is the binding property of the
modified molecule to its antigen. The binding can be determined
quantitatively and/or qualitatively in terms of specificity and/or
affinity and/or avidity as used for quality control purposes.
[0204] The binding properties of the molecules according to the
invention obtained upon modification may further be tuned by
standard techniques, such as affinity maturation. Thereby the
nucleotide sequence within or surrounding the antigen binding site
is further exchanged for modulating the binding properties.
[0205] Moreover, the binding molecule of a kit according to the
present invention may be used for selecting the modified
immunoglobulin with the appropriate potency according to the
present invention from a library consisting of at least 10,
preferably at least 100, more preferably at least 1000, more
preferred at least 10000, especially at least 100000
immunoglobulins with different modifications in the structural
loops.
[0206] Examples have shown that one of the key features is engineer
those immunoglobulin domains or regions which are not normally
involved in the desirable intrinsic functions of a antibody, such
as antigen binding. Thus, modifying in regions other than the CDR
region, including those loops adjacent to the CDR loops, of an
antibody would preserve its antigen binding function. It was
observed that the specific fold of immunoglobulin domains allows
the introduction of random mutations in regions which are
structurally analogous to the CDRs but different in position and
sequence. The regions identified by the present invention are, like
CDRs, loop regions connecting the beta strands of the
immunoglobulin fold.
[0207] More specifically, it is described herein that by
introducing mutations, e.g. random mutations in the loops
connecting beta strands A-B and E-F of a human IgG1 CH3 domain,
mutated CH3 domains were selected that bind specifically to either
Toll like receptor 9-peptide (TLR-9) or to hen egg lysozyme, which
are a peptide and a protein, respectively, that are not normally
recognized and bound by human CH3 domains of IgG1. The mutations
introduced by us include mutations in which selected amino acid
residues in the wildtype sequence were replaced by randomly chosen
residues, and they also include insertions of extra amino acid
residues in the loops mentioned above.
[0208] By analogy the immunoglobulin domains from any class of
immunoglobulins and from immunoglobulins from any species are
amenable to this type of engineering. Furthermore not only the
specific loops targeted in the present invention can be
manipulated, but any loop connecting beta strands in immunoglobulin
domains can be manipulated in the same way.
[0209] Engineered immunoglobulin domains from any organism and from
any class of immunoglobulin can be produced according to the
present invention either as such (as single domains), or as part of
a larger molecule. For example, they can be part of an intact
immunoglobulin, which accordingly would have its "normal" antigen
binding region formed by the 6 CDRs and the new, engineered antigen
binding region. Like this, a multi-specific, e.g. bispecific,
immunoglobulin could be generated. The engineered immunoglobulin
domains can also be part of any fusion protein. The use of these
engineered immunoglobulin domains is in the general field of the
use of immunoglobulins.
[0210] Except where indicated otherwise all numbering of the amino
acid sequences of the immunoglobulins is according to the IMGT
numbering scheme (IMGT, the international ImMunoGeneTics
information system@imgt.cines.fr; http://imgt.cines.fr; Lefranc et
al., 1999, Nucleic Acids Res. 27: 209-212; Ruiz et al., 2000
Nucleic Acids Res. 28: 219-221; Lefranc et al., 2001, Nucleic Acids
Res. 29: 207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:
307-310; Lefranc et al., 2005, Dev Comp Immunol 29:185-203).
TABLE-US-00001 SEQ ID No. 1 PREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGKAAA SEQ ID No. 2 ccatggcccc ccgagaacca caggtgtaca
ccctgccccc atcccgtgac gagctcnnsn nsnnscaagt cagcctgacc tgcctggtca
aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca
actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc tacagcaagc
ttaccgtgnn snnsnnsagg tggnnsnnsg ggaacgtctt ctcatgctcc gtgatgcatg
aggctctgca caaccactac acacagaaga gcctctccct gtctccgggt aaagcggccg
ca // SEQ ID No. 3 MAPREPQVYTLPPSEDELXXXQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVXXXRWXXGNVFSCSVMHE
ALHNHYTQKSLSLSPGKAAA SEQ ID No. 4 cttgccatgg ccccccgaga accacaggtg
tac SEQ ID No. 5 agtcgagctc gtcacgggat gggggcaggg SEQ ID No. 6
gtacgagctc nnsnnsnnsc aagtcagcct gacctgcctg g SEQ ID No. 7
tgccaagctt gctgtagagg aagaaggagc cg SEQ ID No. 8 tgccaagctt
accgtgnnsn nsnnsaggtg gnnsnnsggg aacgtcttct catgctccg SEQ ID No. 9
agttgcggcc gctttacccg gagacaggga gag SEQ ID No. 10
MAPREPQVYTLPPSRDELXXXQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVXXXXXXRWXXGNVFSCSV MHEALHNHYTQKSLSLSPGKAAA SEQ ID No.
11 ccatggcccc ccgagaacca caggtgtaca ccctgccccc atcccgtgac
gagctcnnsn nsnnscaagt cagcctgacc tgcctggtca aaggcttcta tcccagcgac
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
gtgctggact ccgacggctc cttcttcctc tacagcaagc ttaccgtgnn snnsnnsnns
nnsnnsaggt ggnnsnnsgg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac
aaccactaca cacagaagag cctctccctg tctccgggta aagcggccgc a SEQ ID No.
12 tgccaagctt accgtgnnsn nsnnsnnsnn snnsaggtgg nnsnnsggga
acgtcttctc atgctccg SEQ ID No. 13 MAPREPQVYTLPPSRDELXXXQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVXXXXXXXXRWXXGNVFSCSV
MHEALHNHYTQKSLSLSPGKAAA SEQ ID No. 14 ccatggcccc ccgagaacca
caggtgtaca ccctgccccc atcccgtgac gagctcnnsn nsnnscaagt cagcctgacc
tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc
tacagcaagc ttaccgtgnn snnsnnsnns nnsnnsnnsn nsaggtggnn snnsgggaac
gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc
tccctgtctc cgggtaaagc ggccgca SEQ ID No. 15 tgccaagctt accgtgnnsn
nsnnsnnsnn snnsnnsnns aggtggnnsn nsgggaacgt cttctcatgc tccg
Example 1
Construction of the CH3 Library and Phage Surface Display
[0211] The crystal structure of an IgG1 Fc fragment, which is
published in the Brookhaven Database as entry 1OQO.pdb was used to
aid in the design of the mutated CH3 domain.
[0212] The sequence which was used as the basis for construction of
the CH3 library is given in SEQ ID No. 1. In this sequence, the
first amino acid corresponds to Proline 343 of chain A of
Brookhaven database entry 1oqo.pdb. The last residue contained in
1oqo.pdb is Serine 102 of SEQ ID No. 1. After detailed analysis of
the structure of 1oqo.pdb and by visual inspection of the residues
forming the loops which connect the beta strands, it was decided to
randomize residues 17, 18 and 19, which are part of the loop
connecting beta strand A-B as well as 71, 72, 73, 76, and 77, which
are part of the loop connecting beta strand E-F of SEQ ID No. 1.
The engineered gene was produced by a series of PCR reactions
followed by ligation of the resulting PCR products. To facilitate
ligation, some of the codons of the nucleotide sequence coding for
SEQ ID No. 1 were modified to produce restriction sites without
changing the amino acid sequences (silent mutations). For insertion
into the cloning vector pHEN1 (Nucleic Acids Res. 1991 Aug. 11;
19(15):4133-7. Multi-subunit proteins on the surface of filamentous
phage: methodologies for displaying antibody (Fab) heavy and light
chains. Hoogenboom H R, Griffiths A D, Johnson K S, Chiswell D J,
Hudson P, Winter G.) in frame with the pelB secretion signal, extra
nucleotide residues encoding Met-Ala were attached at the 5' end of
the sequence to create an NcoI restriction site. For the randomized
residues, the codon NNS (IUPAC code, where S means C or G) was
chosen which encodes all 20 naturally occurring amino acids, but
avoids 2 out of 3 stop codons. The engineered sequence is given as
a nucleotide sequence in SEQ ID No. 2 and as an amino acid sequence
in SEQ ID No. 3. The Letter X in SEQ ID No. 3 denotes randomized
amino acid residues. The sequences of the PCR primers used for
assembly of the mutated CH3 domain are given in SEQ ID No. 4
through 9.
[0213] cDNA of the heavy chain of the human monoclonal antibody 3D6
(Felgenhauer M, Kohl J, Ruker F. Nucleotide sequences of the cDNAs
encoding the V-regions of H- and L-chains of a human mono-lonal
antibody specific to HIV-1-gp41. Nucleic Acids Res. 1990 Aug. 25;
18(16):4927.) were used as template for the PCR reactions. The 3
PCR products were digested with SacI and/or HindIII respectively
and ligated together. The ligation product was further digested
with NcoI and Not I and ligated into the surface display phagemid
vector pHen1, which had previously been digested with NcoI and
NotI. A number of selected clones were controlled by restriction
analysis and by DNA sequencing and were found to contain the insert
as planned, including the correctly inserted randomized sequences.
For the following steps of phage preparation, standard protocols
were followed. Briefly, the ligation mixture was transformed into
E. coli TG1 cells by electroporation. Subsequently, phage particles
were rescued from E. coli TG1 cells with helper phage M13-KO7.
Phage particles were then precipitated from culture supernatant
with PEG/NaCl in two steps, dissolved in water and used for
selection by panning or, alternatively, they were stored at minus
80.degree. C.
Example 2
Construction of the CH3+3 Library
[0214] This library was constructed and cloned in the same way as
the CH3 library. The amino acid sequence of the construct is given
in SEQ ID No. 10, the corresponding nucleotide sequence in SEQ ID
No. 11, and the primers used for construction were SEQ ID No. 4-7,
SEQ ID No. 9 and SEQ ID No. 12.
Example 3
Construction of the CH3+5 Library
[0215] This library was constructed and cloned in the same way as
the CH3 library. The amino acid sequence of the construct is given
in SEQ ID No. 13, the corresponding nucleotide sequence in SEQ ID
No. 14, and the primers used for construction were SEQ ID No. 4-7,
SEQ ID No. 9 and SEQ ID No. 15.
Example 4
Construction of a CH1 Library
[0216] In the human IgG1 CH1 library, Ser93, Ser94, Ser95, Gly98,
Thr99 and Gln100 were randomized and 3 random residues additionally
inserted using site directed random mutagenesis. Leu96 was not
mutated. In another human IgG1 CH1 library, Pro92, Ser93, Ser94,
Ser95 Leu96 Thr101, Gly98, Thr99 and Gln100 were randomized and 3
random residues additionally inserted using site directed random
mutagenesis. The genes coding for the libraries were cloned in
frame with the pelB leader at the N-terminus and in frame with
protein III from fd phage at the C-terminus using the restriction
sites NcoI and NotI of the phagemid vector pHEN1. Preparation of
phage particles, panning and selection of specifically binding
clones were performed using standard procedures.
Library Sequence:
TABLE-US-00002 [0217] Nucleotide sequence of the first CH1 library:
1 GCCTCCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT CCTCCAAGAG CACCTCTGGG
GGCACAGCGG CCCTGGGCTG CCTGGTCAAG GACTACTTCC 101 CCGAACCGGT
GACGGTGTCG TGGAACTCAG GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT
ACAGTCCTCA GGACTCTACT CCCTCAGCAG 201 CGTGGTGACC GTGCCCNNSN
NSNNSTTGNN SNNSNNSNNS NNSNNSACCT ACATCTGCAA CGTGAATCAC AAGCCCAGCA
ACACCAAGGT GGACAAGAAA 301 GTTGAGCCCA AATCTGCGGC CGCA Amino acid
sequence of the first CH1 library:
MKYLLPTAAAGLLLLAAQPAMAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT-
FPAVLQSSGLYSLSSVVTVPXXXLXXXXX XTYICNVNHKPSNTKVDKKVEPKSAAA
Nucleotide sequence of the second CH1 library: 1 GCCTCCACCA
AGGGCCCATC GGTCTTCCCC CTGGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGGAG
CCCTGGGCTG CCTGGTCAAG GACTACTTCC 101 CCGAACCGGT GACGGTGTCG
TGGAACTCAG GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT GCAGTCCTCA
GGACTCTACT CCCTCAGCAG 201 CGTGGTGACC GTGNNSNNSN NSNNSNNSNN
SNNSNNSNNS NNSNNSNNST ACATCTGCAA CGTGAATCAC AAGCCCAGCA ACACCAAGGT
GGACAAGAAA 301 GTTGAGCCCA AATCTGCGGC CGCT Amino acid sequence of
the second CH1 library:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVXXX-
XXXXXXXXXYICNVNHKPSNTKVDKKVEP KSAAA
Example 4
Construction of a CL Library
[0218] In the human IgG1 CL library, Ser92, Lys93, Ala94, Asp95,
Glu97, Lys98 and His99 were randomized and 3 random residues
additionally inserted between Ser16 and Gly17 using site directed
random mutagenesis. The genes coding for the libraries were cloned
in frame with the pelB leader at the N-terminus and in frame with
protein III from fd phage at the C-terminus using the restriction
sites NcoI and NotI of the phagemid vector pHEN1. Preparation of
phage particles, panning and selection of specifically binding
clones were performed using standard procedures.
TABLE-US-00003 Nucleotide sequence of the CL Library: 1 GTGGCTGCAC
CATCTGTCTT CATCTTCCCG CCATCTGATG AGCAGTTGAA ATCTNNSNNS NNSGGAACTG
CCTCTGTTGT GTGCCTGCTG AATAACTTCT 101 ATCCCAGAGA GGCCAAAGTA
CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC AGGAGAGTGT CACAGAGCAG
GACAGCAAGG ACAGCACCTA 201 CAGCCTCAGG TCGACCCTGA CGCTGNNSNN
SNNSNNSTAC NNSNNSNNSA AAGTCTACGC CTGCGAAGTC ACCCATCAGG GCCTGAGCTC
GCCCGTCACA 301 AAGAGCTTCA ACAGGGGAGA G Amino acid sequence of the
CL library:
VAAPSVFIFPPSDEQLKSXXXGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLRSTLT-
LXXXXYXXXKVYACEVTHQGLSSPVTKSF NRGE
Example 5
Panning of the CH3-Phage Library on Rp10-L Peptide
[0219] 3 panning rounds were performed. Maleimide activated plates
(Pierce) were coated with a synthetic peptide Rp10-L, representing
a mimotope of B-cell molecular marker CD20 (Perosa et al. Ann N Y
Acad. Sci. (2005) 51:672-83). Its deduced amino acid sequence is as
follows: ITPWPHWLERSS. 200 .mu.l of the following solution were
added per well: PBS, pH=7.2, with the following concentrations of
dissolved peptide: [0220] 1.sup.st panning round: 100 .mu.g/ml
[0221] 2.sup.nd panning round: 100 .mu.g/ml [0222] 3.sup.rd panning
round: 50 .mu.g/ml.
[0223] Incubation was overnight at 4.degree. C., followed by
blocking with 10 .mu.g/ml cysteine-HCl in PBS, with 200 .mu.l per
well for 2 h at room temperature.
[0224] The surface display phage library, containing equal
concentration of phage from libraries CH3, CH3+3, CH3+5, and CH3+7,
was then allowed to react with the bound peptide by adding phage
suspension and 2% BSA-PBS up to 200 .mu.l, followed by incubation
for 45 min with shaking and 90 min without shaking at room
temperature.
[0225] Unbound phage particles were washed away as follows; [0226]
after the 1.sup.st panning round: 15.times.200 .mu.l T-PBS,
5.times.200 .mu.l T-PBS [0227] after the 1.sup.st panning round:
15.times.200 .mu.l T-PBS, 10.times.200 .mu.l T-PBS [0228] after the
1.sup.st panning round: 20.times.200 .mu.l T-PBS, 20.times.200
.mu.l T-PBS.
[0229] Elution of bound particles was performed by adding 200 .mu.l
per well of 0.1 M glycine, pH=-2.2, and incubation with shaking for
30 min at room temperature. Subsequently, the phage suspension was
neutralised by the addition of 60 .mu.l 2M Tris-base, followed by
the infection of E. coli TG1 cells by mixing 10 ml exponentially
growing culture with 0.5 ml eluted phage and incubation for 30 min
at 37.degree.. Finally, infected bacteria were plated on TYE medium
with 1% glucose and 100 .mu.g/ml ampicillin, and incubated at
30.degree. C. overnight.
Results of the Panning of the CH3-Phage Library on Rp10-L
Peptide
Phage Titers
TABLE-US-00004 [0230] Panning concentration round Rp10-L input
(phage/ml) output (phage/ml) 1.sup.st 100 .mu.g/ml 2 .times.
10.sup.14 2 .times. 10.sup.10 2.sup.nd 100 .mu.g/ml 3 .times.
10.sup.17 3 .times. 10.sup.10 3.sup.rd 50 .mu.g/ml 6.02 .times.
10.sup.14 1.5 .times. 10.sup.10
Example 6
Cloning of Selected Clones for Soluble Expression
[0231] Altered CH3 domain-encoding sequences, contained within
eluted phage particles, were batch amplified with PCR. After
restriction with NcoI and NotI, they were inserted in pNOTBAD
(Invitrogen vector pBAD with subsequently inserted NotI site).
After transformation into E. coli E104, the cells were selected on
TYE medium with 1% glucose and 100 .mu.g/ml ampicillin at
30.degree. C.
Soluble Expression of Selected Clones and Screening
[0232] 4.times.96 ampicillin resistant colonies were cultured in
200 .mu.l 2xYT medium with ampicillin in microtitre plates on a
shaker overnight at 30.degree. C. They were then induced with
L-arabinose added to end concentration of 0.1%. After another
overnight incubation, the cells were collected by centrifuging 15
min at 2000 rpm at room temperature and their periplasma proteins
were released by resuspending in 100 .mu.l Na-borate buffer (160 mM
Na-borate, 200 mM NaCl, pH=8.0) and incubation for at least 6
hours.
[0233] For screening, 4 maleimide plates were coated with 100
.mu.g/ml solution of 50 .mu.g/ml peptide Rp10-L, dissolved in PBS,
pH=7.2, overnight at 4.degree. C. Plates were then blocked with 10
.mu.g/ml cysteine-HCl in PBS, with 200 .mu.l per well for 2 h at
room temperature.
[0234] Released periplasmic protein was then allowed to react with
the bound peptide by adding 50 .mu.l lysate and 50 .mu.l 2%
BSA-PBS, followed by an overnight incubation at room
temperature.
[0235] Binding of the his-tagged protein was revealed by
90-min-incubation with 100 .mu.l per well solution of antibodies
against tetra-his (QIAgen), diluted 1:1000 in 1% BSA-PBS, and a
90-min-incubation with 100 .mu.l per well solution of goat
anti-mouse antibodies, labelled with HRP (Sigma), diluted 1:1000 in
1% BSA-PBS. Signals were observed after the addition of substrate
OPD (3 mg/ml) in Na-citrate/phosphate buffer, pH=4.5, and 0.4
.mu.l/ml H.sub.2O.sub.2. The reaction was stopped with by adding
100 .mu.l 1.25 M H.sub.2SO.sub.4.
Results of Screening for Binding of Rp10-L on a Single Well Per
Clone
TABLE-US-00005 [0236] Clone A21 A57 B63 B78 C50 C55 D5 D37 D39 D80
D83 D91 A.sub.492/620 0.395 0.039 0.063 0.075 0.190 0.045 0.644
0.071 0.448 0.077 0.426 0.142
Background Reaction
TABLE-US-00006 [0237] Plate A.sub.492/620 A 0.027 B 0.035 C 0.037 D
0.035
Clones revealing a positive signal were cultured in 20 ml 2xYT with
ampicillin, at 30.degree. C. overnight. Then they were inoculated
1:20 into fresh medium, and after 3 h at 30.degree. C. they were
induced with end concentration of 0.1% L-arabinose, and allowed to
express the recombinant CH3-domain overnight at 16.degree. C.
Periplasma of the expressing cells was then lysed in 1 ml of
Na-borate buffer, pH=8.0, for a minimum of 6 h. Periplasmic extract
was allowed to react with Rp10-L peptide and the binding was
revealed exactly as described above.
Results of Screening for Binding of Rp10-L
TABLE-US-00007 [0238] Rp10-L clone .mu.g/ml A21 A57 B63 B78 C50 C55
D5 D37 D39 D80 D83 D91 -- 0.265 0.006 0.006 0.005 0.803 0.006 0.035
0.006 0.469 0.004 0.088 0.009 0.81 0.362 0.005 0.007 0.006 1.202
0.008 0.052 0.008 0.660 0.007 0.106 0.009 1.63 0.383 0.006 0.007
0.006 1.308 0.014 0.050 0.014 0.719 0.008 0.129 0.005 3.13 0.352
0.008 0.010 0.005 1.453 0.006 0.060 0.006 0.719 0.008 0.210 0.006
6.25 0.343 0.005 0.008 0.006 1.516 0.007 0.057 0.007 0.694 0.006
0.114 0.008 12.5 0.315 0.007 0.009 0.006 1.495 0.007 0.064 0.007
0.770 0.007 0.130 0.009 25.0 0.335 0.008 0.010 0.008 1.603 0.009
0.063 0.009 0.868 0.008 0.120 0.007 50.0 0.398 0.009 0.011 0.009
1.632 0.009 0.070 0.009 0.765 0.008 0.125 0.008
Cloning of Selected Clones for Soluble Expression in pET27b
[0239] Altered CH3 domain-encoding sequences, contained within
clones that produced a significant signal on binding to Rp10-L,
were amplified with PCR. After restriction with NcoI and NotI, they
were inserted in pET27b (Novagen). After transformation into E.
coli BL21 (DE3), transformed cells were selected on TYE medium with
1% glucose and 50 .mu.g/ml kanamycin at 30.degree. C.
[0240] Clones revealing a positive signal were cultured in 20 ml
M9ZB medium with 2% glucose and kanamycin, at 30.degree. C.
overnight. Then they were inoculated 1:20 into fresh medium, and
after 3 h at 30.degree. C. they were induced with medium containing
1% glycerin instead of glucose, kanamycin and 1 mM IPTG, and
allowed to express the recombinant CH3-domain overnight at
16.degree. C. Periplasma of the expressing cells was then lysed in
1 ml of Na-borate buffer, pH=8.0, for a minimum of 6 h. Periplasmic
extract was analysed for the presence of recombinant protein with
western blotting and detection with anti tetra-his antibodies
(QIAgen).
Nucleotide Sequences and Inferred Protein Sequences of CD-20
Binding Clones
TABLE-US-00008 [0241] source clone 1.sup.st group 2.sup.nd group
3.sup.rd group library A21 VDG PWGPRD WP CH3 + 3 C50 * LTH ALCRWF
VQ CH3 + 3 D5 ALR FCGGVV GL CH3 + 3 D39 GWW QQKPFA TD CH3 + 3 D83
APP DLVHVA MV CH3 + 3 * an insertion of 2 nucleotides in the
2.sup.nd group of mutated residues causes an insertion of G between
otherwise constant residues R and W separating 2.sup.nd and
3.sup.rd group of mutated residues.
Protein Sequence of CD20 Specific CH3+3 Library Clone D83 (IMGT
Numbering)
TABLE-US-00009 [0242] 15-17 92-94
MAPREPQVYTLPPSRDELAPPQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DLV 97-98 HVARWMVGNVFSCSVMHEALHNHYTQKSLSLSPGKAAA
Analysis of Binding of CD-20, Expressed on Cells, Using FACS
[0243] Approximately 10.sup.5 Daudi cells were washed with PBS (800
rpm, 5 min, room temperature) and the recombinant CH3 domain in 1%
BSA-PBS was allowed to bind for 2 h on ice. Cells were washed again
with PBS and the allowed to react with 2 .mu.g/ml anti penta
His-Alexa fluor 488 antibody (QIAgen), diluted in 1% BSA-PBS, for
30 min on ice. After washing, the cells were analysed in FACS.
Unlabelled cells, wild-type CH3 domain and cell line K562 were used
as controls.
Example 7
Isolation of CH1-Mutant Proteins Binding CD20 Antigen
[0244] 3 panning rounds were performed. Maleimide activated plates
(Pierce) were coated with a synthetic peptide, representing a
mimotope of B-cell molecular marker CD20. 200 .mu.l of the
following solution were added per well: PBS, pH=7.2, with the
following concentrations of dissolved peptide: [0245] 1.sup.st
panning round: 100 .mu.g/ml [0246] 2.sup.nd panning round: 100
.mu.g/ml [0247] 3.sup.rd panning round: 50 .mu.g/ml.
[0248] Incubation was overnight at 4.degree. C., followed by
blocking with 10 .mu.g/ml cysteine-HCl in PBS, with 200 .mu.l per
well for 2 h at room temperature.
[0249] The surface display phage library, displaying mutated CH1
domain, was then allowed to react with the bound peptide by adding
phage suspension and 2% BSA-PBS up to 200 .mu.l, followed by
incubation for 45 min with shaking and 90 min without shaking at
room temperature.
[0250] Unbound phage particles were washed away as follows; [0251]
after the 1.sup.st panning round: 10.times.200 .mu.l T-PBS,
5.times.200 .mu.l T-PBS [0252] after the 1.sup.st panning round:
15.times.200 .mu.l T-PBS, 10.times.200 .mu.l T-PBS [0253] after the
1.sup.st panning round: 20.times.200 .mu.l T-PBS, 20.times.200
.mu.l T-PBS.
[0254] Elution of bound particles was performed by adding 200 .mu.l
per well of 0.1 M glycine, pH=2.2, and incubation with shaking for
30 min at room temperature. Subsequently, the phage suspension was
neutralised by the addition of 60 .mu.l 2M Tris-base, followed by
the infection of E. coli TG1 cells by mixing 10 ml exponentially
growing culture with 0.5 ml eluted phage and incubation for 30 min
at 37.degree.. Finally, infected bacteria were plated on TYE medium
with 1% glucose and 100 .mu.g/ml ampicillin, and incubated at
30.degree. C. overnight.
Results of the Panning of the CH1-Phage Library on Rp10-L
Peptide
Phage Titers
TABLE-US-00010 [0255] Panning concentration round Rp10-L input
(phage/ml) output (phage/ml) 1.sup.st 100 .mu.g/ml 5.6 .times.
10.sup.13 1.6 .times. 10.sup.10 2.sup.nd 100 .mu.g/ml 4.04 .times.
10.sup.14 8.55 .times. 10.sup.8 3.sup.rd 50 .mu.g/ml 3.53 .times.
10.sup.14 1.19 .times. 10.sup.12
Cloning of Selected Clones for Soluble Expression
[0256] Altered CH1 domain-encoding sequences, contained within
eluted phage particles, were batch amplified with PCR. After
restriction with NcoI and NotI, they were inserted in pNOTBAD
(Invitrogen vector pBAD with subsequently inserted NotI site).
After transformation into E. coli E104, the cells were selected on
TYE medium with 1% glucose and 100 .mu.g/ml ampicillin at
30.degree. C.
Soluble Expression of Selected Clones and Screening
[0257] 4.times.96 ampicillin resistant colonies were cultured in
200 .mu.l 2xYT medium with ampicillin in microtitre plates on a
shaker overnight at 30.degree. C. They were then induced with
L-arabinose added to end concentration of 0.1%. After another
overnight incubation, the cells were collected by centrifuging 15
min at 2000 rpm at room temperature and their periplasma proteins
were released by resuspending in 100 .mu.l Na-borate buffer (160 mM
Na-borate, 200 mM NaCl, pH=8.0) and incubation for at least 6
hours.
[0258] For screening, 4 maleimide plates were coated with 100
.mu.g/ml solution of 50 .mu.g/ml peptide Rp10-L, dissolved in PBS,
pH=7.2, overnight at 4.degree. C. Plates were then blocked with 10
.mu.g/ml cysteine-HCl in PBS, with 200 .mu.l per well for 2 h at
room temperature.
[0259] Released periplasmic protein was then allowed to react with
the bound peptide by adding 50 .mu.l lysate and 50 .mu.l 2%
BSA-PBS, followed by an overnight incubation at room
temperature.
[0260] Binding of the his-tagged protein was revealed by
90-min-incubation with 100 .mu.l per well solution of antibodies
against tetra-his (QIAgen), diluted 1:1000 in 1% BSA-PBS, and a
90-min-incubation with 100 .mu.l per well solution of goat
anti-mouse antibodies, labelled with HRP (Sigma), diluted 1:1000 in
1% BSA-PBS. Signals were observed after the addition of substrate
OPD (3 mg/ml) in Na-citrate/phosphate buffer, pH=4.5, and 0.4
.mu.l/ml H.sub.2O.sub.2. The reaction was stopped with by adding
100 .mu.l 1.25 M H.sub.2SO.sub.4.
Results of Screening for Binding of Rp10-L on a Single Well Per
Clone
TABLE-US-00011 [0261] clone A13 A79 A96 B6 B17 B19 B21 B23 A492/620
0.027 0.353 0.023 0.038 0.036 0.037 0.032 0.035 clone C14 C45 C49
C68 C79 C81 D36 D82 A492/620 0.025 0.021 0.044 0.025 0.051 0.021
0.027 0.086
Background Reaction
TABLE-US-00012 [0262] Plate A.sub.492/620 A 0.008 B 0.012 C 0.015 D
0.015
Clones revealing a positive signal were cultured in 20 ml 2xYT with
ampicillin, at 30.degree. C. overnight. Then they were inoculated
1:20 into fresh medium, and after 3 h at 30.degree. C. they were
induced with end concentration of 0.1% L-arabinose, and allowed to
express the recombinant CH1-domain overnight at 16.degree. C.
Periplasma of the expressing cells was then lysed in 1 ml of
Na-borate buffer, pH=8.0, for a minimum of 6 h. Periplasmic extract
was allowed to react with Rp10-L peptide and the binding was
revealed exactly as described above.
Results of Screening for Binding of Rp10-L
TABLE-US-00013 [0263] clone + - A13 -0.002 -0.006 A79 0.004 0.001
A96 0.010 0.006 B6 0.004 0.001 B17 0.002 0.007 B19 -0.002 0.007 B21
0.002 0.001 B23 0.055 0.020 C14 0.015 0.017 C45 0.004 0.001 C49
0.005 -0.001 C68 0.003 0.001 C79 0.005 0.002 C81 0.004 0.002 D36
0.029 0.019 D62 0.137 0.126
Cloning of Selected Clones for Soluble Expression in pET27b
[0264] Altered CH1 domain-encoding sequences, contained within
clones that produced a significant signal on binding to Rp10-L,
were amplified with PCR. After restriction with NcoI and NotI, they
were inserted in pET27b (Novagen). After transformation into E.
coli BL21 (DE3), transformed cells were selected on TYE medium with
1% glucose and 50 .mu.g/ml kanamycin at 30.degree. C.
[0265] Clones revealing a positive signal were cultured in 20 ml
M9ZB medium with 2% glucose and kanamycin, at 30.degree. C.
overnight. Then they were inoculated 1:20 into fresh medium, and
after 3 h at 30.degree. C. they were induced with medium containing
1% glycerin instead of glucose, kanamycin and 1 mM IPTG, and
allowed to express the recombinant CH1-domain overnight at
16.degree. C. Periplasma of the expressing cells was then lysed in
1 ml of Na-borate buffer, pH=8.0, for a minimum of 6 h. Periplasmic
extract was analysed for the presence of recombinant protein with
western blotting and detection with anti tetra-his antibodies
(QIAgen).
Sequence of CD20 Specific CH1 SMID, Clone C45
TABLE-US-00014 [0266] LOCUS C45 324 bp ds-DNA SYN 4-JUL-2006 1
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
61 ggcacagcag ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 121 tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct gcagtcctca 181 ggactctact ccctcagcag cgtggtgacc
gtggcccctc tgggtgttgg tgggcatctc 241 gtcctgcact acatctgcaa
cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 301 gttgagccca
aatctgcggc cgct // ENTRY C45 5 10 15 20 25 30 1 A S T K G P S V F P
L A P S S K S T S G G T A A L G C L V K 31 D Y F P E P V T V S W N
S G A L T S G V H T F P A V L Q S S 61 G L Y S L S S V V T V A P L
G V G G H L V L H Y I C N V N H 91 K P S N T K V D K K V E P K S A
A A
Analysis of Binding of CD-20, Expressed on Cells, Using FACS
[0267] Approximately 10.sup.5 Daudi cells were washed with PBS (800
rpm, 5 min, room temperature) and the recombinant CH3 domain in 1%
BSA-PBS was allowed to bind for 2 h on ice. Cells were washed again
with PBS and the allowed to react with 2 .mu.g/ml anti penta
His-Alexa fluor 488 antibody (QIAgen), diluted in 1% BSA-PBS, for
30 min on ice. After washing, the cells were analysed in FACS.
Unlabelled cells, wild-type CH1 domain and cell line K562 were used
as controls.
Example 8
Isolation of CL-Mutant Proteins Binding CD20 Antigen
[0268] 3 panning rounds were performed. Maleimide activated plates
(Pierce) were coated with a synthetic peptide, representing a
mimotope of B-cell molecular marker CD20. 200 .mu.l of the
following solution were added per well: PBS, pH=7.2, with the
following concentrations of dissolved peptide: [0269] 1.sup.st
panning round: 100 .mu.g/ml [0270] 2.sup.nd panning round: 100
.mu.g/ml [0271] 3.sup.rd panning round: 50 .mu.g/ml.
[0272] Incubation was overnight at 4.degree. C., followed by
blocking with 10 .mu.g/ml cysteine-HCl in PBS, with 200 .mu.l per
well for 2 h at room temperature.
[0273] The surface display phage library, displaying mutated CL
domain, was then allowed to react with the bound peptide by adding
phage suspension and 2% BSA-PBS up to 200 .mu.l, followed by
incubation for 45 min with shaking and 90 min without shaking at
room temperature.
[0274] Unbound phage particles were washed away as follows; [0275]
after the 1.sup.st panning round: 10.times.200 .mu.l T-PBS,
5.times.200 .mu.l T-PBS [0276] after the 1.sup.st panning round:
15.times.200 .mu.l T-PBS, 10.times.200 .mu.l T-PBS [0277] after the
1.sup.st panning round: 20.times.200 .mu.l T-PBS, 20.times.200
.mu.l T-PBS.
[0278] Elution of bound particles was performed by adding 200 .mu.l
per well of 0.1 M glycine, pH=2.2, and incubation with shaking for
30 min at room temperature. Subsequently, the phage suspension was
neutralised by the addition of 60 .mu.l 2M Tris-base, followed by
the infection of E. coli TG1 cells by mixing 10 ml exponentially
growing culture with 0.5 ml eluted phage and incubation for 30 min
at 37.degree.. Finally, infected bacteria were plated on TYE medium
with 1% glucose and 100 .mu.g/ml ampicillin, and incubated at
30.degree. C. overnight.
Results of the Panning of the CL-Phage Library on Rp10-L
Peptide
Phage Titers
TABLE-US-00015 [0279] Panning concentration round Rp10-L input
(phage/ml) output (phage/ml) 1.sup.st 100 .mu.g/ml 2.8 .times.
10.sup.13 3.6 .times. 10.sup.7 2.sup.nd 100 .mu.g/ml 4.29 .times.
10.sup.14 6.88 .times. 10.sup.9 3.sup.rd 50 .mu.g/ml 1 .times.
10.sup.15 .sup. 6.54 .times. 10.sup.11
Cloning of Selected Clones for Soluble Expression
[0280] Altered CL domain-encoding sequences, contained within
eluted phage particles, were batch amplified with PCR. After
restriction with NcoI and NotI, they were inserted in pNOTBAD
(Invitrogen vector pBAD with subsequently inserted NotI site).
After transformation into E. coli E104, the cells were selected on
TYE medium with 1% glucose and 100 .mu.g/ml ampicillin at
30.degree. C.
Soluble Expression of Selected Clones and Screening
[0281] 4.times.96 ampicillin resistant colonies were cultured in
200 .mu.l 2xYT medium with ampicillin in microtitre plates on a
shaker overnight at 30.degree. C. They were then induced with
L-arabinose added to end concentration of 0.1%. After another
overnight incubation, the cells were collected by centrifuging 15
min at 2000 rpm at room temperature and their periplasma proteins
were released by resuspending in 100 .mu.l Na-borate buffer (160 mM
Na-borate, 200 mM NaCl, pH=8.0) and incubation for at least 6
hours.
[0282] For screening, 4 maleimide plates were coated with 100
.mu.g/ml solution of 50 .mu.g/ml peptide Rp10-L, dissolved in PBS,
pH=7.2, overnight at 4.degree. C. Plates were then blocked with 10
.mu.g/ml cysteine-HCl in PBS, with 200 .mu.l per well for 2 h at
room temperature.
[0283] Released periplasmic protein was then allowed to react with
the bound peptide by adding 50 .mu.l lysate and 50 .mu.l 2%
BSA-PBS, followed by an overnight incubation at room
temperature.
[0284] Binding of the his-tagged protein was revealed by
90-min-incubation with 100 .mu.l per well solution of antibodies
against tetra-his (QIAgen), diluted 1:1000 in 1% BSA-PBS, and a
90-min-incubation with 100 .mu.l per well solution of goat
anti-mouse antibodies, labelled with HRP (Sigma), diluted 1:1000 in
1% BSA-PBS. Signals were observed after the addition of substrate
OPD (3 mg/ml) in Na-citrate/phosphate buffer, pH=4.5, and 0.4
.mu.l/ml H.sub.2O.sub.2. The reaction was stopped with by adding
100 .mu.l 1.25 M H.sub.2SO.sub.4.
Results of Screening for Binding of Rp10-L on a Single Well Per
Clone
TABLE-US-00016 [0285] clone A2 A51 A57 A64 B21 B23 B44 B92 A492/620
0.048 0.083 0.035 0.032 0.037 0.036 0.041 0.154 clone C18 C19 C28
C56 C76 D2 D51 D82 A492/620 0.153 0.033 0.042 0.062 0.030 0.016
0.033 0.046
Background Reaction
TABLE-US-00017 [0286] Plate A.sub.492/620 A 0.016 B 0.016 C 0.012 D
0.014
Clones revealing a positive signal were cultured in 20 ml 2xYT with
ampicillin, at 30.degree. C. overnight. Then they were inoculated
1:20 into fresh medium, and after 3 h at 30.degree. C. they were
induced with end concentration of 0.1% L-arabinose, and allowed to
express the recombinant CL-domain overnight at 16.degree. C.
Periplasma of the expressing cells was then lysed in 1 ml of
Na-borate buffer, pH=8.0, for a minimum of 6 h. Periplasmic extract
was allowed to react with Rp10-L peptide and the binding was
revealed exactly as described above.
Results of Screening for Binding of Rp10-L
TABLE-US-00018 [0287] clone + - A2 0.002 0.001 A57 0.006 0.004 A62
0.016 0.005 A64 0.006 0.006 B21 0.005 -0.002 B23 0.004 0.004 B44
0.007 0.002 B92 0.038 0.017 C18 0.025 0.041 C19 0.006 0.003 C28
0.010 0.003 C56 0.026 0.010 C76 0.075 0.034 D2 0.003 0.002 D82
0.007 -0.007
Cloning of Selected Clones for Soluble Expression in pET27b
[0288] Altered CL domain-encoding sequences, contained within
clones that produced a significant signal on binding to Rp10-L,
were amplified with PCR. After restriction with NcoI and NotI, they
were inserted in pET27b (Novagen). After transformation into E.
coli BL21 (DE3), transformed cells were selected on TYE medium with
1% glucose and 50 .mu.g/ml kanamycin at 30.degree. C.
[0289] Clones revealing a positive signal were cultured in 20 ml
M9ZB medium with 2% glucose and kanamycin, at 30.degree. C.
overnight. Then they were inoculated 1:20 into fresh medium, and
after 3 h at 30.degree. C. they were induced with medium containing
1% glycerin instead of glucose, kanamycin and 1 mM IPTG, and
allowed to express the recombinant CL-domain overnight at
16.degree. C. Periplasma of the expressing cells was then lysed in
1 ml of Na-borate buffer, pH=8.0, for a minimum of 6 h. Periplasmic
extract was analysed for the presence of recombinant protein with
western blotting and detection with anti tetra-his antibodies
(QIAgen).
Example 9
Cloning, Expression and Characterisation of an Integrin-Binding
Fcab
[0290] The potentially cyclic peptide CRGDCL was originally
isolated by Koivunen et al 1993 (J. Biol. Chem. 1993 Sep. 25;
268(27):20205-10) from a 6-amino acid peptide library expressed on
filamentous phage and was shown to inhibit the binding of
RGD-expressing phage to .alpha..sub.v.beta..sub.1 integrin or the
attachment of .alpha..sub.v.beta..sub.1-expressing cells to
fibronectin. The peptide also inhibited cell attachment mediated by
the .alpha..sub.v.beta..sub.1, .alpha..sub.v.beta..sub.3, and
.alpha..sub.v.beta..sub.5 integrins.
[0291] We have inserted the sequence GCRGDCL in the structural loop
(the "EF" loop) of the CH3 domain of human IgG1. For that purpose,
residues Asp92 and Lys 93 (IMGT numbering) were mutated to Gly and
Leu respectively, and the 5 residues CRGDC were inserted between
these mutated residues 92 and 93 to create the loop with the
integrin-binding RGD motif, using standard cloning techniques. At
the C-terminus of the insert, the sequence was fused in frame with
the multiple cloning site of the vector so that the HSV-tag and the
His-tag are attached C-terminally to the recombinant protein. The
name of this recombinant protein Fcab-RGD4, or short RGD4. The DNA
sequence coding for Fcab-RGD4 and the translation in amino acid
sequence are shown below.
TABLE-US-00019 ##STR00001## ##STR00002##
The sequences encoding Fcab-RGD4 and Fcab-wt, respectively, were
introduced into the mammalian expression vector pCEP4 by
conventional cloning techniques. HEK 293 cells were transiently
transfected with these expression plasmids and the Fcab containing
culture medium harvested after 3 days and after one week. The Fcabs
were purified via a Protein A column and acidic elution from the
column, followed by immediate neutralisation. The Fcabs were
dialysed against PBS and tested in an ELISA for binding to human
.alpha..sub.v.beta..sub.3 integrin (Chemicon).
[0292] For the integrin ELISA, 1 .mu.g/ml human
.alpha..sub.v.beta..sub.3 integrin in PBS was coated over night on
Maxisorp plates and blocked for 1 h with BSA in PBS containing 1 mM
Ca2+. Fcab-RGD4 and Fcab-wt, respectively, were allowed to bind for
1 h in various dilutions starting from 10 ug/ml purified protein.
Bound Fcabs were detected by HRP labelled protein A and TMB as a
substrate. Binding of RGD4 to integrin (red line) resulted in
significant signals from 10 ug/ml protein down to 0.16 ug/ml. As
negative controls, RGD4 did not bind to the plate in the absence of
integrin (grey line), nor did Fcab-wt bind to the integrin coated
plate (green line). The binding of the commercial mouse anti human
.alpha..sub.v.beta..sub.3 integrin mAb LM609 (Chemicon; blue line)
served as a positive control.
TABLE-US-00020 protein coating BLK LM609 (anti concentration
Fcab-RGD4 Fcab-wt Fcab-RGD4 integrin mAb) ..quadrature.g/ml) (OD
450) (OD 450) (OD 450) (OD 450) 10 3.4513 0.0485 0.0152 0.6475
2.500 1.7446 0.0338 0.0127 0.6443 0.625 0.7068 0.0337 0.0125 0.6570
0.156 0.2384 0.0327 0.0123 0.6257 0.039 0.0829 0.0295 0.0127 0.3907
0.010 0.0388 0.0276 0.0103 0.1567 0.002 0.0303 0.0273 0.0112
0.0770
Table: ELISA data demonstrating the binding of RGD4 and LM609 to
human .alpha..sub.v.beta..sub.3 integrin. The various proteins were
tested in concentrations as indicated in the first column resulting
in the signals at 450 nm in the respective rows. Values for HEK
produced and protein A purified Fcab-RGD4 binding to integrin are
shown in the second column, Fcab-wt negative control in the third,
and Fcab-RGD4 coating blank control in the fourth column. The
values for binding of mouse anti .alpha..sub.v.beta..sub.3 integrin
mAb LM609 are shown in the last column.
* * * * *
References