U.S. patent application number 16/743344 was filed with the patent office on 2020-06-25 for polypeptides.
The applicant listed for this patent is AFFIBODY AB. Invention is credited to Lars Abrahmsen, Caroline Ekblad, Torbjorn Graslund, Elin Gunneriusson, Malin Lindborg, John Lofblom, Johan Seijsing.
Application Number | 20200199197 16/743344 |
Document ID | / |
Family ID | 47891491 |
Filed Date | 2020-06-25 |
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United States Patent
Application |
20200199197 |
Kind Code |
A1 |
Ekblad; Caroline ; et
al. |
June 25, 2020 |
POLYPEPTIDES
Abstract
The present disclosure relates to a class of engineered
polypeptides having a binding affinity for the neonatal Fc receptor
(in the following referred to as FcRn), and provides an FcRn
binding polypeptide comprising the sequence EX.sub.2 X.sub.3
X.sub.4 AX.sub.6 X.sub.7 EIRWLPNL X.sub.16X.sub.17 X.sub.18
QRX.sub.21 AFIX.sub.25 X.sub.26LX.sub.28 X.sub.29 (SEQ ID NO:1075).
The present disclosure also relates to the use of such an FcRn
binding polypeptide as an agent for modifying pharmacokinetic and
pharmacodynamic properties and as a therapeutic agent.
Inventors: |
Ekblad; Caroline;
(Saltsjo-Boo, SE) ; Gunneriusson; Elin;
(Saltsjobaden, SE) ; Lindborg; Malin; (Bromma,
SE) ; Abrahmsen; Lars; (Stockholm, SE) ;
Lofblom; John; (Huddinge, SE) ; Graslund;
Torbjorn; (Hagersten, SE) ; Seijsing; Johan;
(Stockhom, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AFFIBODY AB |
Solna |
|
SE |
|
|
Family ID: |
47891491 |
Appl. No.: |
16/743344 |
Filed: |
January 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15842178 |
Dec 14, 2017 |
10562955 |
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16743344 |
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14776319 |
Sep 14, 2015 |
9975943 |
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PCT/EP2014/055299 |
Mar 17, 2014 |
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15842178 |
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61787305 |
Mar 15, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/70535 20130101; A61P 7/06 20180101; A61P 19/08 20180101;
C07K 14/745 20130101; A61P 9/04 20180101; A61P 9/14 20180101; A61P
19/02 20180101; A61P 7/02 20180101; A61P 29/00 20180101; A61P 17/02
20180101; A61P 31/04 20180101; C07K 14/31 20130101; A61P 17/00
20180101; C07K 14/435 20130101; C07K 14/70539 20130101; A61P 37/00
20180101; A61P 13/12 20180101; C07K 2319/74 20130101; A61P 25/00
20180101; A61P 21/04 20180101 |
International
Class: |
C07K 14/745 20060101
C07K014/745; C07K 14/435 20060101 C07K014/435; C07K 14/31 20060101
C07K014/31; C07K 14/74 20060101 C07K014/74; C07K 14/735 20060101
C07K014/735 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 15, 2013 |
EP |
13159500.1 |
Claims
1. An FcRn binding polypeptide, comprising an FcRn binding motif
BM, which motif consists of the amino acid sequence TABLE-US-00032
(SEQ ID NO: 1075) EX.sub.2 X.sub.3 X.sub.4 AX.sub.6 X.sub.7 EIR
WLPNLX.sub.16X.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25
X.sub.26LX.sub.28 X.sub.29
wherein, independently from each other, X.sub.2 is selected from A,
D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X.sub.3 is selected
from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y;
X.sub.4 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T,
V, W and Y; X.sub.6 is selected from A, E, F, G, H, I, K, Q, R, S
and V; X.sub.7 is selected from A, F, H, K, N, Q, R, S and V;
X.sub.16 is selected from N and T; X.sub.17 is selected from F, W
and Y; X.sub.18 is selected from A, D, E and N; X.sub.21 is
selected from A, S, V and W; X.sub.25 is selected from D, E, G, H,
I, K, L, N, Q, R, S, T, V, W and Y; X.sub.26 is selected from K and
S, X.sub.28 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T,
V, W and Y; and X.sub.29 is selected from D and R.
2. The FcRn binding polypeptide according to claim 1, wherein the
BM consists of an amino acid sequence selected from TABLE-US-00033
i) (SEQ ID NO: 1076) EX.sub.2 X.sub.3 X.sub.4 AX.sub.6 HEIR
WLPNLTX.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25 KLX.sub.28 D
wherein, independently from each other, X.sub.2 is selected from A,
D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X.sub.3 is selected
from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y; X.sub.4 is
selected from A, D, E, F, G, I, K, L, N, Q, R, S, T, V and Y;
X.sub.6 is selected from A, G, K, R, S and V; X.sub.17 is selected
from F, W and Y; X.sub.18 is selected from A, D, E and N; X.sub.21
is selected from A, S, V and W; X.sub.25 is selected from D, G, H,
K, L, N, R, V and W; X.sub.28 is selected from A, D, E, H, K, L, N,
Q, R, S, T, W and Y; and ii) an amino acid sequence which has at
least 96% identity to said sequence.
3. The FcRn binding polypeptide according to claim 1, wherein
sequence i) fulfills at least three of the six conditions I-VI: I.
X.sub.6 is selected from A, G, K and S, II. X.sub.7 is H; III.
X.sub.17 is selected from F and Y; IV. X.sub.18 is D; V. X.sub.21
is selected from V and W; VI. X.sub.25 is selected from H and
R.
4. The FcRn binding polypeptide according to claim 1, wherein the
sequence is selected from the group consisting of SEQ ID
NO:1-353.
5. The FcRn binding polypeptide according to claim 1, wherein said
FcRn binding motif forms part of a three-helix bundle protein
domain.
6. The FcRn binding polypeptide according to claim 1, which
comprises an amino acid sequence selected from: TABLE-US-00034 iii)
(SEQ ID NO: 1077) K-[BM]-DPSQS X.sub.aX.sub.bLLX.sub.c EAKKL
X.sub.dX.sub.eX.sub.fQ;
wherein [BM] is an FcRn binding motif as defined herein, provided
that X.sub.29 is D; X.sub.a is selected from A and S, X.sub.b is
selected from N and E; X.sub.c is selected from A, S and C; X.sub.d
is selected from E, N and S, X.sub.e is selected from D, E and S,
X.sub.f is selected from A and S, and iv) an amino acid sequence
which has at least 93% identity to a sequence defined by iii).
7. The FcRn binding polypeptide according to claim 6, wherein
sequence iii) is selected from the group consisting of SEQ ID
NO:354-706.
8. The FcRn binding polypeptide according to claim 1, which
comprises an amino acid sequence selected from: TABLE-US-00035 xi)
(SEQ ID NO: 1092) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is an FcRn binding motif as defined in claim 1; and
xii) an amino acid sequence which has at least 94% identity to the
sequence defined in xi).
9. The FcRn binding polypeptide according to claim 8, in which
sequence xi) is selected from the group consisting of SEQ ID
NO:1060-1062.
10. The FcRn binding polypeptide according to claim 1, which
comprises an amino acid sequence selected from: TABLE-US-00036
xiii) (SEQ ID NO: 1095) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,
wherein [BM] is an FcRn binding motif as defined in claim 1; and
xiv) an amino acid sequence which has at least 94% identity to the
sequence defined in xiii).
11. The FcRn binding polypeptide according to claim 10, in which
sequence xiii) is selected from the group consisting of SEQ ID
NO:707-1059.
12. The FcRn binding polypeptide according to claim 1, which is
capable of binding to FcRn at pH 6.0 such that the K.sub.D value of
the interaction is at most 1.times.10.sup.-6 M.
13. The FcRn binding polypeptide according to claim 1, wherein the
K.sub.D value of the interaction between FcRn binding polypeptide
and FcRn at pH 7.4 is higher than the K.sub.D value of said
interaction at pH 6.0.
14. The FcRn binding polypeptide according to claim 1, wherein the
K.sub.D value of the interaction between FcRn binding polypeptide
and FcRn at pH 7.4 is at least 1.times.10.sup.-8 M.
15. The FcRn binding polypeptide according to claim 1, wherein the
K.sub.D value of the interaction between FcRn binding polypeptide
and FcRn at pH 7.4 is at most 1.times.10.sup.-6 M.
16. A fusion protein or conjugate comprising a first moiety
consisting of an FcRn binding polypeptide according to claim 1; and
a second moiety consisting of a polypeptide having a desired
biological activity.
17. An FcRn binding polypeptide, fusion protein or conjugate
according to claim 1, which inhibits binding of IgG to FcRn.
18. A polynucleotide encoding a polypeptide according to claim
1.
19. A composition comprising an FcRn binding polypeptide, fusion
protein or conjugate according to claim 1 and at least one
pharmaceutically acceptable excipient or carrier.
20. An FcRn binding polypeptide, fusion protein or conjugate
according to claim 1 for use as a medicament.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is Continuation of U.S. application Ser.
No. 15/842,178 filed Dec. 14, 2017 which is a Continuation of U.S.
application Ser. No. 14/776,319 filed Sep. 14, 2015, now U.S. Pat.
No. 9,975,943, which is a U.S. National Stage Application of
PCT/EP2014/055299 filed Mar. 17, 2014, which claims priority from
European Patent Application No.: 13159500.1, filed Mar. 15, 2013,
and U.S. Provisional Application Ser. No. 61/787,305 filed Mar. 15,
2013, all of which are incorporated by reference in their
entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] The present disclosure relates to a class of engineered
polypeptides having a binding affinity for the neonatal Fc receptor
(in the following referred to as FcRn). The present disclosure also
relates to the use of such an FcRn binding polypeptide as an agent
for modifying pharmacokinetic and pharmacodynamic properties of a
biomolecule, e.g. a pharmaceutical, and as a therapeutic agent.
BACKGROUND
[0003] The neonatal Fc receptor (FcRn) is a heterodimeric protein
consisting of a transmembrane MHC class I-like heavy chain (FcRn
.alpha.-chain) and the .beta.2-microglobulin light chain, the
latter also forming a part of MHC class I molecules (Simister and
Mostov (1989) Nature 337:184-7; Burmeister et al. (1994) Nature
372:379-83).
[0004] FcRn is predominantly located in endosomes and is able to
bind to serum albumin and immunoglobulin G (IgG) at pH.gtoreq.6.5
and release them at pH.gtoreq.7.0 (reviewed in Roopenian (2007) Nat
Rev Immunol 7:715-25).
[0005] FcRn carries out several distinct tasks in mammals (reviewed
in Roopenian, supra). FcRn is involved in recycling of endocytosed
IgG and serum albumin, thus avoiding their degradation in the
lysosome, giving them longer half-life and higher availability in
the blood than other serum proteins. When IgG, serum albumin and
other serum proteins are passively pinocytosed by cells in contact
with blood, the pH becomes gradually lower in the formed endosomes,
which permits the binding of IgG and serum albumin to FcRn. The
receptor is then, together with its bound ligand, transported via
recycling endosomes back to the plasma membrane. After returning to
the plasma membrane, the pH increases to above 7, at which point
the bound ligand is released.
[0006] FcRn is also recognized for its ability to transport IgG
over barriers such as the placenta, the upper airway epithelium,
the blood-brain barrier and the proximal small intestine.
[0007] In mammals, the properties of FcRn are used to transcytose
IgG from a mother to a fetus via the placenta, and to transcytose
IgG from a mother's milk to the blood stream of an infant in the
proximal small intestine.
[0008] The expression pattern of FcRn differs between species.
However, FcRn is widely expressed by cells in the blood brain
barrier, upper airway epithelium, kidneys and vascular endothelia,
and by antigen presenting cells as well as by other cells of
hematopoietic origin in most species (reviewed in Roopenian (2007),
supra).
[0009] Antibodies and peptides with affinity towards FcRn (Liu et
al. (2007) J Immunol 179:2999-3011, Mezo et al. (2008) Proc Natl
Acad Sci USA 105:2337-42) and .beta.2-microglobulin (Getman and
Balthasar (2005) J Pharm Sci 94:718-29) have been developed with a
view to inhibit the binding between endogenous IgG and FcRn.
Another approach has been to mutate the Fc region of the IgG to get
a higher affinity for FcRn (Petkova et al. (2006) Int Immunol
18:1759-69, Vaccaro et al. (2005) Nat Biotechnol 23:1283-8).
[0010] Fusion to the Fc domain or to albumin is a widely used
strategy to increase the in vivo half-life of proteins. However,
the large size of such fusion proteins adversly affects tissue
penetration and reduces the specificity to the fusion partner
(Valles et al. (2011) J Interferon Cytokine Res 32:178-184). On the
other hand, mutations have been made in the Fc fragment of
antibodies administered to non human primates to prolong half-life
(Hinton et al. (2004) J Biol Chem 279:6213-6). However, this
approach is only limited in use to therapeutic antibodies, and
cannot be extrapolated to other therapeutic proteins unless the
proteins in question are fused to Fc fragments, which also results
in large size molecules. A number of chemical and recombinant
methods have been devised to improve protein half-life, such as
PEGylation and genetic fusions of the protein to the Fc domain of
IgG or albumin (reviewed in Schellenberger et al. (2009) Nat
Biotechnol 21:1186-1190). PEGylation of proteins has been reported
to decrease their potency and contribute to their
immunoreactivity.
[0011] Fc-fusion proteins have also been used for oral and
pulmonary delivery mediated by the FcRn (Low et al., (2005) Human
reproduction July; 20(7):1805-13), however similar problems
relating to tissue penetration and reduced specificity remain, due
to the size of the fusion molecules.
[0012] Hence, there is large need in the field for the continued
provision of molecules with high affinity for FcRn. In particular,
small binding molecules are needed that, when present as a fusion
partner, do not adversely affect the properties of the molecules
they are fused to and do not contribute to immunoreactivity.
SUMMARY OF THE INVENTION
[0013] It is an object of the present disclosure to provide new
FcRn binding agents for use in modifying pharmacokinetic and/or
pharmacodynamic properties of a biomolecule, e.g. a
pharmaceutical.
[0014] It is also an object of the present disclosure to provide
new FcRn binding agents for use as therapeutic agents in their own
right, alone or as combination treatment.
[0015] It is an object of the present disclosure to provide a
molecule allowing for efficient targeting of FcRn, while
alleviating the above-mentioned and other drawbacks of current
therapies.
[0016] These and other objects which are evident to the skilled
person from the present disclosure are met by different aspects of
the invention as claimed in the appended claims and as generally
disclosed herein.
[0017] Thus, in the first aspect of the disclosure, there is
provided a neonatal Fc receptor (FcRn) binding polypeptide,
comprising an FcRn binding motif, BM, which motif consists of the
amino acid sequence
TABLE-US-00001 (SEQ ID NO: 1075) EX.sub.2 X.sub.3 X.sub.4 AX.sub.6
X.sub.7 EIR WLPNLX.sub.16 X.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25
X.sub.26LX.sub.28 X.sub.29
wherein, independently from each other,
[0018] X.sub.2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S,
T, V, W and Y;
[0019] X.sub.3 is selected from A, D, E, F, G, H, I, K, L, M, N, Q,
R, S, T, V, W and Y;
[0020] X.sub.4 is selected from A, D, E, F, G, H, I, K, L, N, Q, R,
S, T, V, W and Y;
[0021] X.sub.6 is selected from A, D, E, F, G, H, I, K, L, N, Q, R,
S, T, V, W and Y;
[0022] X.sub.7 is selected from A, F, H, I, K, L, N, Q, R, S, T, V,
W and Y;
[0023] X.sub.16 is selected from N and T;
[0024] X.sub.17 is selected from F, W and Y;
[0025] X.sub.18 is selected from A, D, E and N;
[0026] X.sub.21 is selected from A, S, V and W;
[0027] X.sub.25 is selected from A, D, E, F, G, H, I, K, L, N, Q,
R, S, T, V, W and Y;
[0028] X.sub.26 is selected from K and S,
[0029] X.sub.28 is selected from A, D, E, F, H, I, K, L, N, Q, R,
S, T, V, W and Y; and
[0030] X.sub.29 is selected from D and R.
[0031] The above definition of a class of sequence related, FcRn
binding polypeptides is based on a statistical analysis of a number
of random polypeptide variants of a parent scaffold, that were
selected for their interaction with FcRn in several different
selection experiments. The identified FcRn binding motif, or "BM",
corresponds to the target binding region of the parent scaffold,
which region constitutes two alpha helices within a three-helical
bundle protein domain. In the parent scaffold, the varied amino
acid residues of the two BM helices constitute a binding surface
for interaction with the constant Fc part of antibodies. In the
present disclosure, the random variation of binding surface
residues and subsequent selection of variants have replaced the Fc
interaction capacity with a capacity for interaction with FcRn.
[0032] In one embodiment of said FcRn binding polypeptide, the BM
consists of the amino acid sequence
TABLE-US-00002 (SEQ ID NO: 1076) EX.sub.2 X.sub.3 X.sub.4 AX.sub.6
X.sub.7 EIR WLPNLTX.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25
KLX.sub.28 D
wherein, independently from each other,
[0033] X.sub.2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S,
T, V, W and Y;
[0034] X.sub.3 is selected from A, D, E, F, G, H, I, K, L, M, N, Q,
R, S, T, V, W and Y;
[0035] X.sub.4 is selected from A, D, E, F, G, H, I, K, L, N, Q, R,
S, T, V, W and Y;
[0036] X.sub.6 is selected from A, D, E, F, G, H, I, K, L, N, Q, R,
S, T, V, W and Y;
[0037] X.sub.7 is selected from A, F, H, I, K, L, N, Q, R, S, T, V,
W and Y;
[0038] X.sub.17 is selected from F, W and Y;
[0039] X.sub.18 is selected from A, D, E and N;
[0040] X.sub.21 is selected from A, S, V and W;
[0041] X.sub.25 is selected from A, D, E, F, G, H, I, K, L, N, Q,
R, S, T, V, W and Y; and
[0042] X.sub.28 is selected from A, D, E, F, H, I, K, L, N, Q, R,
S, T, V, W and Y.
[0043] In another embodiment of the first aspect of the disclosure,
said neonatal Fc receptor (FcRn) binding polypeptide comprises an
FcRn binding motif, BM, which motif consists of the amino acid
sequence
TABLE-US-00003 (SEQ ID NO: 1075) EX.sub.2 X.sub.3 X.sub.4 AX.sub.6
X.sub.7 EIR WLPNLX.sub.16X.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25
X.sub.26LX.sub.28 X.sub.29
wherein, independently from each other,
[0044] X.sub.2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S,
T, V, W and Y;
[0045] X.sub.3 is selected from A, D, E, F, G, H, I, K, L, M, N, Q,
R, S, T, V, W and Y;
[0046] X.sub.4 is selected from A, D, E, F, G, H, I, K, L, N, Q, R,
S, T, V, W and Y;
[0047] X.sub.6 is selected from A, E, F, G, H, I, K, Q, R, S and
V;
[0048] X.sub.7 is selected from A, F, H, K, N, Q, R, S and V;
[0049] X.sub.16 is selected from N and T;
[0050] X.sub.17 is selected from F, W and Y;
[0051] X.sub.18 is selected from A, D, E and N;
[0052] X.sub.21 is selected from A, S, V and W;
[0053] X.sub.25 is selected from D, E, G, H, I, K, L, N, Q, R, S,
T, V, W and Y;
[0054] X.sub.26 is selected from K and S,
[0055] X.sub.28 is selected from A, D, E, F, H, I, K, L, N, Q, R,
S, T, V, W and Y; and
[0056] X.sub.29 is selected from D and R.
[0057] In another embodiment of the first aspect, there is provided
an FcRn binding polypeptide, wherein, independently from each
other,
[0058] X.sub.2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S,
T, V, W and Y;
[0059] X.sub.3 is selected from A, D, E, F, H, I, K, L, M, N, Q, R,
S, T, V, W and Y;
[0060] X.sub.4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S,
T, V, W and Y;
[0061] X.sub.6 is selected from A, E, F, G, H, I, K, Q, R and
S,
[0062] X.sub.7 is selected from A, F, H, K, N, Q, R, S and V;
[0063] X.sub.16 is selected from N and T;
[0064] X.sub.17 is selected from F and Y;
[0065] X.sub.18 is D;
[0066] X.sub.21 is V;
[0067] X.sub.25 is selected from D, E, H, I, K, L, N, Q, R, S, T,
V, W and Y;
[0068] X.sub.26 is selected from K and S,
[0069] X.sub.28 is selected from A, D, E, F, H, I, K, L, N, Q, R,
S, T, V and W and.
[0070] X.sub.29 is selected from D and R.
[0071] In another embodiment of the first aspect, the BM consists
of an amino acid sequence selected from
TABLE-US-00004 i) (SEQ ID NO: 1076) EX.sub.2 X.sub.3 X.sub.4
AX.sub.6 HEIR WLPNLTX.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25
KLX.sub.28 D
wherein, independently from each other,
[0072] X.sub.2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S,
T, V, W and Y;
[0073] X.sub.3 is selected from A, D, E, G, H, K, L, M, N, Q, R, S,
T, V and Y;
[0074] X.sub.4 is selected from A, D, E, F, G, I, K, L, N, Q, R, S,
T, V and Y;
[0075] X.sub.6 is selected from A, G, K, R, S and V;
[0076] X.sub.17 is selected from F, W and Y;
[0077] X.sub.18 is selected from A, D, E and N;
[0078] X.sub.21 is selected from A, S, V and W;
[0079] X.sub.25 is selected from D, G, H, K, L, N, R, V and W;
[0080] X.sub.28 is selected from A, D, E, H, K, L, N, Q, R, S, T, W
and Y; and [0081] ii) an amino acid sequence which has at least 96%
identity to said sequence.
[0082] In yet another embodiment of said aspect, the BM in sequence
i) consists of an amino acid sequence selected from
TABLE-US-00005 (SEQ ID NO: 1076) EX.sub.2 X.sub.3 X.sub.4 AX.sub.6
HEIR WLPNLTX.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25 KLX.sub.28
D
wherein, independently from each other,
[0083] X.sub.2 is selected from A, D, E, F, N, Q, R, S and W;
[0084] X.sub.3 is selected from D, E, G, H, K, M, N, Q, S and
T;
[0085] X.sub.4 is selected from A, D, E, G, N, Q, R, S, T, V and
Y;
[0086] X.sub.6 is selected from A, G, S and V;
[0087] X.sub.17 is selected from F, W and Y;
[0088] X.sub.18 is selected from A, D, E and N;
[0089] X.sub.21 is selected from A, S, V and W;
[0090] X.sub.25 is selected from D, G, H, K, L, N, R and V; and
[0091] X.sub.28 is selected from A, E, H, L, N, Q, R, S, T, W and
Y.
[0092] As the skilled person will realize, the function of any
polypeptide, including the FcRn binding capacity of the polypeptide
of the present disclosure, is dependent on the tertiary structure
of the polypeptide. It is therefore possible to make minor changes
to the sequence of amino acids in a polypeptide without affecting
the function thereof. Thus, the disclosure encompasses modified
variants of the FcRn binding polypeptide, which are such that the
FcRn binding characteristics are retained.
[0093] Therefore, as described above, also encompassed by the
present disclosure is a FcRn binding polypeptide comprising an
amino acid sequence with 96% or greater identity to a polypeptide
as defined in i).
[0094] In some embodiments, such changes may be made in all
positions of the sequences of the FcRn binding polypeptide as
disclosed herein. In other embodiments, such changes may be made
only in the non-variable positions, also denoted as scaffold amino
acid residues. In such cases, changes are not allowed in the
variable positions, i.e. positions denoted with an "X" in sequence
i). For example, it is possible that an amino acid residue
belonging to a certain functional grouping of amino acid residues
(e.g. hydrophobic, hydrophilic, polar etc) could be exchanged for
another amino acid residue from the same functional group.
[0095] The term "% identity", as used throughout the specification,
may for example be calculated as follows. The query sequence is
aligned to the target sequence using the CLUSTAL W algorithm
(Thompson et al. (1994) Nucleic Acids Research 22:4673-4680). A
comparison is made over the window corresponding to the shortest of
the aligned sequences. The shortest of the aligned sequences may in
some instances be the target sequence. In other instances, the
query sequence may constitute the shortest of the aligned
sequences. The amino acid residues at each position are compared,
and the percentage of positions in the query sequence that have
identical correspondences in the target sequence is reported as %
identity.
[0096] Below follows a list of embodiments which further specify
amino acid residue X.sub.n, wherein n is an integer which denotes
the position of said residue within the polypeptide described
herein. To clarify, in cases where the BM comprised in the
polypeptide may consist of either a given amino acid sequence or an
amino acid sequence with at least a given % identity to said given
amino acid sequence, the X.sub.n as used herein refers to an amino
acid residue in said given amino acid sequence. For example, when
applicable, X.sub.n refers to an amino acid residue in sequence i)
above.
[0097] In one embodiment, X.sub.2 is selected from A, D, E, F, I,
L, N, Q, R, S, T, V, W and Y.
[0098] In one embodiment, X.sub.2 is selected from A, D, F, I, L,
N, Q, R, S, T, V, W and Y.
[0099] In one embodiment, X.sub.2 is selected from A, D, F, I, L,
N, Q, R, S, V and
[0100] W.
[0101] In one embodiment, X.sub.2 is selected from A, I, L, N, Q,
R, S, T, V, W and Y.
[0102] In one embodiment, X.sub.2 is selected from A, I, L, N, Q,
S, T, V and W.
[0103] In one embodiment, X.sub.2 is selected from A, I, L, N, Q, V
and W.
[0104] In one embodiment, X.sub.2 is selected from A, I, L, Q, V
and W.
[0105] In one embodiment, X.sub.2 is selected from A, I, L and
Q.
[0106] In one embodiment, X.sub.2 is selected from I, L and Q.
[0107] In one embodiment, X.sub.2 is selected from I and Q.
[0108] In one embodiment, X.sub.2 is I.
[0109] In one embodiment, X.sub.2 is Q.
[0110] In one embodiment, X.sub.3 is selected from A, D, E, G, H,
K, L, M, N, Q, R, S, T, V and Y.
[0111] In one embodiment, X.sub.3 is selected from A, D, E, H, K,
L, M, N, Q, R, S, T, V and Y.
[0112] In one embodiment, X.sub.3 is selected from A, D, E, G, H,
K, L, M, N, Q, R, S and T.
[0113] In one embodiment, X.sub.3 is selected from A, D, E, G, H,
K, M, N, Q, S and T.
[0114] In one embodiment, X.sub.3 is selected from A, D, E, G, H,
M, N, Q, S and T.
[0115] In one embodiment, X.sub.3 is selected from A, D, E, K, N,
Q, S and T.
[0116] In one embodiment, X.sub.3 is selected from A, D, E, K, Q,
and T.
[0117] In one embodiment, X.sub.3 is selected from A, D, E, Q and
T.
[0118] In one embodiment, X.sub.3 is selected from D, E and T.
[0119] In one embodiment, X.sub.3 is selected from D and E.
[0120] In one embodiment, X.sub.3 is D.
[0121] In one embodiment, X.sub.3 is E.
[0122] In one embodiment, X.sub.4 is selected from A, D, E, F, G,
I, K, L, N, Q, R, S, T, V and Y.
[0123] In one embodiment, X.sub.4 is selected from A, D, E, G, N,
Q, R, S, T and V.
[0124] In one embodiment, X.sub.4 is selected from A, D, E, F, I,
K, L, N, Q, R, S, T and V.
[0125] In one embodiment, X.sub.4 is selected from A, D, E, I, K,
N, Q, R, S and T.
[0126] In one embodiment, X.sub.4 is selected from A, D, E, I, K,
Q, S and T.
[0127] In one embodiment, X.sub.4 is selected from A, D, I, K, Q
and S.
[0128] In one embodiment, X.sub.4 is selected from A, D, E, K and
S.
[0129] In one embodiment, X.sub.4 is selected from A, D, K and
S.
[0130] In one embodiment, X.sub.4 is selected from A, D, E and
K.
[0131] In one embodiment, X.sub.4 is selected from A, D and K.
[0132] In one embodiment, X.sub.4 is selected from A and D.
[0133] In one embodiment, X.sub.4 is selected from A and E.
[0134] In one embodiment, X.sub.4 is A.
[0135] In one embodiment, X.sub.4 is D.
[0136] In one embodiment, X.sub.4 is E.
[0137] In one embodiment, X.sub.6 is selected from A, G, K, Q, R, S
and V.
[0138] In one embodiment, X.sub.6 is selected from A, G, K, R, S
and V.
[0139] In one embodiment, X.sub.6 is selected from A, G, K, R and
S.
[0140] In one embodiment, X.sub.6 is selected from A, G, K, S and
V.
[0141] In one embodiment, X.sub.6 is selected from A, G, K and
V.
[0142] In one embodiment, X.sub.6 is selected from A, G, K and
S.
[0143] In one embodiment, X.sub.6 is selected from A, G and K.
[0144] In one embodiment, X.sub.6 is selected from A, G and V.
[0145] In one embodiment, X.sub.6 is selected from A and G.
[0146] In one embodiment, X.sub.6 is A.
[0147] In one embodiment, X.sub.6 is G.
[0148] In one embodiment, X.sub.7 is selected from A and H.
[0149] In one embodiment, X.sub.7 is H.
[0150] In one embodiment, X.sub.16 is T.
[0151] In one embodiment, X.sub.16 is N.
[0152] In one embodiment, X.sub.17 is selected from F and Y.
[0153] In one embodiment, X.sub.17 is F.
[0154] In one embodiment, X.sub.18 is selected from A, D and E.
[0155] In one embodiment, X.sub.18 is selected from A and D.
[0156] In one embodiment, X.sub.18 is D.
[0157] In one embodiment, X.sub.21 is selected from V and W.
[0158] In one embodiment, X.sub.21 is V.
[0159] In one embodiment, X.sub.25 is selected from D, E, G, H, K,
L, N, Q, R, V and W.
[0160] In one embodiment, X.sub.25 is selected from D, G, H, K, L,
N, R, V and W.
[0161] In one embodiment, X.sub.25 is selected from D, G, H, K, L,
N, R and V.
[0162] In one embodiment, X.sub.25 is selected from H, L, R, V and
W.
[0163] In one embodiment, X.sub.25 is selected from H, R, V and
W.
[0164] In one embodiment, X.sub.25 is selected from H, R and V.
[0165] In one embodiment, X.sub.25 is selected from H, L and R.
[0166] In one embodiment, X.sub.25 is selected from H and R.
[0167] In one embodiment, X.sub.25 is selected from H and V.
[0168] In one embodiment, X.sub.25 is H.
[0169] In one embodiment, X.sub.26 is K.
[0170] In one embodiment, X.sub.26 is S.
[0171] In one embodiment, X.sub.28 is selected from A, D, E, H, K,
L, N, Q, R, S, T, W and Y.
[0172] In one embodiment, X.sub.28 is selected from A, D, E, K, L,
N, Q, R, S, T, W and Y.
[0173] In one embodiment, X.sub.28 is selected from A, D, E, L, R,
S, T, W and Y.
[0174] In one embodiment, X.sub.28 is selected from A, D, K, L, N,
Q, R, S, T and W.
[0175] In one embodiment, X.sub.28 is selected from A, D and R.
[0176] In one embodiment, X.sub.28 is selected from A and R.
[0177] In one embodiment, X.sub.28 is selected from D and R.
[0178] In one embodiment, X.sub.28 is A.
[0179] In one embodiment, X.sub.28 is R.
[0180] In one embodiment, X.sub.29 is D.
[0181] In one embodiment, X.sub.29 is R.
[0182] In one embodiment, X.sub.6X.sub.7 is selected from AH and
GH.
[0183] In one embodiment, X.sub.6X.sub.7 is AH.
[0184] In one embodiment, X.sub.6X.sub.7 is GH.
[0185] In one embodiment, X.sub.17X.sub.18 is selected from FD and
YD.
[0186] In one embodiment, X.sub.17X.sub.18 is FD.
[0187] In a more specific embodiment defining a sub-class of the
FcRn binding polypeptide, the sequence fulfills at least three of
the six conditions I-VI: [0188] I. X.sub.6 is selected from A, G, K
and S, such as in particular A; [0189] II. X.sub.7 is H, [0190]
III. X.sub.17 is selected from F and Y, such as in particular F;
[0191] IV. X.sub.18 is D; [0192] V. X.sub.21 is selected from V and
W, such as in particular V; [0193] VI. X.sub.25 is selected from H
and R, such as in particular H.
[0194] In some examples of an FcRn binding polypeptide according to
the first aspect, said sequence fulfills at least four of the six
conditions I-VI. More specifically, the sequence may fulfill at
least five of the six conditions I-VI, such as all of the six
conditions I-VI.
[0195] As described in detail in the experimental section to
follow, the selection of FcRn binding polypeptide variants has led
to the identification of a number of individual FcRn binding motif
(BM) sequences. These sequences constitute individual embodiments
according to this aspect. The sequences of individual FcRn binding
motifs are presented in FIGS. 1A-1MM and as SEQ ID NO:1-353. Hence,
in one embodiment of the FcRn binding polypeptide according to this
aspect, the sequence is selected from the group consisting of SEQ
ID NO:1-353. In one embodiment, the sequence is selected from the
group consisting of SEQ ID NO:1-15, SEQ ID NO:17-140 and SEQ ID
NO:353. In one embodiment, the sequence is selected from the group
consisting of SEQ ID NO:1-2 and SEQ ID NO:17-140. In one
embodiment, the sequence is selected from the group consisting of
SEQ ID NO:1-2, SEQ ID NO:17-92, SEQ ID NO:94-103, SEQ ID NO:105-125
and SEQ ID NO:127-140. In one embodiment, the sequence is selected
from the group consisting of SEQ ID NO:1-8, SEQ ID NO:13 SEQ ID
NO:19-20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44,
SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:75-77 and SEQ
ID NO:353. In another embodiment, the sequence is selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:23, SEQ ID NO:28, SEQ ID
NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:73 and SEQ ID
NO:75-77. In yet another embodiment, the sequence is selected from
SEQ ID NO:1, SEQ ID NO:23, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75
and SEQ ID NO:77. In one embodiment, the sequence is selected from
SEQ ID NO:1, SEQ ID NO:23 and SEQ ID NO:75. In one embodiment, the
sequence is SEQ ID NO:1.
[0196] In some embodiments of the present disclosure, the BM as
defined above "forms part of" a three-helix bundle protein domain.
This is understood to mean that the sequence of the BM is
"inserted" into or "grafted" onto the sequence of the original
three-helix bundle domain, such that the BM replaces a similar
structural motif in the original domain. For example, without
wishing to be bound by theory, the BM is thought to constitute two
of the three helices of a three-helix bundle, and can therefore
replace such a two-helix motif within any three-helix bundle. As
the skilled person will realize, the replacement of two helices of
the three-helix bundle domain by the two BM helices has to be
performed so as not to affect the basic structure of the
polypeptide. That is, the overall folding of the Ca backbone of the
polypeptide according to this embodiment of the invention is
substantially the same as that of the three-helix bundle protein
domain of which it forms a part, e.g. having the same elements of
secondary structure in the same order etc. Thus, a BM according to
the disclosure "forms part" of a three-helix bundle domain if the
polypeptide according to this embodiment of the aspect has the same
fold as the original domain, implying that the basic structural
properties are shared, those properties e.g. resulting in similar
CD spectra. The skilled person is aware of other parameters that
are relevant.
[0197] In particular embodiments, the FcRn binding motif (BM) thus
forms part of a three-helix bundle protein domain. For example, the
BM may essentially constitute two alpha helices with an
interconnecting loop, within said three-helix bundle protein
domain. In particular embodiments, said three-helix bundle protein
domain is selected from domains of bacterial receptor proteins.
Non-limiting examples of such domains are the five different
three-helical domains of Protein A from Staphylococcus aureus, such
as domain B, and derivatives thereof. In some embodiments, the
three-helical bundle protein domain is a variant of protein Z,
which is derived from domain B of staphylococcal Protein A.
[0198] In embodiments where the FcRn binding polypeptide of the
invention forms part of a three-helix bundle protein domain, the
FcRn binding polypeptide may comprise an amino acid sequence
selected from:
TABLE-US-00006 iii) (SEQ ID NO: 1077) K-[BM]-DPSQS
X.sub.aX.sub.bLLX.sub.c EAKKL X.sub.dX.sub.eX.sub.fQ;
wherein
[0199] [BM] is an FcRn binding motif as defined herein, provided
that X.sub.29 is D;
[0200] X.sub.a is selected from A and S,
[0201] X.sub.b is selected from N and E;
[0202] X.sub.c is selected from A, S and C;
[0203] X.sub.d is selected from E, N and S,
[0204] X.sub.e is selected from D, E and S,
[0205] X.sub.f is selected from A and S,
and [0206] iv) an amino acid sequence which has at least 93%
identity to a sequence defined by iii).
[0207] In embodiments where the FcRn binding polypeptide of the
invention forms part of a three-helix bundle protein domain, the
FcRn binding polypeptide may comprise an amino acid sequence
selected from:
TABLE-US-00007 v) (SEQ ID NO: 1080) K-[BM]-QPEQS
X.sub.aX.sub.bLLX.sub.c EAKKL X.sub.dX.sub.eX.sub.fQ;
wherein
[0208] [BM] is an FcRn binding motif as defined herein, provided
that X.sub.29 is R;
[0209] X.sub.a is selected from A and S,
[0210] X.sub.b is selected from N and E;
[0211] X.sub.c is selected from A, S and C;
[0212] X.sub.d is selected from E, N and S,
[0213] X.sub.e is selected from D, E and S,
[0214] X.sub.f is selected from A and S, and [0215] vi) an amino
acid sequence which has at least 93% identity to a sequence defined
by v).
[0216] As discussed above, polypeptides comprising minor changes as
compared to the above amino acid sequences which do not largely
affect the tertiary structure and the function thereof are also
within the scope of the present disclosure. Thus, in some
embodiments, sequence iv) or sequence vi) has at least 95%, for
example at least 97% identity to a sequence defined by iii) and v),
respectively.
[0217] In one embodiment, X.sub.a in sequence iii) or v) is A. In
an alternative embodiment, X.sub.a in sequence iii) or v) is S.
[0218] In one embodiment, X.sub.b in sequence iii) or v) is N. In
an alternative embodiment, X.sub.b in sequence iii) or v) is E.
[0219] In one embodiment, X.sub.c in sequence iii) or v) is A. In
an alternative embodiment, X.sub.6 in sequence iii) or v) is S. In
yet another alternative embodiment, X.sub.c in sequence iii) or v)
is C.
[0220] In one embodiment, X.sub.d in sequence iii) or v) is E.
[0221] In one embodiment, X.sub.d in sequence iii) or v) is N.
[0222] In one embodiment, X.sub.d in sequence iii) or v) is S.
[0223] In one embodiment, X.sub.e in sequence iii) or v) is D.
[0224] In one embodiment, X.sub.6 in sequence iii) or v) is E.
[0225] In one embodiment, X.sub.e in sequence iii) or v) is S.
[0226] In one embodiment, X.sub.dX.sub.e in sequence iii) or v) is
selected from EE, ES, SE and SS.
[0227] In one embodiment, X.sub.dX.sub.e in sequence iii) or v) is
ES.
[0228] In one embodiment, X.sub.dX.sub.e in sequence iii) or v) is
SE.
[0229] In one embodiment, X.sub.f in sequence iii) or v) is A. In
an alternative embodiment, X.sub.f in sequence iii) or v) is S.
[0230] In one embodiment, in sequence iii) or v), X.sub.a is A;
X.sub.b is N; X.sub.c is A and X.sub.f is A.
[0231] In one embodiment, in sequence iii) or v), X.sub.a is A;
X.sub.b is N; X.sub.c is C and X.sub.f is A.
[0232] In one embodiment, in sequence iii) or v), X.sub.a is S,
X.sub.b is E; X.sub.c is S and X.sub.f is S.
[0233] In one embodiment, in sequence iii) or v), X.sub.a is S,
X.sub.b is E; X.sub.c is C and X.sub.f is S.
[0234] In one embodiment, in sequence iii) or v), X.sub.a is A;
X.sub.b is N; X.sub.c is A; X.sub.dX.sub.e is ND and X.sub.f is
A.
[0235] In one embodiment, in sequence iii) or v), X.sub.a is A;
X.sub.b is N; X.sub.c is C; X.sub.dX.sub.e is ND and X.sub.f is
A.
[0236] In one embodiment, in sequence iii) or v), X.sub.a is S,
X.sub.b is E; X.sub.c is S, X.sub.dX.sub.e is ND and X.sub.f is
S.
[0237] In one embodiment, in sequence iii) or v), X.sub.a is S,
X.sub.b is E; X.sub.c is C; X.sub.dX.sub.e is ND and X.sub.f is
S.
[0238] In one embodiment, in sequence iii) or v), X.sub.a is A;
X.sub.b is N; X.sub.c is A; X.sub.dX.sub.e is SE and X.sub.f is
A.
[0239] In one embodiment, in sequence iii) or v), X.sub.a is A;
X.sub.b is N; X.sub.c is C; X.sub.dX.sub.e is SE and X.sub.f is
A.
[0240] In one embodiment, in sequence iii) or v), X.sub.a is S,
X.sub.b is E; X.sub.c is S, X.sub.dX.sub.e is SE and X.sub.f is
S.
[0241] In one embodiment, in sequence iii) or v), X.sub.a is S,
X.sub.b is E; X.sub.c is C; X.sub.dX.sub.e is SE and X.sub.f is
S.
[0242] In one embodiment, in sequence iii) or v), X.sub.a is A;
X.sub.b is N; X.sub.c is A; X.sub.dX.sub.e is ES and X.sub.f is
A.
[0243] In one embodiment, in sequence iii) or v), X.sub.a is A;
X.sub.b is N; X.sub.c is C; X.sub.dX.sub.e is ES and X.sub.f is
A.
[0244] In one embodiment, in sequence iii) or v), X.sub.a is S,
X.sub.b is E; X.sub.c is S, X.sub.dX.sub.e is ES and X.sub.f is
S.
[0245] In one embodiment, in sequence iii) or v), X.sub.a is S,
X.sub.b is E; X.sub.c is C; X.sub.dX.sub.e is ES and X.sub.f is
S.
[0246] In yet a further embodiment, sequence iii) in the definition
of FcRn binding polypeptides above is selected from the group
consisting of SEQ ID NO:354-706. In one embodiment, sequence iii)
is selected from the group consisting of SEQ ID NO:354-368, SEQ ID
NO:370-493 and SEQ ID NO:706. In one embodiment, sequence iii) is
selected from the group consisting of SEQ ID NO:354-355 and SEQ ID
NO:370-493. In one embodiment, sequence iii) is selected from the
group consisting of SEQ ID NO:354-355, SEQ ID NO:370-445, SEQ ID
NO:447-456, SEQ ID NO:458-478 and SEQ ID NO:480-493. In one
embodiment, sequence iii) is selected from the group consisting of
SEQ ID NO:354-361, SEQ ID NO:366, SEQ ID NO:372-373, SEQ ID NO:376,
SEQ ID NO:381, SEQ ID NO:394, SEQ ID NO:397, SEQ ID NO:418, SEQ ID
NO:423, SEQ ID NO:426, SEQ ID NO:428-430 and SEQ ID NO:706. In
another embodiment, sequence iii) is selected from the group
consisting of SEQ ID NO:354, SEQ ID NO:376, SEQ ID NO:381, SEQ ID
NO:394, SEQ ID NO:397, SEQ ID NO:418, SEQ ID NO:426 and SEQ ID
NO:428-430. In yet another embodiment, sequence iii) is selected
from SEQ ID NO:354, SEQ ID NO:376, SEQ ID NO:397, SEQ ID NO:418,
SEQ ID NO:428 and SEQ ID NO:430. In one embodiment, sequence iii)
is selected from SEQ ID NO:354, SEQ ID NO:376 and SEQ ID NO:428. In
one embodiment, sequence iii) is SEQ ID NO:354.
[0247] Also, in a further embodiment, there is provided an FcRn
binding polypeptide as defined above, which comprises an amino acid
sequence selected from:
TABLE-US-00008 vii) (SEQ ID NO: 1081) YAK-[BM]-DPSQS SELLX.sub.c
EAKKL NDSQA P;
wherein [BM] is an FcRn binding motif as defined above and X.sub.c
is selected from A, S and C; and [0248] viii) an amino acid
sequence which has at least 94% identity to a sequence defined by
vii).
[0249] Alternatively, there is provided an FcRn binding polypeptide
as defined above, which comprises an amino acid sequence selected
from:
TABLE-US-00009 ix) (SEQ ID NO: 1082) FNK-[BM]-DPSQS ANLLX.sub.c
EAKKL NDAQA P;
wherein [BM] is an FcRn binding motif as defined above and X.sub.c
is selected from A and C; and [0250] x) an amino acid sequence
which has at least 94% identity to a sequence defined by ix).
[0251] As discussed above, polypeptides comprising minor changes as
compared to the above amino acid sequences that do not largely
affect the tertiary structure and the function thereof are also
within the scope of the present disclosure. Thus, in some
embodiments, the FcRn binding polypeptide as defined above may
comprise a sequence which is at least 96%, such as at least 98%
identical to a sequence defined by vii) or ix).
[0252] In some embodiments, the FcRn binding motif may form part of
a polypeptide comprising an amino acid sequence selected from
TABLE-US-00010 (SEQ ID NO: 1083)
ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK; (SEQ ID NO: 1084)
ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 1085)
ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK; (SEQ ID NO: 1086)
ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK; (SEQ ID NO: 1087)
AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK; (SEQ ID NO: 1088)
VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 1089)
AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK; (SEQ ID NO: 1090)
VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; (SEQ ID NO: 1091)
VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK; (SEQ ID NO: 1092)
AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 1093)
AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 1094)
AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; (SEQ ID NO: 1095)
VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 1096)
VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 1097)
VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; (SEQ ID NO: 1098)
VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and (SEQ ID NO: 1099)
AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK;
wherein [BM] is an FcRn binding motif as defined above.
[0253] In one embodiment, the FcRn binding polypeptide comprises an
amino acid sequence selected from:
TABLE-US-00011 xi) (SEQ ID NO: 1078)
AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is an FcRn binding motif as defined above; and [0254]
xii) an amino acid sequence which has at least 94% identity to the
sequence defined in xi).
[0255] In one embodiment, sequence xi) is selected from the group
consisting of SEQ ID NO:1060-1062.
[0256] In one embodiment, the FcRn binding polypeptide comprises an
amino acid sequence selected from:
TABLE-US-00012 xiii) (SEQ ID NO: 1079)
VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is an FcRn binding motif as defined above; and [0257]
xiv) an amino acid sequence which has at least 94% identity to the
sequence defined in xiii).
[0258] Sequence xiii) in such a polypeptide may for example be
selected from the group consisting of SEQ ID NO:707-1059. In one
embodiment, sequence xiii) is selected from the group consisting of
SEQ ID NO:707-721, SEQ ID NO:723-846 and SEQ ID NO:1059. In one
embodiment, sequence xiii) is selected from the group consisting of
SEQ ID NO:707-708 and SEQ ID NO:723-846. In one embodiment,
sequence xiii) is selected from the group consisting of SEQ ID
NO:707-708, SEQ ID NO:723-798, SEQ ID NO:800-809, SEQ ID NO:811-831
and SEQ ID NO:833-846. In one embodiment, sequence xiii) is
selected from the group consisting of SEQ ID NO:707-714, SEQ ID
NO:719, SEQ ID NO:725-726, SEQ ID NO:729, SEQ ID NO:734, SEQ ID
NO:747, SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:776, SEQ ID NO:779,
SEQ ID NO:781-783 and SEQ ID NO:1059. In another embodiment,
sequence xiii) is selected from the group consisting of SEQ ID
NO:707, SEQ ID NO:729, SEQ ID NO:734, SEQ ID NO:747, SEQ ID NO:750,
SEQ ID NO:771, SEQ ID NO:779 and SEQ ID NO:781-783. In yet another
embodiment, sequence xiii) is selected from SEQ ID NO:707, SEQ ID
NO:729, SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:781 and SEQ ID
NO:783. In one embodiment, sequence xiii) is selected from SEQ ID
NO:707, SEQ ID NO:729 and SEQ ID NO:781. In one embodiment,
sequence xiii) is SEQ ID NO:707.
[0259] Again, polypeptides comprising minor changes as compared to
the above amino acid sequences which do not largely affect the
tertiary structure and the function thereof are also within the
scope of the present disclosure. Thus, in some embodiments, the
FcRn binding polypeptide as defined above may comprise a sequence
which is at least 96%, such as at least 98% identical to a sequence
defined by xi) or xiii).
[0260] The terms "FcRn binding" and "binding affinity for FcRn" as
used in this specification refer to a property of a polypeptide
which may be tested for example by the use of surface plasmon
resonance (SPR) technology or ELISA.
[0261] For example as described in the examples below, FcRn binding
affinity may be tested in an experiment in which FcRn, or a
correctly folded fragment thereof, is immobilized on a sensor chip
of the instrument, and the sample containing the polypeptide to be
tested is passed over the chip. Alternatively, the polypeptide to
be tested is immobilized on a sensor chip of the instrument, and a
sample containing FcRn, or a correctly folded fragment thereof, is
passed over the chip. The skilled person may then interpret the
results obtained by such experiments to establish at least a
qualitative measure of the binding affinity of the polypeptide for
FcRn. If a quantitative measure is desired, for example to
determine a K.sub.D value for the interaction, surface plasmon
resonance methods may also be used. Binding values may for example
be defined in a Biacore (GE Healthcare) or ProteOn XPR 36 (Bio-Rad)
instrument. FcRn is suitably immobilized on a sensor chip of the
instrument, and samples of the polypeptide whose affinity is to be
determined are prepared by serial dilution and injected in random
order. K.sub.D values may then be calculated from the results using
for example the 1:1 Langmuir binding model of the BIAevaluation 4.1
software, or other suitable software, provided by the instrument
manufacturer.
[0262] Alternatively, as described in the examples below, FcRn
binding affinity may be tested in an experiment in which samples of
the polypeptide are captured on antibody coated ELISA plates, and
biotinylated FcRn is added followed by streptavidin conjugated HRP.
TMB substrate is added and the absorbance at 450 nm is measured
using a multi-well plate reader, such as Victor.sup.3 (Perkin
Elmer). The skilled person may then interpret the results obtained
by such experiments to establish at least a qualitative measure of
the binding affinity of the polypeptide for FcRn. If a quantitative
measure is desired, for example to determine the K.sub.D value (the
half maximal effective concentration) for the interaction, ELISA
may also be used. The response of the polypeptides against a
dilution series of biotinylated FcRn are measured using ELISA as
described above. The skilled person may then interpret the results
obtained by such experiments and K.sub.D values may be calculated
from the results using for example GraphPad Prism 5 and non-linear
regression.
[0263] In one embodiment, there is provided an FcRn binding
polypeptide, which is capable of binding to FcRn at pH 6.0 such
that the K.sub.D value of the interaction is at most
1.times.10.sup.-6 M, such as at most 1.times.10.sup.-7 M, such as
at most 1.times.10.sup.-8 M, such as at most 1.times.10.sup.-9 M,
such as at most 1.times.10.sup.-10 M. An FcRn binding polypeptide
according to this embodiment would bind, or remain bound, to FcRn
in acidic pH conditions, such as pH 6.0, for example in a lysosome.
If such a polypeptide were to enter an increasingly acidic
intracellular environment, it would be recycled to the plasma
membrane through its interaction with FcRn, and thus avoid
degradation.
[0264] In one embodiment, the K.sub.D value of the interaction
between FcRn binding polypeptide and FcRn at pH 7.4 is higher than
the K.sub.D value of said interaction at pH 6.0. Thus, the FcRn
binding polypeptide would bind to FcRn with higher affinity at pH
6.0 than at pH 7.4. In one embodiment, the K.sub.D value of said
interaction at pH 7.4 is at least 2 times higher, such as at least
5 times higher, such as at least 10 times higher, such as at least
50 times higher, such as at least 100 times higher than the K.sub.D
value of said interaction at pH 6.0.
[0265] In one embodiment, the K.sub.D value of the interaction
between FcRn binding polypeptide and FcRn at pH 7.4 is at least
1.times.10.sup.-8 M, such as at least 1.times.10.sup.-7 M, such as
at least 1.times.10.sup.-6 M, such as at least 1.times.10.sup.-5 M.
In some embodiments, the only criterion for the interaction between
FcRn binding polypeptide and FcRn at pH 7.4 is that any FcRn
binding polypeptide which has bound to FcRn during more acidic
conditions is released more rapidly from FcRn when the pH value
increases.
[0266] In an alternative embodiment, there is provided an FcRn
binding polypeptide, for which the K.sub.D of said interaction at
pH 7.4 is the same as or lower than the K.sub.D of said interaction
at pH 6.0. An FcRn binding polypeptide according to this embodiment
would bind or remain bound to FcRn in acidic pH conditions (i.e.
would have an off-rate at pH 6.0 which is sufficiently slow to
avoid release), for example in the lysosome, as well as in neutral
or slightly basic pH conditions, for example on the plasma
membrane. In a more specific embodiment, the K.sub.D value of said
interaction at pH 7.4 is at least 2 times lower, such as at least 5
times lower, such as at least 10 times lower, such as at least 50
times lower, such as at least 100 times lower than the K.sub.D
value of said interaction at pH 6.0.
[0267] In another embodiment, there is provided an FcRn binding
polypeptide, which is capable of binding to FcRn at pH 7.4 such
that the K.sub.D value of the interaction is at most
1.times.10.sup.-6 M, such as at most 1.times.10.sup.-7 M, such as
at most 1.times.10.sup.-8 M, such as at most 1.times.10.sup.-9 M,
such as at most 1.times.10.sup.-10 M. An FcRn binding polypeptide
according to this embodiment would bind or remain bound for an
extended time to FcRn in neutral or slightly basic pH conditions,
such as pH 7.4, for example on the plasma membrane. The term
"remain bound" should be understood to mean an interaction having a
slow off-rate at given conditions.
[0268] In general, the skilled person knows that the K.sub.D value
of an interaction is defined as the ratio between the off-rate
(k.sub.off) and the on-rate (k.sub.on). Thus, a high K.sub.D value
may be due to either a high k.sub.off, a low k.sub.on or both, and
conversely, a low K.sub.D value may be due to either a low
k.sub.off, a high k.sub.on or both.
[0269] The skilled person will understand that various
modifications and/or additions can be made to an FcRn binding
polypeptide according to any aspect disclosed herein in order to
tailor the polypeptide to a specific application without departing
from the scope of the present disclosure.
[0270] For example, in one embodiment there is provided an FcRn
binding polypeptide as described herein, which polypeptide has been
extended by one or more amino acids at the C terminal and/or N
terminal end. Such a polypeptide should be understood as a
polypeptide having one or more additional amino acid residues at
the very first and/or the very last position in the polypeptide
chain. Thus, an FcRn binding polypeptide may comprise any suitable
number of additional amino acid residues, for example at least one
additional amino acid residue. Each additional amino acid residue
may individually or collectively be added in order to, for example,
improve production, purification, stabilization in vivo or in
vitro, coupling, or detection of the polypeptide. Such additional
amino acid residues may comprise one or more amino acid residues
added for the purpose of chemical coupling. One example of this is
the addition of a cysteine residue. Such additional amino acid
residues may also provide a "tag" for purification or detection of
the polypeptide, such as a His.sub.6 tag or a "myc" (c-myc) tag or
a "FLAG" tag for interaction with antibodies specific to the tag or
immobilized metal affinity chromatography (IMAC) in the case of the
hexahistidine tag.
[0271] The further amino acids as discussed above may be coupled to
the FcRn binding polypeptide by means of chemical conjugation
(using known organic chemistry methods) or by any other means, such
as expression of the FcRn binding polypeptide as a fusion protein
or joined in any other fashion, either directly or via a linker,
for example an amino acid linker.
[0272] The further amino acids as discussed above may for example
comprise one or more polypeptide domain(s). A further polypeptide
domain may provide the FcRn binding polypeptide with another
function, such as for example another binding function, or an
enzymatic function, or a toxic function or a fluorescent signaling
function, or combinations thereof.
[0273] A further polypeptide domain may moreover provide another
FcRn binding moiety with the same FcRn binding function. Thus, in a
further embodiment, there is provided an FcRn binding polypeptide
in a multimeric form. Said multimer is understood to comprise at
least two FcRn binding polypeptides as disclosed herein as monomer
units, the amino acid sequences of which may be the same or
different. Multimeric forms of the polypeptides may comprise a
suitable number of domains, each having an FcRn binding motif, and
each forming a monomer within the multimer. These domains may have
the same amino acid sequence, but alternatively, they may have
different amino acid sequences. In other words, the FcRn binding
polypeptide of the invention may form homo- or heteromultimers, for
example homo- or heterodimers. In one embodiment, there is provided
an FcRn binding polypeptide, wherein said monomeric units are
covalently coupled together. In another embodiment, said FcRn
binding polypeptide monomer units are expressed as a fusion
protein. In one embodiment, there is provided an FcRn binding
polypeptide in dimeric form.
[0274] Additionally, "heterogenic" fusion polypeptides or proteins,
or conjugates, in which an FcRn binding polypeptide described
herein, or multimer thereof, constitutes a first domain, or first
moiety, and the second and further moieties have other functions
than binding FcRn, are also contemplated and fall within the ambit
of the present disclosure. The second and further moiety/moieties
of the fusion polypeptide or conjugate in such a protein suitably
have a desired biological activity.
[0275] Thus, in a second aspect of the present disclosure, there is
provided a fusion protein or a conjugate, comprising a first moiety
consisting of an FcRn binding polypeptide according to the first
aspect, and a second moiety consisting of a polypeptide having a
desired biological activity. In another embodiment, said fusion
protein or conjugate may additionally comprise further moieties,
comprising desired biological activities that can be either the
same or different from the biological activity of the second
moiety.
[0276] Such heterogenic fusion polypeptides could also be used to
create heteromultimeric complexes of higher order. One example is a
heterodimeric complex of two fusion polypeptides, each comprising
an FcRn binding polypeptide according to the present disclosure in
fusion with another moiety. Such a complex could for example form a
heterodimer in vivo or in vitro and be held together by
non-covalent and/or covalent interactions. A specific example of
such a complex is a Fab fragment, in which both the light chain and
heavy chain are produced in fusion with one FcRn binding
polypeptide each, and which may include an inter-domain disulphide
bond. Many biologically relevant, heterodimeric complexes known to
the skilled person may be constructed using FcRn binding fusion
proteins as monomer units.
[0277] In one embodiment of said fusion protein or conjugate, the
total size of the molecule is below the threshold for efficient
renal clearance upon administration to a mammalian subject.
[0278] In another embodiment of said fusion protein or conjugate,
the total size of the molecule is above the threshold for efficient
renal clearance upon administration to a mammalian subject.
[0279] In one embodiment, there is provided a fusion protein or
conjugate, wherein the in vivo half-life of said fusion protein or
conjugate is longer than the in vivo half-life of the polypeptide
having the desired biological activity per se.
[0280] Non-limiting examples of a desired biological activity
comprise a therapeutic activity, a binding activity, and an
enzymatic activity.
[0281] In one embodiment, said desired biological activity is a
binding activity to a selected target.
[0282] One example of such a binding activity is a binding
activity, which increases the in vivo half-life of a fusion protein
or conjugate. This fusion protein or conjugate may comprise at
least one further moiety. In one particular embodiment, said target
is albumin, binding to which increases the in vivo half-life of
said fusion protein or conjugate. In one embodiment, said albumin
binding activity is provided by an albumin binding domain (ABD) of
streptococcal protein G or a derivative thereof. For example, said
fusion protein or conjugate, comprising at least one further
moiety, may comprise [FcRn binding polypeptide moiety]-[albumin
binding moiety]-[moiety with affinity for selected target]. It is
to be understood that the three moieties in this example may be
arranged in any order from the N- to the C-terminal of the
polypeptide.
[0283] In one embodiment, when a complex between a target and the
fusion protein or conjugate as described herein is formed (or
maintained) at acidic pH, such as pH 6.0, the target is rescued
from elimination by lysosomal degradation. Thus, target half-life
is extended. Half-life extension implies that the elimination rate
of a target is lower when interacting with said fusion protein or
conjugate than the elimination rate of the target molecule in the
absence of said fusion protein or conjugate. Furthermore, it is
desirable in this embodiment that the binding of target by the
fusion protein or conjugate should not interfere substantially with
the function of the target.
[0284] On the other hand, when a complex between the target and the
fusion protein or conjugate as described herein is not maintained
or not formed at acidic pH, the target is directed to the
subcellular lysosomes where it is degraded.
[0285] In one embodiment, there is provided a fusion protein or
conjugate, wherein the rate of elimination of a selected,
undesirable target from the subject is increased. Increased
elimination of an undesirable target implies increased elimination
rate of the target from the body of the multicellular organism, as
compared to a "normal" elimination rate of the target molecule per
se, i.e. without previous interaction with the fusion protein or
conjugate.
[0286] In another embodiment, binding of a selected undesirable
target could inactivate the function of the target, thereby
blocking its biological activity in situations where this is
desirable. Such biological activity may for example be activation
or blocking of receptors or an enzymatic or otherwise toxic or
undesirable activity. Such undesirable target may be an endogenous
hormone, enzyme, cytokine, chemokine or a target having some other
biological activity. By using an inactivating target binding, the
biological activity is blocked until the target is delivered for
degradation and released at a low pH value, and the target binding
fusion protein is recycled to circulation. This recycling of the
target binding fusion protein (via its FcRn binding moiety) enables
it to "catalyze" the removal of more than one molecule of the
selected undesirable target.
[0287] Undesirable targets may for example be foreign proteins and
compounds, or naturally expressed proteins that display elevated
levels in plasma following a medical condition and where a
therapeutic effect may be achieved by elimination of said protein.
The undesired target is not necessarily evenly distributed in the
plasma but may be concentrated in certain regions, for example
around a tumor or at sites of inflammation.
[0288] Non-limiting examples of targets are targets selected from
the group consisting of allergens, amyloids, antibodies,
auto-antigens, blood clotting factors, hormones, tumor cells, drug
molecules, cytokines, chemokines, proteases, hypersensitivity
mediators, proinflammatory factors, toxins such as bacterial toxins
and snake venoms; pollutants, metals and anti-oxidants.
[0289] Under certain conditions, such as in certain cancer
diseases, it is desired to remove endogenous molecules, for example
VEGF, PDGF, HGF and other growth stimulatory hormones. Such
molecules could also be targeted by a binding function in said
fusion protein or conjugate.
[0290] Under other conditions, such as in certain immunological
diseases, it may be desirable to remove endogenous molecules
transiently, such as selected interleukines or TNF. Such molecules
could also be targeted by a binding function in said fusion protein
or conjugate.
[0291] In one embodiment, the second moiety having a desired
biological activity is a therapeutically active polypeptide.
Non-limiting examples of therapeutically active polypeptides are
biomolecules, such as molecules selected from the group consisting
of enzymes, for example algasidase a and .beta.,
glucocerebrosidase, laronidase, arylsulphatase,
aglucosidase-.alpha., asparaginase, Factor VII, Factor VIII, Factor
IX and Factor Xa, hormones and growth factors, for example growth
hormone, transforming growth factor-.beta.2, erythropoietin,
insulin, insulin-like growth factor-1, myostatin, bone-derived
growth factor and glucagon-like peptide-1; chemokines, for example
CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1 and CXC3CL1, and
cytokines, for example interleukin (IL)-2, IL-4, IL-7, IL-10,
IL-12, IL-15, IL-18, IL-22, IL-27, interferon (IFN)-.alpha.,
IFN-.beta., IFN-.gamma., tumor necrosis factor, granulocyte-colony
stimulating factor (G-CSF), macrophage-CSF, and
granulocyte/macrophage-CSF.
[0292] As the skilled person understands, the FcRn binding
polypeptide according to the first aspect may be useful in a fusion
protein or as a conjugate partner to any other moiety. Therefore,
the above lists of therapeutically active polypeptides should not
be construed as limiting in any way.
[0293] Other possibilities for the creation of fusion polypeptides
or conjugates are also contemplated. Thus, an FcRn binding
polypeptide according to the first aspect of the invention may be
covalently coupled to a second or further moiety or moieties, which
in addition to or instead of target binding exhibit other
functions. One example is a fusion between one or more FcRn binding
polypeptide(s) and an enzymatically active polypeptide serving as a
reporter or effector moiety.
[0294] With regard to the description above of fusion proteins or
conjugates incorporating an FcRn binding polypeptide according to
the disclosure, it is to be noted that the designation of first,
second and further moieties is made for clarity reasons to
distinguish between FcRn binding polypeptide or polypeptides
according to the disclosure on the one hand, and moieties
exhibiting other functions on the other hand. These designations
are not intended to refer to the actual order of the different
domains in the polypeptide chain of the fusion protein or
conjugate. Thus, for example, said first moiety may without
restriction appear at the N-terminal end, in the middle, or at the
C-terminal end of the fusion protein or conjugate.
[0295] In one embodiment, there is provided an FcRn binding
polypeptide, fusion protein or conjugate, which binds to FcRn such
that binding of IgG to FcRn is at least partially inhibited. This
inhibition may be due to binding of the FcRn binding polypeptide,
fusion protein or conjugate to the same, or an at least partially
overlapping, region of FcRn as IgG. Alternatively, the FcRn binding
polypeptide, fusion protein or conjugate may bind to a different
region of FcRn than IgG but sterically hinder the binding of IgG to
FcRn. Thus, the rate of elimination or clearance of IgG from the
circulatory system would increase due to increased lysosomal
degradation of IgG, because the FcRn mediated recycling of IgG
would be wholly or partially unavailable due to the occupation of
FcRn binding sites by the FcRn binding polypeptide according to the
present disclosure. In other words, administration of FcRn binding
polypeptide, fusion protein or conjugate or composition according
to the present disclosure will act to increase the catabolism of
circulating IgG antibodies.
[0296] In one embodiment, the K.sub.D value of the interaction
between the FcRn binding polypeptide, fusion protein or conjugate
and FcRn is lower than the K.sub.D of the interaction between IgG
and FcRn. This relationship may be true at both pH 6.0 and pH 7.4,
or at pH 6.0 only.
[0297] The above aspects furthermore encompass polypeptides in
which the FcRn binding polypeptide according to the first aspect,
or the FcRn binding polypeptide as comprised in a fusion protein or
conjugate according to the second aspect, further comprises a
label, such as a label selected from the group consisting of
fluorescent dyes and metals, chromophoric dyes, chemiluminescent
compounds and bioluminescent proteins, enzymes, radionuclides and
particles. Such labels may for example be used for detection of the
polypeptide.
[0298] In other embodiments, the labeled FcRn binding polypeptide
is present as a moiety in a fusion protein or conjugate also
comprising a second moiety having a desired biological activity
and/or comprising a binding function as described above. The label
may in some instances be coupled only to the FcRn binding
polypeptide, and in some instances both to the FcRn binding
polypeptide and to the second moiety of the conjugate or fusion
protein. Furthermore, it is also possible that the label may be
coupled to a second moiety only and not to the FcRn binding moiety.
Hence, in yet another embodiment there is provided an FcRn binding
polypeptide comprising a second moiety, wherein said label is
coupled to the second moiety only.
[0299] When reference is made to a labeled polypeptide, this should
be understood as a reference to all aspects of polypeptides as
described herein, including fusion proteins and conjugates
comprising an FcRn binding polypeptide and a second and optionally
further moieties. Thus, a labeled polypeptide may contain only the
FcRn binding polypeptide and e.g. a therapeutic radionuclide, which
may be chelated or covalently coupled to the FcRn binding
polypeptide, or contain the FcRn binding polypeptide, a therapeutic
radionuclide and a second moiety such as a small molecule having a
desired biological activity, for example resulting in a therapeutic
efficacy.
[0300] In embodiments where the FcRn binding polypeptide, fusion
protein or conjugate is radiolabeled, such a radiolabeled
polypeptide may comprise a radionuclide. A majority of
radionuclides have a metallic nature, are used in the ionic form,
and are typically incapable of forming stable covalent bonds with
elements presented in proteins and peptides. For this reason,
labeling of proteins and peptides with radioactive metals is
performed with the use of chelators, i.e. multidentate ligands,
which form non-covalent compounds, called chelates, with the metal
ions. In an embodiment of the FcRn binding polypeptide, fusion
protein or conjugate, the incorporation of a radionuclide is
enabled through the provision of a chelating environment, through
which the radionuclide may be coordinated, chelated or complexed to
the polypeptide.
[0301] One example of a chelator is the polyaminopolycarboxylate
type of chelator. Two classes of such polyaminopolycarboxylate
chelators can be distinguished: macrocyclic and acyclic
chelators.
[0302] In one embodiment, the FcRn binding polypeptide, fusion
protein or conjugate comprises a chelating environment provided by
a polyaminopolycarboxylate chelator conjugated to the FcRn binding
polypeptide via a thiol group of a cysteine residue or an epsilon
amine group of a lysine residue.
[0303] The most commonly used macrocyclic chelators for
radioisotopes of indium, gallium, yttrium, bismuth, radioactinides
and radiolanthanides are different derivatives of DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). In one
embodiment, a chelating environment of the FcRn binding
polypeptide, fusion protein or conjugate is provided by DOTA or a
derivative thereof. More specifically, in one embodiment, the
chelating polypeptides encompassed by the present disclosure are
obtained by reacting the DOTA derivative
1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic
acid-10-maleimidoethylacetamide (maleimidomonoamide-DOTA) with said
polypeptide.
[0304] Additionally, 1,4,7-triazacyclononane-1,4,7-triacetic acid
(NOTA) and derivatives thereof may be used as chelators. Hence, in
one embodiment, there is provided an FcRn binding polypeptide,
fusion protein or conjugate, wherein the polyaminopolycarboxylate
chelator is 1,4,7-triazacyclononane-1,4,7-triacetic acid or a
derivative thereof.
[0305] The most commonly used acyclic polyaminopolycarboxylate
chelators are different derivatives of DTPA
(diethylenetriamine-pentaacetic acid). Hence, polypeptides having a
chelating environment provided by diethylenetriaminepentaacetic
acid or derivatives thereof are also encompassed by the present
disclosure.
[0306] In a further embodiment, the FcRn binding polypeptide,
produced recombinantly through expression of a polynucleotide or
synthetically, is conjugated to one or more synthetic polymers, in
order for example to increase its hydrodynamic radius. Polyethylene
glycol (PEG) is commonly used for this purpose, but other polymers
have also been used in the art. Such "PEGylation" may be used to
increase the size of the FcRn binding polypeptide of any of the
types described herein to a size above the threshold for effective
renal excretion.
[0307] In one embodiment, a synthetic polymer is conjugated to one
or more chemically synthesized, monomeric FcRn binding
polypeptides. Other functionalities may also be conjugated to the
same synthetic polymer. If the FcRn binding polypeptide and other
components are chemically synthesized, none of the components will
have to be made in a biological system if this is not desired.
[0308] In a preferred embodiment, one or more synthetically or
biologically manufactured FcRn binding polypeptides are conjugated
to a synthetic polymer, to achieve a size exceeding the size
associated with efficient renal clearance and used for blocking
binding of IgG to FcRn. A unique cysteine in each FcRn binding
polypeptide may be used for site specific conjugation, for example
a C-terminally located cysteine introduced for this purpose. With a
branched synthetic polymer, more than two FcRn binding moieties may
be conjugated to the same polymer, to enhance the avidity and
therefore the blocking potency.
[0309] In a third aspect of the present disclosure, there is
provided a polynucleotide encoding an FcRn binding polypeptide or a
fusion protein as described herein. Also encompassed by this
disclosure is a method of producing a polypeptide or fusion protein
as described above comprising expressing a polynucleotide; an
expression vector comprising the polynucleotide; and a host cell
comprising the expression vector.
[0310] Also encompassed is a method of producing a polypeptide,
comprising culturing said host cell under conditions permissive of
expression of said polypeptide from its expression vector, and
isolating the polypeptide.
[0311] The FcRn binding polypeptide of the present disclosure may
alternatively be produced by non-biological peptide synthesis using
amino acids and/or amino acid derivatives having protected reactive
side-chains, the non-biological peptide synthesis comprising [0312]
step-wise coupling of the amino acids and/or the amino acid
derivatives to form a polypeptide according to the first aspect
having protected reactive side-chains, [0313] removal of the
protecting groups from the reactive side-chains of the polypeptide,
and [0314] folding of the polypeptide in aqueous solution.
[0315] In a fourth aspect of the disclosure, there is provided a
composition comprising an FcRn binding polypeptide, fusion protein
or conjugate as described herein and at least one pharmaceutically
acceptable excipient or carrier. In one embodiment thereof, said
composition further comprises at least one additional active agent,
such as at least two additional active agents, such as at least
three additional active agents. Non-limiting examples of additional
active agents that may prove useful in such a combination are
immunosuppressing agents, anti-inflammatory agents, anti-microbial
agents and enzymes.
[0316] In one embodiment of this aspect, said composition is
adapted for administration by a route selected from the group
consisting of oral administration, intranasal administration,
pulmonar administration, vaginal administration, rectal
administration, intravenous injection, intraperitoneal injection,
intramuscular injection, subcutaneous injection and intradermal
injection.
[0317] As used herein, the term "systemic administration" refers to
a route of administration such the substance of interest enters
into the circulatory system so that the entire body is affected.
The skilled person is aware that systemic administration can take
place via enteral administration (absorption of the drug through
the gastrointestinal tract) or parenteral administration (generally
injection, infusion or implantation).
[0318] In one embodiment, said composition is adapted for
administration systemically or locally. In certain embodiments,
systemic administration of said compound may be used. In another
embodiment, said composition is adapted for administration by a
local route. For example, local administration may be topical in an
ointment, paste, foam or cream. In another embodiment, said
composition is adapted for administration across an endothelial or
epithelial layer. Here, the composition may be transcytosed across
said layer.
[0319] In one embodiment, the rate of uptake of a composition
comprising a fusion protein or conjugate as described herein is
higher than the rate of uptake of polypeptides corresponding to
second or further moieties per se. In one embodiment, the rate of
uptake is at least 2 times higher, such as at least 5 times higher,
such as at least 10 times higher, such as at least 25 times higher
than the rate of uptake of the at second or further moieties per
se.
[0320] It should be understood from the above disclosure that the
FcRn binding polypeptide fusion protein or conjugate or the
composition as described herein may for example be useful as a
therapeutic agent, and/or as a means for extending the in vivo
half-life of a fusion partner, and/or as a means for increasing the
rate of elimination of undesirable targets.
[0321] Hence, in a fifth aspect of the present disclosure, there is
provided an FcRn binding polypeptide, fusion protein, conjugate or
composition as disclosed herein for use as a medicament.
[0322] In a related, sixth, aspect of the present disclosure, there
is provided a method of treatment of a subject in need thereof,
comprising the step of administrating a therapeutically active
amount of an FcRn binding polypeptide, fusion protein, conjugate or
composition as disclosed herein.
[0323] In one embodiment of any one of these two latter aspects,
the medicament or method is intended for treatment in which the
capacity of the FcRn binding polypeptide to at least partially
block binding of IgG to FcRn is exploited, i.e. treatment in which
increased catabolism of IgG antibodies is desired. In one
embodiment, a condition in which such treatement may be indicated
is an auto-immune condition. As non-limiting examples of indicated
conditions, mention is made of myasthenia gravis, Guillain-Barre
syndrome, autoimmune limbic encephalitis, pediatric autoimmune
neuropsychiatric disorders associated with streptococcal infection
(PANDAS), neuromyotonia (Isaac's syndrome), morvan syndrome,
multiple sclerosis, pemphigus vulgaris, foliaceus, bullous
pemphigoid, epidermolysis bullosa acquisita, pemphigoid
gestationis, mucous membrane pemphigoid, lichen sclerosus,
antiphospholipid syndrome, erlapsing polychondritis, autoimmune
anemia, idiopathic trombocytic purpura, autoimmune Grave's disease,
dilated cardiomyopathy, vasculitis, Goodpasture's syndrome,
idiopathic membranous nephropathy, rheumatoid arthritis and
systemic lupus erythematosus.
[0324] In another embodiment, there is provided an FcRn binding
polypeptide, fusion protein, conjugate or composition as described
herein for use in blocking or removal of an undesirable target from
the circulation. In one embodiment, said undesirable target is
selected from the group comprising allergens, amyloids, antibodies,
auto-antigens, blood clotting factors, hormones, tumor cells, drug
molecules, cytokines, chemokines, hypersensitivity mediators,
pro-inflammatory factors, toxins such as bacterial toxins and snake
venoms, pollutants, metals and anti-oxidants.
[0325] While the invention has been described with reference to
various exemplary aspects and embodiments, it will be understood by
those skilled in the art that various changes may be made and
equivalents may be substituted for elements thereof without
departing from the scope of the invention. In addition, many
modifications may be made to adapt a particular situation or
molecule to the teachings of the invention without departing from
the essential scope thereof. Therefore, it is intended that the
invention not be limited to any particular embodiment contemplated,
but that the invention will include all embodiments falling within
the scope of the appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0326] FIG. 1A-1MM is a listing of the amino acid sequences of
examples of FcRn binding motifs comprised in FcRn binding
polypeptides of the invention (SEQ ID NO:1-353), examples of 49-mer
FcRn binding polypeptides according to the disclosure (SEQ ID
NO:354-706), examples of 58-mer FcRn binding polypeptides according
to the disclosure (SEQ ID NO:707-1062) as well as the amino acid
sequences of the albumin binding polypeptide variant PP013 (SEQ ID
NO:1063), Taq polymerase binding Z variant Z03638 (SEQ ID NO:1064),
human .alpha.FcRn (SEQ ID NO:1065), murine .alpha.FcRn (SEQ ID
NO:1070), human .delta.2-microglobulin (SEQ ID NO:1066), murine
.beta.2-microglobulin (SEQ ID NO:1067), human .alpha.FcRn (SEQ ID
NO:1068) when in human FcRn-eGFP and murine .alpha.FcRn (SEQ ID
NO:1069) when in murine FcRn-eGFP.
[0327] FIGS. 2A-2E show the binding to human FcRn at pH 6.0 and
dissociations at pH 6.0 and 7.4 for His.sub.6-tagged Z variants and
for IgG as described in Example 3. Overlays of sensorgrams obtained
from a Biacore instrument representing injection at pH 6.0 followed
by dissociation at pH 6.0 (solid line) and injection at pH 6.0
followed by dissociation at pH 7.4 (dashed line) are displayed for
(A) Z07918 (SEQ ID NO:707), (B) Z07960 (SEQ ID NO:710), (C) Z10109
(SEQ ID NO:709), (D) Z10193 (SEQ ID NO:708) and (E) IgG.
[0328] FIG. 3 shows dot plots from a flow cytometry analysis of
binding of FcRn binding Z variant to human (upper panel) and mouse
(lower panel) FcRn-eGFP HeLa cells, as described in Example 4. Due
to heterogeneous expression of FcRn-eGFP by HeLa cells, cells were
gated according to FcRn-eGFP expression level. Cells in gate H are
considered to be FcRn-eGFP negative and cells in gate I are
considered to be positive. Incubation with Alexa647 labeled Z
variants resulted in a population positive both for Alexa647 and
eGFP, whereas incubation with buffer (buffer control) did not. The
figure shows that the three variants Z07960 (SEQ ID NO:710), Z07930
(SEQ ID NO:712) and Z07918 (SEQ ID NO:707) bind to human FcRn and
mouse FcRn. The y-axis shows Alexa647 intensity and the x-axis
shows eGFP activity.
[0329] FIG. 4 shows mean fluorescence intensity (MFI) values of
Alexa647 labeled Z07960 (SEQ ID NO:710), Z07930 (SEQ ID NO:712) and
Z07918 (SEQ ID NO:707), measured in the cell binding assay
described in Example 4. Diagram (A) shows MFI from HeLa cells
transduced with human FcRn-eGFP and diagram (B) shows MFI from HeLa
cells transduced with mouse FcRn-eGFP.
[0330] FIG. 5 shows dot plots from flow cytometry analysis of human
or mouse IgG Alexa647 binding to human (upper panel) and mouse
(lower panel) FcRn-eGFP HeLa cells, as described in Example 5. Due
to heterogeneous expression of FcRn-eGFP by HeLa cells, cells were
gated according to the abundance of FcRn-eGFP on the cell surface.
Cells in gate M are considered to be FcRn-eGFP negative and cells
in gate N are considered to be positive. Binding of 100 nM human or
mouse IgG-Alexa647 to FcRn transduced HeLa cells are shown in the
left panel (0 nM). The figure shows that IgG binding was blocked by
His.sub.6-tagged Z07918 (SEQ ID NO:707) in a dose dependent manner
(1, 10, 100 and 1000 nM). The y-axis shows Alexa647 intensity and
the x-axis shows eGFP activity.
[0331] FIG. 6 shows mean fluorescence intensity (MFI) values
resulting from FcRn binding of IgG Alexa647 in the presence of
different concentrations of His.sub.6-tagged Z07918 (SEQ ID NO:707)
on (A) human FcRn-eGFP transduced HeLa cells and (B) mouse
FcRn-eGFP transduced HeLa cells, as described in Example 5. The
figure shows dose dependent blocking of the IgG-FcRn binding by the
Z variant.
[0332] FIGS. 7A-7C show kinetics of binding of three Z variants to
human FcRn at pH 6.0, as described in Example 6, using a Biacore
instrument. Sensorgrams for a concentration series of (A) Z11948
(SEQ ID NO:1060), (B) Z11946 (SEQ ID NO:1061) and (C) Z11947 (SEQ
ID NO:1062), respectively, in fusion with the albumin binding
polypeptide PP013 (SEQ ID NO:1063) and the control Z variant
molecule Z03638 (SEQ ID NO:1064, not specific for FcRn), are
displayed. Curves from 640 nM (dashed line), 160 nM (dotted line)
and 40 nM (solid grey line) were subjected to kinetic analysis
using the Langmuir 1:1 binding model. Kinetic parameters and
affinities were calculated from fitted curves (solid black lines)
and are shown in Table 5.
[0333] FIG. 8 shows the pharmacokinetic profiles for three FcRn
binding Z variants fused to the albumin binding polypeptide PP013
obtained as described in Example 6. The Z variants Z11947 (SEQ ID
NO: 1062, open squares), Z11946 (SEQ ID NO:1061, open triangles)
and Z11948 (SEQ ID NO:1060, open diamonds) all displayed prolonged
half-life compared to the negative control PP013-Z03638 (open
circles).
[0334] FIG. 9 shows the blocking of human IgG to human FcRn by
His.sub.6-Z07918 (SEQ ID NO:707; black circles), IVIg (grey
squares) and SCIg (grey triangles), respectively, assayed as
described in Example 10.
[0335] FIG. 10 shows that blocking of the IgG-FcRn interactions
with FcRn specific Z variants in mice results in reduced levels of
IgG. As further described in Example 11, mice were treated with
five daily injections of Vehicle (+), the ABD fused Z variant
Z07918-PP013 (open square) and Z11948 (SEQ ID NO:1060, closed
circle). The concentration of endogenous IgG was measured by ELISA.
The concentration of IgG in individual mice at 24, 72, 120 and 168
h were related to the level at 0 h and the results are therefore
presented as percentage of IgG at 0 h.
EXAMPLES
Summary
[0336] The following Examples disclose the development of novel Z
variant molecules targeting the neonatal Fc receptor (FcRn). The Z
variants were obtained using phage display technology. The genes
encoding FcRn binding polypeptides described herein were sequenced,
and the corresponding amino acid sequences are listed in FIG.
1A-1MM, and denoted by the identifiers SEQ ID NO:707-1059. Also,
the deduced binding motifs of these selected binding variants are
listed in FIG. 1A-1MM with sequence identifiers SEQ ID
NO:1-353.
Example 1
Production of Human .alpha.FcRn and Human .beta.2-Microglobulin
(B2M)
[0337] In this Example, the extracellular domain (ECD) of human
.alpha.FcRn (SEQ ID NO:1065) in complex with human
.beta.2-microglobulin (SEQ ID NO:1066) (complex denoted FcRn) and
human .beta.2-microglobulin in non-complexed form (denoted B2M)
were produced as soluble proteins. Human FcRn and B2M produced in
this Example were used for phage selection, ELISA and Biacore
assays in Examples 2 and 3.
Materials and Methods
[0338] Construction of Plasmids Containing the Genes for Human
.alpha.FcRn and Human .beta.2-Microglobulin to be Used for
Co-Expression:
[0339] The genes encoding human .alpha.FcRn (Genbank BC008734.2)
and human .beta.2-microglobulin (B2M) (Genbank BC032589.1) were
obtained from OpenBiosystems. Using PCR overlap extension, a gene
fragment encoding amino acids 24-290 of human .alpha.FcRn
(.alpha.FcRn.sub.ECD) (SEQ ID NO:1065) was amplified to a construct
consisting of attB1-site/Kozak sequence followed by a gene
encoding: an Ig kappa chain leader sequence, hFcRn.sub.ECD, a
GS-linker and a flag tag, followed by an attB2 site. A similar
construct was made containing a gene fragment encoding amino acids
21-119 of human B2M (SEQ ID NO:1066), except that a His.sub.6 tag
replaced the flag tag. The constructs were inserted into the
plasmid pDONOR221 (Invitrogen, cat. no. 12536-017) by recombination
using the Gateway system (Invitrogen, cat. no. 11789020, GATEWAY BP
CLONASE II Enzyme mix), according to the manufacturer's
recommendations. After verification of correct sequences, the human
.alpha.FcRn.sub.ECD construct was inserted into 2K7.sub.bsd (Suter
et al. (2006) Stem Cells 24:615-623) using multi-site gateway
cloning together with the promoter-containing plasmid pENTR-CMV
(Tai et al. (2012) PLoS One 7(9):e46269), resulting in the vector
2K7.sub.bsd-CMV-hFcRn.sub.ECD. The human B2M gene construct was
similarly inserted into 2K7.sub.neo (Suter et al., supra), giving
the vector 2K7.sub.neo-CMV-hB2M.
[0340] Cell Culture, Preparation of Recombinant Lentiviral Vectors
and Gene Insertions into SKOV-3 Cell Line:
[0341] The HEK293T and SKOV-3 cell lines were obtained from ATCC.
Cells were grown at 37.degree. C. in a humidified incubator in the
presence of 5% CO.sub.2. Complete medium for the HEK293T cell line
was Dulbeccos modified eagle medium (DMEM) supplemented with 10%
fetal bovine serum (FBS), 1% Antibiotic Antimycotic Solution (AA)
and 1% MEM Non-essential Amino Acid Solution (NEAA). Complete
medium for the SKOV-3 cell line was McCoy's 5A medium supplemented
with 10% FBS and 1% AA.
[0342] The plasmids 2K7.sub.bsd-CMV-hFcRn.sub.ECD and
2K7.sub.neo-CMV-hB2M were separately co-transfected together with
VSV-G envelope and gag/pol packaging plasmid into HEK293T cells
using calcium chloride transfection (Zufferey et al. (1997) Nat
Biotechnol 15(9):871-5, Jakobsson et al. (2006) J Neurosci Res
84:58-67). HEK293 culture supernatants containing formed lentiviral
particles with human .alpha.FcRn.sub.ECD and human B2M transgenes,
respectively, were cleared from cell debris by centrifugation and
filtration. The two types of lentiviral particles were used to
sequentially transduce SKOV-3 cells. Successful double integrants
containing both the human .alpha.FcRn.sub.ECD and the B2M genes
were selected for by the addition of blasticidin (Invitrogen) and
G418 sulfate (Invitrogen) to culture medium while passaging the
cells for two weeks. The resulting, stably transduced SKOV-3 cell
line was denoted SKOV-3 hFcRn.sub.ECD/hB2M.
[0343] Expression of Recombinant Human FcRn:
[0344] SKOV-3 cells, co-expressing human .alpha.FcRn.sub.ECD and
B2M resulting in human FcRn, were expanded and 1.5.times.10.sup.7
cells were seeded in a HYPERFIask (Corning) in 560 ml complete
growth medium. After five days, when the cells had settled and
multiplied, the medium was changed to complete growth medium
without FBS. After five days, the culture was terminated and the
supernatant was collected, passed through a 45 .mu.m filter and
frozen at -80.degree. C.
[0345] Purification of Recombinant Human FcRn Using Human IgG
Chromatography:
[0346] Protein purification was carried out in an AKTA Explorer
system (GE Healthcare). Human IgG (Pharmacia), 1 ml in 0.2 M
NaHCO.sub.3, 0.5 M NaCl pH 8.3 at a concentration of 10 mg/ml, was
coupled to a 1 ml HiTrap NHS-activated HP column (GE Healthcare)
according to the manufacturer's instruction. The supernatant
containing recombinant human FcRn from SKOV-3 cells was thawed and
the pH was adjusted to 5.8 with HCl. The supernatant was
subsequently loaded in batches of 100 ml onto the column previously
equilibrated with 20 mM Bis-Tris pH 5.8. The column was washed with
20 ml of 20 mM Bis-Tris pH 5.8 and eluted in fractions of 1 ml
using 50 mM Tris, pH 8.1. Buffer exchange to PBS (phosphate
buffered saline, 10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4)
was performed using dialysis.
[0347] SDS-PAGE and Western Blot:
[0348] The purity of the eluted fractions from the protein
purification was analyzed by SDS-PAGE and staining with GelCode
Blue Stain Reagent (Pierce) and SILVERXPRESS Silver Staining Kit
(Invitrogen). Western blotting was carried out using an Amersham
HYBOND-C Extra nitrocellulose membrane (GE Healthcare). The
membrane was blocked with 5% non-fat dry milk (Semper) in TBS+T (50
mM Trizma base, 150 mM NaCl, 0.05% Tween-20, pH 8) for 1 hour, then
probed with a mixture of rabbit anti-FCGRT polyclonal antibody
(Atlas Antibodies) at a concentration of 0.15 .mu.g/ml and rabbit
anti-B2M polyclonal antibody (Atlas Antibodies) at a concentration
of 0.23 .mu.g/ml in TBS+T. The membrane was subsequently incubated
with stabilized goat anti-rabbit antibody conjugated with horse
radish peroxidase (Pierce) diluted 1:10,000 in TBS+T. After
addition of TMB Substrate (Pierce), an image of the membrane was
acquired on Amersham Hyperfilm ECL (GE Healthcare). The Hyperfilm
was processed using GBX developer and GBX fixer
(Sigma-Aldrich).
[0349] Production of a Non-Complexed Form of Human B2M:
[0350] Human B2M was produced in E. coli. The expression and
purification was performed essentially as described in Sandalova et
al. (2005) Acta Chryst F61:1090-1093 and Michaelsson et al. (2001)
J Immunol 166:7327-7334. The purified protein, consisting of amino
acids 21-119 of human B2M, in urea was subjected to arginine
refolding as follows; 0.5 mg of B2M was rapidly added to 2 ml
refolding buffer (20 ml 1 M Tris-HCl pH 8.0, 16.87 g L-Arginine
(buffered with HCl), 0.8 ml 0.5 M EDTA, 61 mg GSSG, 307 mg GSH and
milli-Q water to a final volume of 200 ml, pH 8.0, and supplemented
with protease inhibitor (Roche, cat. no. 11 873 580 001)). The
refolding procedure was performed at 4.degree. C. during 4 hours.
Refolded B2M protein was buffer exchanged to PBS using a PD-10
column (GE Healthcare).
Results
[0351] Construction of Plasmids Containing the Genes for Human
.alpha.FcRn and Human .beta.2-Microglobulin to be Used for
Co-Expression:
[0352] Genes encoding the extracellular domain of the .alpha.-chain
of human FcRn (.alpha.FcRn.sub.ECD) and human B2M were inserted
into the lentiviral transfer plasmids 2K7.sub.bsd and 2K7.sub.neo,
respectively. In both cases, the inserted gene is under the control
of a CMV promoter. The genes were extended so that the resulting
proteins would have an Ig kappa chain leader sequence in the
N-terminus to target the protein for export through the endoplasmic
reticulum to the culture medium (the signal sequence was cleaved
upon secretion). In addition, .alpha.FcRn.sub.ECD had a C-terminal
spacer sequence followed by a FLAG-tag for potential detection.
Human B2M had a C-terminal spacer sequence followed by a His.sub.6
tag for potential detection. The spacer sequence was added to
enhance accessibility of the tag. The lentiviral transfer plasmids
also contained two different antibiotic resistance genes to allow
selection of cells where both constructs had been inserted.
[0353] Expression and Purification of Recombinant Human FcRn:
[0354] The genes encoding .alpha.FcRn.sub.ECD and B2M were inserted
into the genome of SKOV-3 by lentiviruses, and the resulting FcRn
protein was secreted into the culture medium. To capture only FcRn
having retained pH-dependent IgG binding, affinity chromatography
using immobilized IgG was used where the receptor was captured at
pH 5.8 and eluted at pH 8.1. Captured protein was eluted in three
fractions.
[0355] SDS-PAGE and Western Blot:
[0356] To investigate the presence of two peptide chains
(.alpha.FcRn.sub.ECD and B2M) of the produced FcRn protein, and to
analyze the purity of the eluted material, an SDS-PAGE analysis was
performed on the eluted fractions. For the gel stained with GelCode
Blue Stain, two bands were detected with molecular weights of 12
and 36 kDa, respectively. This corresponds approximately to the
theoretical molecular weights of the non-glycosylated peptide
chains of 12 kDa for B2M and 31 kDa for .alpha.FcRn.sub.ECD. The
.alpha.FcRn.sub.ECD part of the protein contains one glycosylation
site and it was therefore expected that its molecular mass would be
higher than 31 kDa. The gel was also silver stained to increase
sensitivity and possibly detect impurities. A band of approximately
66 kDa was detected in the first eluted fraction, which could
correspond to BSA (bovine serum albumin) originating from cell
attachment. The total amount of protein recovered in fraction 2 and
3 corresponded to 1.4 mg/l culture medium. A western blot analysis
on the pooled material was carried out, which showed essentially
only the two major bands and in addition a very weak band below 12
kDa which might correspond to a degradation product.
Example 2
Selection and ELISA Binding of FcRn Binding Z Variants
[0357] In this Example, human FcRn was used as target in phage
display selections using a phage library of Z variants. Selected
clones were DNA sequenced, produced in E. coli periplasmic
fractions and assayed against FcRn in ELISA (enzyme-linked
immunosorbent assay).
Materials and Methods
[0358] Biotinylation of Target Protein FcRn and of B2M:
[0359] Human FcRn and human B2M, produced as described in Example
1, were biotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin
(Pierce, cat. no. 21327) at a 31.times.(FcRn) and 10.times.(B2M)
molar excess, respectively, according to the manufacturer's
recommendations. The reactions were performed at room temperature
(RT) for 30 min. Subsequent buffer exchange to PBS was performed
using Slide-a-lyzer dialysis cassettes (FcRn; Pierce, cat. no.
66380, 10,000 MWCO and B2M; Pierce, cat. no. 66333, 3,500 MWCO),
according to the manufacturer's instructions.
[0360] Phage Display Selection of FcRn Binding Z Variants:
[0361] A library of random variants of protein Z displayed on
bacteriophage, constructed in phagemid pAY02592 essentially as
described in Gronwall et al. (2007) J Biotechnol, 128:162-183, was
used to select FcRn binding Z variants. In this library, an albumin
binding domain (ABD, GA3 of protein G from Streptococcus strain
G148) is used as fusion partner to the Z variants. The library is
denoted Zlib006Naive.II and has a size of 1.5.times.10.sup.10
library members (Z variants). E. coli RRI.DELTA.M15 cells (Ruther
et al., (1982) Nucleic Acids Res 10:5765-5772) from a glycerol
stock containing the phagemid library Zlib006Naive.II, were
inoculated in 20 l of a defined proline free medium [dipotassium
hydrogenphosphate 7 g/l, trisodium citrate dihydrate 1 g/l, uracil
0.02 g/l, YNB (DIFCO Yeast Nitrogen Base w/o amino acids, Becton
Dickinson) 6.7 g/l, glucose monohydrate 5.5 g/l, L-alanine 0.3 g/l,
L-arginine monohydrochloride 0.24 g/l, L-asparagine monohydrate
0.11 g/l, L-cysteine 0.1 g/l, L-glutamic acid 0.3 g/l, L-glutamine
0.1 g/l, glycine 0.2 g/l, L-histidine 0.05 g/l, L-isoleucine 0.1
g/l, L-leucine 0.1 g/l, L-lysine monohydrochloride 0.25 g/l,
L-methionine 0.1 g/l, L-phenylalanine 0.2 g/l, L-serine 0.3 g/l,
L-threonine 0.2 g/I, L-tryptophane 0.1 g/l, L-tyrosine 0.05 g/l,
L-valine 0.1 g/l supplemented with 100 .mu.g/ml ampicillin. The
cultivations were grown at 37.degree. C. in a fermenter (Belach
Bioteknik, BR20). When the cells reached an optical density at 600
nm (OD600) of 0.75, approximately 2.6 l of the cultivation was
infected using a 10 .times. molar excess of M13K07 helper phage
(New England Biolabs, cat. no. N0315S). The cells were incubated
for 30 minutes, whereupon the fermenter was filled up to 20 l with
TSB-YE (Tryptic Soy Broth-Yeast Extract; 30 g/l TSB, 5 g/l yeast
extract) supplemented with 100 .mu.M
isopropyl-.beta.-D-1-thiogalactopyranoside (IPTG) for induction of
expression and with 25 .mu.g/ml kanamycin and 12.5 .mu.g/ml
carbenicillin and grown at 30.degree. C. for 22 h. The cells in the
cultivation were pelleted by centrifugation at 15,900 g. The phage
particles were precipitated from the supernatant twice in PEG/NaCl
(polyethylene glycol/sodium chloride), filtered and dissolved in
PBS and glycerol as described in Gronwall et al., supra. Phage
stocks were stored at -80.degree. C. before use.
[0362] Selections against biotinylated human FcRn were performed in
four cycles divided in two different tracks. Phage stock
preparation and selection procedure were performed essentially as
described for selection against another biotinylated target in
WO2009/077175. The amplification of phage between the selection
cycles was performed by infecting E. coli RRI.DELTA.M15 with phage,
then performing cultivation in solution as follows. Eluted phage
and 10.times. excess of M13K07 helper phage compared to bacteria
were allowed to simultaneously infect log phase bacteria at
37.degree. C. for 30 min without rotation, followed by 30 min with
slow rotation. Prior to infection, bacteria were grown to log phase
in the defined proline free medium described above. Infected
bacteria were pelleted by centrifugation at 4,300 g for 10 min and
resuspended in 200 ml TSB+YE medium supplemented with 0.1 mM IPTG,
25 .mu.g/ml kanamycin and 100 .mu.g/ml ampicillin and cultivated at
30.degree. C. overnight for phage production.
[0363] The selection buffer consisted of 100 mM sodium phosphate
and 150 mM sodium chloride adjusted to pH 5.5 with hydrogen
chloride and supplemented with 0.1% gelatin and 0.1% Tween-20. At
selection, human serum albumin (HSA, Albucult, Novozymes) was added
to the selection buffer to a final concentration of 1.5 .mu.M. In
order to reduce the amount of background binders, pre-selection was
performed by incubation of phage stock with DYNABEADS M-280
Streptavidin (SA-beads, Dynal, cat. no. 112.06) for 1 hour at RT. A
second pre-selection was performed during 30 min at RT against
human B2M immobilized in immunotubes (Nunc, cat. no. 444474). 5
.mu.g/ml of human B2M in carbonate buffer (Sigma, cat. no.
068K8214) was immobilized in the tube at 7.degree. C. for >1 h.
After washing twice with tap water, the tubes were blocked with
PBS+0.5% casein (Sigma, cat. no. C8654) for 30 min at RT before
use. All tubes and beads used in the selection were pre-blocked
with PBS+0.1% gelatin. Selection was performed in solution at RT,
followed by capture of target-phage complexes on SA-beads where 1
mg beads per 2.9 .mu.g biotinylated FcRn were used. In cycle 1 of
the selections, 100 nM biotinylated FcRn was used and two washes of
two min each were performed using selection buffer. An increased
stringency, using a lowered target concentration and an increased
number of washes, was applied in the subsequent cycles: 50 nM/5
washes, 25 nM/8 washes and 10 nM/12 washes were applied in cycle 2,
3 and 4, respectively. After the washes, bound phage was eluted
from the two selection tracks using two different procedures; 1)
500 .mu.l 0.1 M glycine-HCl, pH 2.2, followed by immediate
neutralization with 50 .mu.l 1 M Tris-HCl, pH 8.0, and 450 .mu.l
PBS, or; 2) 500 .mu.l of 100 mM sodium phosphate and 150 mM sodium
chloride, pH 8.0 and neutralization with 500 .mu.l PBS.
[0364] Sequencing:
[0365] PCR fragments were amplified from single colonies using a
standard PCR program and the primers AFFI-21
(5'-tgcttccggctcgtatgttgtgtg (SEQ ID NO:1071)) and AFFI-22
(5'-cggaaccagagccaccaccgg (SEQ ID NO:1072)). Sequencing of
amplified fragments was performed using the biotinylated
oligonucleotide AFFI-72 (5'-biotin-cggaaccagagccaccaccgg (SEQ ID
NO:1073)) and a BIGDYE Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems), used in accordance with the manufacturer's
protocol. The sequencing reactions were purified by binding to
magnetic streptavidin coated beads (Detach Streptavidin Beads,
Nordiag, cat. no. 2012-01) using a Magnatrix 8000 (Magnetic
Biosolution), and analyzed on ABI (PRISM) 3130xl Genetic Analyzer
(PE Applied Biosystems).
[0366] Production of Z Variants for ELISA:
[0367] Sequenced Z variants were produced by inoculating single
colonies from the selections into 10 ml TSB-YE medium supplemented
with 100 .mu.g/ml ampicillin and 0.1 mM IPTG and incubating for 24
h at 37.degree. C. Cells were pelleted by centrifugation,
re-suspended in 2 ml PBST (PBS supplemented with 0.05% Tween-20),
frozen at -80.degree. C. and thawed in a water bath, to release the
periplasmic fraction of the cells. The freeze-thawing procedure was
repeated seven times and cells were then pelleted by
centrifugation. The supernatant of the periplasmic extract
contained the Z variants as fusions to ABD, expressed as
AQHDEALE-[Z #####]-VDYV-[ABD]-YVPG (SEQ ID NO: 1100) (Gronwall et
al., supra). Z ##### refers to individual, 58 amino acid residue Z
variants.
[0368] ELISA K.sub.D Analysis of Z Variants:
[0369] The binding of Z variants to FcRn was analyzed in ELISA
assays. Half-area 96-well ELISA plates were coated with 2 .mu.g/ml
of an anti-ABD goat antibody (produced in-house) diluted in coating
buffer (50 mM sodium carbonate, pH 9.6) at 4.degree. C. overnight.
The antibody solution was poured off and the wells were blocked
with 100 .mu.l of PBSC (PBS supplemented with 0.5% casein) for 1.5
h at RT. The blocking solution was discarded and 50 .mu.l
periplasmic solution, diluted 1:4, was added to the wells and
incubated for 1.5 h at RT under slow shaking. The solutions were
poured off and the wells were washed four times with either 0.05%
PCT buffer, pH 6.0 (Mcllvaines phosphate-citrate buffer, pH 6.0,
supplemented with 0.05% Tween-20) or 0.05% PCT buffer, pH 7.4
(Mcllvaines phosphate-citrate buffer, pH 7.4, supplemented with
0.05% Tween-20). The target protein, biotinylated human FcRn, was
added to the wells in a 1:3 diluted concentration series from 2
.mu.g/ml (45 nM) to 0.3 ng/ml (6.9 .mu.M) diluted in PCC buffer, pH
6.0 or pH 7.4, (Mcllvaines phosphate-citrate buffer, pH 6.0 or pH
7.4, supplemented with 0.5% casein), respectively. The plates were
incubated for 1.5 h at RT followed by washes as described above.
Streptavidin conjugated HRP (Thermo Scientific, cat. no. N100) was
diluted 1:30 000 in PCC buffer, pH 6.0 or pH 7.4, respectively, and
added to the wells followed by 45 min incubation. After washing as
described above, 50 .mu.l ImmunoPure TMB substrate (Thermo
Scientific, cat. no. 34021) was added to the wells and the plates
were treated according to the manufacturer's recommendations.
Absorbance was measured at 450 nm using a multi-well plate reader,
Victor.sup.3 (Perkin Elmer). A Z variant binding an irrelevant
protein was used as negative control and a blank was created by
omitting the periplasmic step. A Z variant which bound to FcRn in a
pre-experiment (Z07918, SEQ ID NO:707) was used as positive
control. Measured values were analyzed using GraphPad Prism 5
(GraphPad Software, LaJolla, Calif., USA) and non-linear regression
in order to determine the affinities (K.sub.D) of the
interactions.
[0370] ELISA Specificity Analysis of Z Variants:
[0371] In another ELISA experiment, the specificities of the Z
variants were tested by assaying them against 2 .mu.g/ml
biotinylated human proteins B2M, PSMA (in house produced) and IgG
(polyclonal, Pharmacia, Sweden) and against PCC buffer pH 6.0 or pH
7.4, respectively. The assay was performed at pH 6.0 and at pH 7.4,
respectively, as described above. The biotinylated proteins or
buffer were added to the wells instead of FcRn in the target
protein step.
Results
[0372] Phage Display Selection of FcRn Binding Z Variants:
[0373] Individual clones were obtained after four cycles of phage
display selections against biotinylated human FcRn.
[0374] Sequencing:
[0375] Sequencing was performed on clones picked at random from
selection round four. Each Z variant was given a unique
identification number ##### and individual variants are referred to
as Z #####. The amino acid sequences of the 58 amino acid residues
long Z variants are listed in FIG. 1A-1MM as SEQ ID NO:707-722 and
SEQ ID NO:1059. The deduced FcRn binding motifs of these Z variants
are listed in FIG. 1A-1MM as SEQ ID NO:1-16 and SEQ ID NO:353. The
amino acid sequences of the 49 amino acid residues long
polypeptides predicted to constitute the complete three-helix
bundle within each of these Z variants are listed in FIG. 1A-1MM as
SEQ ID NO:354-369 and SEQ ID NO:706.
[0376] ELISA Assays with Z Variants:
[0377] Sixteen clones were produced as ABD fusion proteins in E.
coli. The periplasmic fractions were used in an ELISA against a
dilution series of human FcRn. The clones were: Z07909 (SEQ ID
NO:719), Z07918 (SEQ ID NO:707), Z07930 (SEQ ID NO:712), Z07960
(SEQ ID NO:710), Z10109 (SEQ ID NO:709), Z10111 (SEQ ID NO:714),
Z10127 (SEQ ID NO:718), Z10129 (SEQ ID NO:715), Z10140 (SEQ ID
NO:711), Z10141 (SEQ ID NO:716), Z10145 (SEQ ID NO:721), Z10152
(SEQ ID NO:720), Z10156 (SEQ ID NO:717), Z10161 (SEQ ID NO:722),
Z10183 (SEQ ID NO:713) and Z10193 (SEQ ID NO:708). K.sub.D values
were determined for all variants at pH 6.0 and for three variants
at pH 7.4 (Table 1). For thirteen variants, data was not obtained
for a K.sub.D analysis at pH 7.4. None of the sixteen variants
displayed non-specific binding when assayed against human B2M, IgG
or PSMA.
TABLE-US-00013 TABLE 1 ELISA K.sub.D analysis of Z-ABD variants in
E. coli periplasmic fractions. Z variant SEQ ID NO: K.sub.D pH 6.0
(M) K.sub.D pH 7.4 (M) Z07909 719 24.5 .times. 10.sup.-9 n.d.
Z07918 707 2.0 .times. 10.sup.-9 10.9 .times. 10.sup.-9 Z07930 712
10.4 .times. 10.sup.-9 n.d. Z07960 710 6.0 .times. 10.sup.-9 n.d.
Z10109 709 3.9 .times. 10.sup.-9 23.9 .times. 10.sup.-9 Z10111 714
11.4 .times. 10.sup.-9 n.d. Z10127 718 21.3 .times. 10.sup.-9 n.d.
Z10129 715 17.6 .times. 10.sup.-9 n.d. Z10140 711 8.8 .times.
10.sup.-9 n.d. Z10141 716 21.2 .times. 10.sup.-9 n.d. Z10145 721
42.0 .times. 10.sup.-9 n.d. Z10152 720 24.6 .times. 10.sup.-9 n.d.
Z10156 717 21.3 .times. 10.sup.-9 n.d. Z10161 722 163.0 .times.
10.sup.-9 n.d. Z10183 713 10.9 .times. 10.sup.-9 n.d. Z10193 708
2.3 .times. 10.sup.-9 25.9 .times. 10.sup.-9 n.d. = not
determinable
Example 3
Production and Characterization of FcRn Binding Z Variants
[0378] In this Example, seventeen Z variants were produced in E.
coli, purified and assayed against human FcRn in Biacore. A subset
of said variants was also assayed against mouse FcRn. Circular
dichroism (CD) spectroscopy was performed for a subset of Z
variants for investigation of their secondary structure.
Materials and Methods
[0379] Subcloning of Z Variants:
[0380] The DNA of seventeen FcRn binding Z variants (SEQ ID
NO:707-722 and SEQ ID NO:1059) was amplified from the library
vector pAY02592. A subcloning strategy for construction of
monomeric Z variant molecules with N-terminal His.sub.6 tag was
applied using standard molecular biology techniques (essentially as
described in detail in WO2009/077175 for Z variants binding another
target). The Z gene fragments were subcloned into the expression
vector pAY01448 resulting in the encoded sequence MGSSHHHHHHLQ-[Z
#####]-VD (SEQ ID NO: 1101).
[0381] In addition, the FcRn binding variant Z07918 (SEQ ID
NO:707), but starting with the amino acids AE instead of VD and
denoted Z11948 (SEQ ID NO:1060), was cloned as homodimeric
constructs with two different linkers between the Z variants and
followed by a C-terminal His.sub.6 tag. This was performed using
conventional molecular biology methods including DNA amplification,
restriction with suitable restriction enzymes and ligation of the
DNA. The two linkers were obtained from Thermo Fisher Scientific.
The Z gene fragments were subcloned into the expression vector
(pET-26 origin, Novagen) resulting in the encoded sequence [Z
#####]-GT-(G.sub.4S)-PR-[Z #####]-LEHHHHHH (SEQ ID NO: 1102) and [Z
#####]-GT-(G.sub.4S).sub.3-[Z #####]-LEHHHHHH (SEQ ID NO: 1103),
respectively.
[0382] Cultivation and purification: E. coli BL21(DE3) cells
(Novagen) were transformed with plasmids containing the gene
fragment of each respective FcRn binding Z variant and cultivated
at 37.degree. C. in 800 or 1000 ml of TSB-YE medium supplemented
with 50 .mu.g/ml kanamycin. At OD.sub.600=2, IPTG was added to
induce expression at a final concentration of 0.17 or 0.2 mM and
the culture was incubated at 37.degree. C. for another 5 h. The
cells were harvested by centrifugation.
[0383] Approximately 2-5 g of each cell pellet was resuspended in
10-25 ml binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM
imidazole, pH 7.4) supplemented with BENZONASE (Merck, cat. no.
1.01654.0001) to a concentration of 15 U/ml and Lysozyme (Sigma,
cat. no. L-7651) to a concentration of 0.5 mg/ml. After cell
disruption by three freeze-thawing cycles or sonication, cell
debris was removed by centrifugation and each supernatant was
applied on a 1 ml His GraviTrap IMAC column (GE Healthcare, cat.
no. 11-0033-99). Contaminants were removed by washing with wash
buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 or 60 mM imidazole,
pH 7.4), and the FcRn binding Z variants were subsequently eluted
with elution buffer 1 (20 mM sodium phosphate, 0.5 M sodium
chloride, 250 mM imidazole, pH 7.4) or elution buffer 2 (0.1 M
acetic acid, 0.5 M sodium chloride, pH 4.5). Purified Z variants
were buffer exchanged to PBS using PD-10 columns (GE Healthcare),
according to the manufacturer's protocol. Protein concentrations
were determined by measuring the absorbance at 280 nm, using a
NANODROP ND-1000 spectrophotometer, and using the extinction
coefficient of the respective protein. The purity of the FcRn
binding Z variants was analyzed by SDS-PAGE stained with Coomassie
Blue. The identity of each purified FcRn binding Z variant was
confirmed using LC/MS analysis.
[0384] Cd Analysis:
[0385] Purified His.sub.6-tagged Z variants were diluted to 0.5
mg/ml in PBS. For each diluted Z variant, a CD spectrum at 250-195
nm or 250-190 nm was obtained at 20.degree. C. In addition, a
variable temperature measurement (VTM) was performed to determine
the melting temperature (Tm). In the VTM, the absorbance was
measured at 221 nm while the temperature was raised from 20 to
90.degree. C., with a temperature slope of 5.degree. C./min. A new
CD spectrum was obtained at 20.degree. C. after the heating
procedure in order to study the refolding ability of the Z
variants. The CD measurements were performed on a Jasco J-810
spectropolarimeter (Jasco Scandinavia AB) using a cell with an
optical path-length of 1 mm.
[0386] Biacore Binding and Kinetic Analysis:
[0387] The interaction of FcRn binding His.sub.6-tagged Z variants
with human FcRn was analyzed in a Biacore 2000 instrument (GE
Healthcare). Human FcRn was immobilized in a flow cell on the
carboxylated dextran layer of a CM5 chip surface (GE Healthcare).
The immobilization was performed using amine coupling chemistry
according to the manufacturer's protocol and using HBS-EP (GE
Healthcare) as running buffer. One flow cell surface on the chip
was activated and deactivated for use as blank during analyte
injections. In the two binding experiments presented below,
Mcllvaines phosphate-citrate buffer pH 6.0 supplemented with 0.005%
Tween-20 (0.005% PCT) was used as running buffer. In all
experiments, a flow rate of 50 .mu.l/min was used.
[0388] In one experiment, the dissociation at pH 6.0 was compared
to the dissociation at pH 7.4. His.sub.6-tagged Z variants and a
human monoclonal IgG1 were diluted in running buffer to a final
concentration of 250 nM or 2.5 nM, respectively, and injected over
the FcRn chip for 1 minute using the co-inject procedure. The
second injection of the co-inject procedure, representing the
dissociation phase of the interactions, contained either running
buffer (pH 6.0) or 0.005% PCT pH 7.4. The Z variants were allowed
to dissociate for 1 minute, except for Z07918 and Z10193, which
were allowed to dissociate for 4 minutes, before a surface
equilibration during 5 minutes in running buffer. IgG was allowed
to dissociate for 4 minutes before equilibration. Buffer injections
were performed in a similar way; co-injection of buffer pH 6.0
followed by pH 6.0 or co-injection of buffer pH 6.0 followed by pH
7.4. The results were analyzed in BiaEvaluation software 4.1 (GE
Healthcare). Curves of the blank surface were subtracted from the
curves of the ligand surface. In addition, curves of buffer
injections were subtracted from the Z variant curves and from the
IgG curves to adjust for the buffer effects.
[0389] In another experiment, approximate kinetic constants
(k.sub.on and k.sub.off) and affinities (K.sub.D) were determined
for a subset of His.sub.6-tagged Z variants. Three concentrations
of the Z variants were injected for 1 minute followed by
dissociation in running buffer for 1 minute. The surfaces were
equilibrated with running buffer during 7.5 minutes before the
start of next cycle. Injected concentrations were either 675 nM,
225 nM and 75 nM (Z10140, Z10156 and Z10183) or 225 nM, 75 nM and
25 nM (Z07918 and Z10193). Kinetic constants were calculated from
the sensorgrams using the Langmuir 1:1 model of BiaEvaluation
software 4.1 (GE Healthcare).
[0390] In a separate experiment, the affinity of the interactions
of Z variants to hFcRn (SEQ ID NO:1065) and mFcRn (SEQ ID NO:1070),
respectively, was measured at both pH 6.0 and pH 7.4 on a Biacore
3000 instrument (GE Healthcare). hFcRn and mFcRn were produced
essentially as described in Example 1 but using mouse 3T3 cells
instead of human SKOV-3 cells for production of mFcRn, and
immobilized on separate flow cells on a CM5 chip in acetate buffer
at pH 4.65. The immobilization level was approximately 1000 RU for
both receptors. A reference flow cell was created by activation and
deactivation. 0.005% PCT pH 6.0 or 7.4 was used as running buffer
and for dilution of the analytes. All analyses were performed at
25.degree. C. The affinity constants for the His.sub.6-tagged Z
variants Z07918 (SEQ ID NO:707), Z07960 (SEQ ID NO:710) and Z10193
(SEQ ID NO:708) were determined by injecting a dilution series from
1024 nM to 0.5 nM (pH 6.0) or from 10240 nM to 5 nM (pH 7.4). The
affinities were derived using GraphPad Prism 5 software, using a
one site binding saturation model.
[0391] AlphaLISA Blocking Assay:
[0392] The potential of Z variants to inhibit binding of IgG to
FcRn was analyzed in an AlphaLISA assay with an EnSpire multiplate
reader 2300 (Perkin Elmer). Human IgG (Roactemra) was immobilized
on AlphaLISA acceptor beads (Perkin Elmer, cat. no. 6772002)
according to the manufacturer's recommendations. Stepwise serial
dilutions 1:3 of His-tagged Z variants to final concentrations of
250 nM to 38 .mu.M were made in a 384-well plate (Perkin Elmer,
cat. no. G6005350) and incubated for 45 min with 10 nM biotinylated
human FcRn (Biorbyt, cat. no. orb84388, biotinylated essentially as
described in Example 2) in AlphaLISA buffer (Perkin Elmer, cat. no.
AL000F) adjusted to pH 6.0 using HCl. IgG-coated Acceptor beads
were added to a final concentration of 10 .mu.M and incubated for
45 min. Finally, streptavidin coated Donor beads (Perkin Elmer,
cat. no. 6772002) were added to a final concentration of 40
.mu.g/ml and incubated for 30 min. All incubations were performed
at RT in the dark. The plate was analyzed in the EnSpire instrument
and the IC50 values were calculated using GraphPad Prism 5.
Results
[0393] Cultivation and Purification:
[0394] The seventeen FcRn binding Z variants (SEQ ID NO: 707-722
and SEQ ID NO:1059), constructed with an N-terminal His.sub.6 tag,
were produced in E. coli. The amount of IMAC-purified protein from
approximately 2-5 g bacterial pellets, determined
spectrophotometrically by measuring the absorbance at 280 nm,
ranged from approximately 10 mg to 20 mg for the different FcRn
binding Z variants. SDS-PAGE analysis of each final protein
preparation showed that these predominantly contained the FcRn
binding Z variant. The correct identity and molecular weight of
each FcRn binding Z variant was confirmed by HPLC-MS analysis.
[0395] Cd Analysis:
[0396] The CD spectra determined for six Z variants showed that
each had an .alpha.-helical structure at 20.degree. C. This result
was also verified in the variable temperature measurements, wherein
melting temperatures (Tm) were determined (Table 2). A reversible
folding was seen for the six Z variants when overlaying spectra
measured before and after heating to 90.degree. C.
TABLE-US-00014 TABLE 2 Melting temperatures for a selection of Z
variants. Z variant SEQ ID NO: Tm (.degree. C.) Z07909 719 56
Z07918 707 49 Z07930 712 56 Z07960 710 58 Z10109 709 61 Z10193 708
59
[0397] Biacore Binding and Kinetic Analyses:
[0398] The binding of seventeen Z variants to human FcRn and the
dissociation at different pH were tested in a Biacore instrument by
sequentially injecting each of the Z variants at pH 6.0 and either
buffer pH 6.0 or pH 7.4 over a chip surface containing FcRn. The
ligand immobilization level of the surface was 1668 RU human FcRn.
The seventeen Z variants showed binding to FcRn at pH 6.0, and for
all variants, faster off-rates were seen at pH 7.4 compared to pH
6.0. The result for IgG was similar, displaying a faster off-rate
at pH 7.4. The variants Z07918 and Z10193 showed the slowest
dissociation curves. Sensorgrams for a subset of variants and IgG
are displayed in FIG. 2 A-E.
TABLE-US-00015 TABLE 3 Biacore kinetic constants and affinities for
hFcRn binding at pH 6.0. Z variant SEQ ID NO: k.sub.on
(M.sup.-1s.sup.-1) k.sub.off (s.sup.-1) K.sub.D (M) Z07918 707 1.4
.times. 10.sup.6 0.022 1.6 .times. 10.sup.-8 Z10140 711 1.4 .times.
10.sup.6 0.12 8.6 .times. 10.sup.-8 Z10156 717 7.6 .times. 10.sup.5
0.28 3.7 .times. 10.sup.-7 Z10183 713 1.0 .times. 10.sup.6 0.13 1.3
.times. 10.sup.-7 Z10193 708 1.5 .times. 10.sup.6 0.033 2.2 .times.
10.sup.-8
[0399] The kinetic constants of five Z variants interacting with
FcRn at pH 6.0 were determined (see Table 3). The immobilization
level of the surface was 2015 RU human FcRn. For each Z variant,
kinetic constants were calculated using a curve set of three
injected concentrations.
[0400] Affinity (K.sub.D) constants were also determined for
His.sub.6-tagged Z variants Z07918 (SEQ ID NO:707), Z07960 (SEQ ID
NO:710) and Z10193 (SEQ ID NO:708) interacting with human and mouse
FcRn at pH 6.0 and pH 7.4 (Table 4). For all three variants,
K.sub.D values were lower at pH 6.0 compared to pH 7.4.
TABLE-US-00016 TABLE 4 Biacore affinities for hFcRn and mFcRn at pH
6.0 and pH 7.4. SEQ ID K.sub.D (M) hFcRn K.sub.D (M) mFcRn Z
variant NO: pH 6.0 pH 7.4 pH 6.0 pH 7.4 Z07918 707 1.2 .times.
10.sup.-8 >5 .times. 10.sup.-7 9.0 .times. 10.sup.-8 >5
.times. 10.sup.-7 Z07960 710 5.0 .times. 10.sup.-8 >1 .times.
10.sup.-6 3.5 .times. 10.sup.-7 >5 .times. 10.sup.-6 Z10193 708
1.4 .times. 10.sup.-8 >5 .times. 10.sup.-7 9.5 .times. 10.sup.-8
>5 .times. 10.sup.-7
TABLE-US-00017 TABLE 5 Calculated IC50 values from AlphaLISA
blocking assay. SEQ ID NO of IC50 Z variant Z variant (M) Z07909
719 4.6 .times. 10.sup.-8 Z07918 707 2.1 .times. 10.sup.-9 Z07930
712 4.2 .times. 10.sup.-8 Z07960 710 4.2 .times. 10.sup.-8 Z10109
709 5.7 .times. 10.sup.-8 Z10111 714 4.6 .times. 10.sup.-8 Z10140
711 5.6 .times. 10.sup.-8 Z10183 713 3.9 .times. 10.sup.-8 Z10193
708 1.2 .times. 10.sup.-8 Z13993 1059 1.3 .times. 10.sup.-7
Z11948-G.sub.4S-Z11948 1060 .sup. 3.8 .times. 10.sup.-10
Z11948-(G.sub.4S).sub.3-Z11948 1060 .sup. 4.1 .times.
10.sup.-10
[0401] AlphaLISA Blocking Assay:
[0402] The ability of seventeen His.sub.6-tagged monomeric Z
variants (SEQ ID NO:707-722 and SEQ ID NO:1059) and two dimeric
variant, Z11948-G.sub.4S-Z11948 and Z11948-(G.sub.4S).sub.3-Z11948
to inhibit IgG binding to FcRn was tested in an AlphaLISA blocking
assay. Serial dilutions of the Z variants were incubated with
biotinylated human FcRn and the blocking ability of each respective
variant was measured after addition of IgG coated Acceptor beads
and subsequently streptavidin coated Donor beads. Inhibition could
be measured as a decrease in AlphaLISA counts for positive Z
variants. The calculated IC50 values for the ten monomeric variants
and the two dimeric variants that were shown to block IgG binding
to FcRn in this assay are shown in Table 5.
Example 4
Binding of FcRn Binding Z Variants to Human or Mouse FcRn/eGFP
Transfected HeLa Cells
[0403] In this example, the binding ability of FcRn binding Z
variants was investigated. The production of HeLa cells expressing
human and murine FcRn-eGFP gene transgene and the use of these
cells for flow cytometry analysis with Alexa647 labeled Z variants
is described.
Materials and Methods
[0404] Cloning of FcRn-eGFP and B2M Viral Vectors:
[0405] The genes encoding murine FcRn (mFcRn, Genbank BC003786.1,
OpenBiosystems) and murine B2M (mB2M, Genbank BC085164.1,
OpenBiosystems) were amplified in a similar way as the genes for
human FcRn and human B2M as described in Example 1. Human and
murine FcRn and B2M genes were amplified as follows: for hFcRn, the
sequence encoding amino acids 1-365 (SEQ ID NO:1068) was amplified;
for hB2M, the sequence encoding amino acids 21-119 (SEQ ID NO:1066)
was amplified; for mFcRn, the sequence encoding amino acids 1-369
(SEQ ID NO:1069) was amplified; and for mB2M, the sequence encoding
amino acids 21-119 (SEQ ID NO:1067) was amplified. The vector
pHR-cPPT-CMV-EGFP (Jakobsson et al. (2003) J Neurosci Res
73:876-85) and FcRn PCR amplicons (human and murine) were cut using
the restriction enzymes BamHI (human) or BclI (murine) and MluI
(New England Biolabs, cat. nos. R0136M, R0160L and R0198L,
respectively), and ligated using T4 DNA Ligase (New England
Biolabs, cat. no. M0202M). The ligation mix was chemically
transformed into E. coli RRI.DELTA.M15 and spread on ampicillin
plates. Colonies were picked and screened with suitable primer
pairs. The construct encoding the original signal peptide, human or
murine FcRn and eGFP at the cytoplasmic tail were verified by
sequencing and denoted pHR-cPPT-CMV-hFcRn-eGFP and
pHR-cPPT-CMV-mFcRn-eGFP, respectively.
[0406] The human and murine B2M PCR amplicons were inserted into
the plasmid pDONOR221 (Invitrogen, cat. no. 12536-017) by
recombination using the Gateway system (Invitrogen, cat. no.
11789020, GATEWAY BP CLONASE II Enzyme mix) according to the
manufacturer's recommendations. After verification of correct
sequences, human or murine B2M was inserted into p2k7_gtc (Suter et
al., supra) using a multi-site gateway cloning system (Invitrogen,
cat. no. 11791020, GATEWAY LR CLONASE II Enzyme mix) together with
the promoter containing plasmid pENTR-CMV (Tai et al. supra),
resulting in the vectors 2k7.sub.neo-CMV-hB2M and
2k7.sub.neo-CMV-mB2M, respectively.
[0407] Lentiviral Transduction of HeLa Cells:
[0408] The vector pairs 2k7.sub.neo-CMV-hB2M and
pHR-cPPT-CMV-hFcRn-eGFP or 2k7.sub.neo-CMV-mB2M and
pHR-cPPT-CMV-mFcRn-eGFP were co-transfected together with VSV-G
envelope and gag/pol packaging plasmid into HEK293T cells using
calcium chloride transfection (Zufferey et al., supra; Jakobsson et
al. (2006) supra). HEK293T culture supernatants containing formed
lentiviral particles with FcRn and B2M transgenes respectively were
used to sequentially transduce HeLa Cervix adenocarcinoma cells
(Cell Line Service) at low passage number. The resulting two stably
transduced HeLa cell lines are in the following denoted hFcRn-eGFP
(transduced with genes for human FcRn-eGFP and hB2M) and mFcRn-eGFP
(transduced with genes for mouse FcRn-eGFP and mB2M).
[0409] Alexa647 Labeling of FcRn Binding Z Variants:
[0410] The three His.sub.6-tagged Z variants Z07918, Z07930 and
Z07960 were labeled with ALEXA FLUOR 647 Carboxylic Acid
Succinimidyl Ester (Invitrogen cat. no. A20106). Before labeling,
buffer was exchanged to 0.2 M carbonate buffer, pH 8.3, using
Vivaspin500 centrifugal filter units (10 kDa MWCO, Vivaproducts
cat. no. 512-2838) spun at 10,000 g. The labeling was performed in
the Vivaspin500 and 1 .mu.l of Alexa647 Succinimidyl Ester dye (40
.mu.g/.mu.l in DMSO corresponding to 1.3.times. molar excess) was
added to 200 .mu.g/25 .mu.l Z variant. The mixes were incubated at
RT in the dark for 40 minutes in a wiggling rota mixer. The
reaction mixes were subsequently put on ice for 3.5 hours and free
dye was removed by washing with 15.times.100 .mu.l PBS in the
Vivaspin500.
[0411] Immunofluorescence Staining of Human and Mouse FcRn-eGFP
Transfected HeLa-Cells with FcRn Binding Z Variants:
[0412] hFcRn-eGFP and mFcRn-eGFP HeLa cells were harvested by
trypsination and washed twice in PBS at pH 6.0 before counting.
100,000 cells were pipetted per well of a v-bottomed 96 well plate
(Nunc, cat no 277143) and the cells in the plate were subsequently
pelleted at 1,700 rpm for 4 min at 4.degree. C. The supernatants
were removed and the cells were fixed with 50 .mu.l of 2%
formaldehyde (Sigma Aldrich, cat. no. F8775) in PBS at pH 6.0 for
10 min at RT. Cells were thereafter washed with 2.times.100 .mu.l
PBS pH 6.0, saturated with casein (PBSC), and resuspended in PBSC
plus 0.1% saponin (AppliChem, cat no A4518.0100) containing 620 nM
of Alexa647 labeled His.sub.6-tagged Z variants; Z07960, Z07930 and
Z07918. Transduced HeLa cells, incubated with buffer alone, were
used as control. The cells were incubated for 1 h at 8.degree. C.
on a shaker in the dark, washed with 2.times.100 .mu.l PBSC and
resuspended in 180 .mu.l of PBS pH 6.0 plus 1% BSA (fraction V,
Merck, cat. no. 1.12018.0100). 10,000 cells/well were analyzed in a
Gallios Flow Cytometer (Beckman Coulter) and the data was analyzed
using Kaluza software (Beckman Coulter).
Results
[0413] Flow cytometry analysis was utilized to determine whether
the FcRn binding Z variants could bind to human and/or mouse FcRn
on human or mouse FcRn/eGFP transduced HeLa cells. The experiment
was performed at pH 6.0 with Alexa647 labeled Z07960, Z07930 and
Z07918 (SEQ ID NO:710, 712 and 707, respectively). Dot plot
analysis (y-axis: Alexa647, x-axis: eGFP) showed that the
transduced cell population could be divided into FcRn-eGFP negative
and positive population (FIG. 3, gate H and I, respectively)
indicating heterogeneous expression of the FcRn-eGFP fusion protein
by HeLa cells (FIG. 3). Accordingly, the mean fluorescence
intensity (MFI) values for Alexa647 in gate I were subtracted by
background MFI values of Alexa647 in gate H. The calculated MFI
values are presented in FIG. 4. The results show that Z07960,
Z07930 and Z07918 are capable of binding HeLa cells displaying
human (FIG. 4, Panel A) or murine (FIG. 4, Panel B) FcRn-eGFP.
Example 5
Blocking of IgG Binding to FcRn with the FcRn Binding Z Variant
Z07918
[0414] In this example, the potential competition of FcRn binding Z
variants with IgG for binding to FcRn was investigated in a cell
based assay. Such binding will result in blocking of the IgG-FcRn
interaction.
Materials and Methods
[0415] Blocking of IgG-FcRn Immunofluorescence Staining:
[0416] Human or murine FcRn-eGFP transduced HeLa cells were
prepared as described in Example 4. Fixed cells were resuspended in
50 .mu.l of a mix of either 100 nM Alexa647-conjugated human or
mouse IgG (Jackson laboratories, cat. no. 009-600-003 and
015-600-003, respectively) and 1000, 100, 10, 1 or 0 (buffer
control) nM His.sub.6-tagged Z07918 diluted in PBS-casein, pH 6.0,
plus 0.1% saponin (AppliChem). The cells were incubated for 1 h at
37.degree. C. on a shaker in the dark, washed with 2.times.100
.mu.l PBS-casein pH 6.0 and re-suspended in 180 .mu.l of PBS, pH
6.0, plus 1% BSA. Data from 10,000 cells/well (except somewhat
fewer cells for mouse 100 nM mIgG-Alexa647) were obtained using a
Gallios Flow Cytometer (Beckman Coulter) and the data was analyzed
using Kaluza software (Beckman Coulter).
Results
[0417] The experiment was performed to determine if the FcRn
binding Z variant Z07918 (SEQ ID NO:707) blocks the IgG-FcRn
interaction. Human or murine FcRn-eGFP transduced HeLa cells were
incubated with human or mouse Alexa647-conjugated IgG. The binding
was blocked with unlabeled Z07918 at different concentrations. Due
to the heterogeneous expression of FcRn by the transduced HeLa
cells (described in Example 4), the MFI values for Alexa647 in gate
N of each sample was subtracted by the corresponding MFI values in
gate M (FIG. 5). The percent IgG Alexa647 binding was calculated by
dividing the different MFI values with the MFI for the blank
control. The results showed that Z07918 effectively blocked hIgG
binding to hFcRn (FIG. 6, Panel A) in a dose dependent manner.
Furthermore, Z07918 also blocked mIgG binding to mFcRn (FIG. 6,
Panel B) although less efficiently compared to hIgG-binding.
Example 6
Pharmacokinetic Study of Three FcRn Binding Z Variants
[0418] In this example, the ability of FcRn binding Z variants to
prolong serum half-life of a non-specific Z variant was
investigated by a pharmacokinetic study performed in mice.
Materials and Methods
[0419] Subcloning of Z Variants:
[0420] A subset of Z variants (Z07918, Z07960 and Z10193) was
submitted to a second subcloning. DNA from the subcloned
His.sub.6-tagged variants in Example 3 was used as template. First,
PCR amplification using suitable primer pairs was performed to
create genes encoding Z variants starting with the amino acids AE
instead of VD. The mutated Z variants are listed in FIG. 1A-1MM and
were denoted Z11948 (SEQ ID NO:1060), Z11946 (SEQ ID NO:1061) and
Z11947 (SEQ ID NO:1062), corresponding to mutated Z07918, Z07960
and Z10193, respectively. Genes encoding the new Z variants were
restriction cleaved and ligated into a vector harboring the genes
encoding albumin binding variant PP013 (SEQ ID NO:1063) and Z03638
(SEQ ID NO:1064) with spacer sequences resulting in a gene fusion
encoding [Z
#####]-GAP(G4S).sub.4TS-[PP013]-GT(G.sub.4S).sub.4PR-[Z03638] (SEQ
ID NO: 1104) (also denoted "Z #####-PP013-Z03638" or "Z variant in
fusion with PP013-Z03638"). The negative control molecule
[Z03638]-GAP(G.sub.4S).sub.4TS-[PP013] (SEQ ID NO: 1105) was
subcloned in a similar way by ligating Z03638 into a vector
containing a (G.sub.4S).sub.4 linker and the sequence for PP013.
The subsequent steps for vector transformation into E. coli were
performed as in Example 3.
[0421] Cultivation and Purification:
[0422] Z variants in fusion with PP013-Z03638 were produced in E.
coli as described in Example 3. Approximately 3 g of each cell
pellet was re-suspended in 30 ml TST-buffer (25 mM Tris-HCl, 1 mM
EDTA, 200 mM NaCl, 0.05% Tween20, pH 8.0) supplemented with
BENZONASE (Merck). After cell disruption by sonication and
clarification by centrifugation, each supernatant was applied on a
gravity flow column with 5 ml agarose immobilized with an anti-ABD
ligand (produced in-house). After washing with TST-buffer and 5 mM
NH.sub.4Ac buffer, pH 5.5, the Z variants were eluted with 0.1 M
HAc. Acetonitrile (ACN) was added to a final concentration of 10%
to the eluted fractions from the anti-ABD agarose affinity
chromatography purification step and the samples were loaded on a 3
ml Resource 15RPC column (GE Healthcare), previously equilibrated
with RPC solvent A (0.1% trifluoroacetic acid (TFA), 10% ACN, 90%
water). After column wash with RPC solvent A, bound protein was
eluted with a linear gradient 0-50% RPC solvent B (0.1% TFA, 80%
ACN, 20% water) during 60 ml. Fractions containing pure Z variant
were identified by SDS-PAGE analysis and pooled. After the RPC
purification, the buffer of the pools was exchanged to PBS using a
HiPrep 26/10 Desalting column (GE Healthcare). Finally, the Z
variants were purified on 1 ml EndoTrap red columns (Hyglos, cat.
no. 321063) to ensure low endotoxin content.
[0423] Protein concentrations, purities and the identity of each
purified Z variant were analyzed as described in Example 3.
[0424] Biacore Analysis:
[0425] Expressed and purified Z variants fused to PP013-Z03638 were
assayed against human FcRn at pH 6.0 essentially as described for
the kinetic analysis in Example 3. The Z variants and the negative
control Z03638-PP013 were injected at 40 nM, 160 nM and 640 nM
during 1 minute followed by dissociation for 2.5 minutes and
equilibration for 1 minute. Kinetic constants and affinities were
determined for the Z variants using the BiaEvaluation software.
[0426] Pharmacokinetic Study:
[0427] Z11947, Z11946 and Z11948 fused to PP013-Z03638 were
administered intravenously (i.v.) to male NMRI mice (Charles River,
Germany) at a dose of 92 nmol/kg body weight. Sera from groups of
three mice were obtained at 0.08, 6, 18, 78, 120, 168 and 240
hours. The concentration of respective Z variant was determined by
ELISA.
[0428] ELISA:
[0429] Half-area 96-well ELISA plates were coated at 4.degree. C.
overnight with 50 .mu.l/well of an Z specific goat antibody
(produced in-house) diluted to 4 .mu.g/ml in coating buffer (50 mM
sodium carbonate, pH 9.6). The antibody solution was poured off and
the wells were blocked with 100 .mu.l of PBSC for 1.5 h at RT. The
sera were diluted in PBSC plus 1% mouse serum (matrix) from 1:100
to 1:51,200 in a two-fold dilution series in a dilutions plate. A
standard titration for respective Z variant and four quality
controls (very low, low, medium and high control) diluted in matrix
were included on each plate. 50 .mu.l of the dilutions were
transferred per well and the ELISA plates were incubated for 1.5 h
at RT. The plates were washed four times with PBST. Bound Z
variants were detected with 50 .mu.l/well of rabbit anti-PP013 Ig
(produced in-house) diluted to 4 .mu.g/ml in PBSC. The plates were
subsequently incubated for 1.5 h at RT followed by washes as
described above. HRP conjugated donkey anti-rabbit HRP obtained
from Jackson laboratories (cat. no. 711-035-152), diluted 1:20,000
in PBSC, was added and the plates were incubated for 1 hour. After
washing as described above, 50 .mu.l of ImmunoPure TMB substrate
was added to the wells and the plates were developed according to
the manufacturer's recommendations. After 15 minutes of
development, the absorbance was measured at 450 nm using a
multi-well plate reader (Victor.sup.3). The absorbance values were
analyzed using GraphPad Prism 5 to determine the concentrations
(cubic-spline curve fit) and area under curve (AUC). The
concentrations were then plotted as their natural logarithms
against time. The resulting curves followed a two compartment model
and the terminal half-life was calculated as In2 divided by the
slope based on the last three time points.
Results
[0430] Cultivation and Purification:
[0431] The three FcRn binding Z variants Z11947, Z11946 and Z11948
(SEQ ID NO:1062, 1061 and 1060), constructed as Z
#####-PP013-Z03638, and the negative control Z03638-PP013, were
produced in E. coli. The amount of purified protein from
approximately 3 g bacterial pellets, determined
spectrophotometrically by measuring the absorbance at 280 nm,
ranged from approximately 10 to 25 mg for the different FcRn
binding Z variants. SDS-PAGE analysis of each final protein
preparation showed that they predominantly contained respective
FcRn binding Z variant. The correct molecular weight of each FcRn
binding Z variant was confirmed by LC/MS analysis.
TABLE-US-00018 TABLE 6 Kinetic constants and affinities for FcRn at
pH 6.0 of Z variants produced as fusions to PP013-Z03638. Z variant
SEQ ID NO: k.sub.on (M.sup.-1s.sup.-1) k.sub.off (s.sup.-1) K.sub.D
(M) Z11948 1060 7.73 .times. 105 0.047 6.2 .times. 10.sup.-8 Z11946
1061 3.35 .times. 105 0.275 8.2 .times. 10.sup.-7 Z11947 1062 6.54
.times. 105 0.064 9.8 .times. 10.sup.-8
[0432] Biacore Analysis:
[0433] The binding to FcRn was analyzed for the three PP013-Z03638
fused Z variants. The immobilization level of the surface was 548
RU of human FcRn. The resulting rough kinetic constants and
affinities for the target binding at pH 6.0 are displayed in Table
6. Fitted curves are displayed in FIG. 7A-C. The negative control
Z03638-PP013 was negative against FcRn.
[0434] Pharmacokinetic Study:
[0435] The pharmacokinetic profiles of the above-mentioned
constructs of Z variants fused to PP013-Z03638 were compared to the
negative control Z03638-PP013 in a mouse pharmacokinetic study. In
previous work, e.g. as described in PCT application WO2009/016043,
it is shown that ABD fusion proteins have a long half-life in
serum, caused by ABD binding to serum albumin. In accordance with
the previous results, terminal half-life of ABD-fused Z variant
molecule (Z03638-PP013) was approximately 43 hours, which is
comparable to half-life of mouse albumin (35 hours). The terminal
half-lives of the constructs containing FcRn binding Z variant
molecule in addition to ABD were two- to three-fold longer (FIG.
8). The calculated terminal half-lives were 99 hours (Z11947), 69
hours (Z11946) and 58 hours (Z11948), suggesting that FcRn binding
of the Z variants contributed to the prolonged half-life.
Example 7
Design and Construction of a Maturation Library of FcRn Binding Z
Variants
[0436] In this Example, a maturated library was constructed. The
library was used for selections of FcRn binding Z variants.
Selections from maturated libraries are usually expected to result
in binders with increased affinity (Orlova et al., (2006) Cancer
Res 66(8):4339-48). In this study, randomized single stranded
linkers were generated using split-pool synthesis enabling
incorporation of defined codons in desired positions in the
synthesis.
Materials and Methods
[0437] Library Design:
[0438] The library was based on the sixteen sequences of the human
FcRn binding Z variants in Table 1 and further described in
Examples 2-6. In the new library, 13 variable positions in the Z
molecule scaffold were biased towards certain amino acid residues,
according to a strategy mainly based on the binding motifs of the Z
variants defined in SEQ ID NO:707-722. A DNA linker was generated
using split-pool synthesis containing the 147 bp partially
randomized helix 1 and 2 of the amino acid sequence: 5'-AA ATA AAT
CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN GCG NNN NNN GAG
ATC NNN NNN TTA CCT AAC TTA ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN
AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3' (SEQ ID
NO:1074, randomized codons are illustrated as NNN) flanked by
restriction sites XhoI and SacI, was ordered from DNA 2.0 (Menlo
Park, Calif., USA). The theoretical distributions of amino acid
residues in the new library, including eight variable amino acid
positions (9, 10, 11, 13, 14, 24, 32 and 35) and five constant
amino acid positions (17, 18, 25, 27 and 28) in the Z molecule
scaffold are given in Table 8. The resulting theoretical library
size is 5.3.times.10.sup.8 variants.
TABLE-US-00019 TABLE 7 Design of library for maturation. Amino acid
No of position in the Randomization amino Z variant (amino acid
abbreviations) acids Proportion 9 A,D,E,F,H,I,K,L,N,Q,R,S,T,V,W,Y
16 1/16 10 A,D,E,F,H,I,K,L,M,N,Q,R,S,T,V,W,Y 17 1/17 11
A,D,E,F,H,I,K,L,N,Q,R,S,T,V,W,Y 16 1/16 13
A,D,E,F,G,H,I,K,L,N,Q,R,S,T,V,W,Y 17 1/17 14 A,F,H
(25%),I,K,L,N,Q,R,S,T,V,W,Y 14 3/52, 13/52 (H) 17 R 1 1 18 W 1 1 24
F,Y 2 1/2 25 D 1 1 27 R 1 1 28 V 1 1 32
A,D,E,F,H,I,K,L,N,Q,R,S,T,V,W,Y 16 1/16 35
A,D,E,F,H,I,K,L,N,Q,R,S,T,V,W,Y 16 1/16
[0439] Library construction: The library was amplified using
AmpliTaq Gold polymerase (Applied Biosystems, cat. no. 4311816)
during 12 cycles of PCR and pooled products were purified with
QIAquick PCR Purification Kit (QIAGEN, cat. no. 28106) according to
the supplier's recommendations. The purified pool of randomized
library fragments was digested with restriction enzymes XhoI and
SacI-HF (New England Biolabs, cat. no. R0146L, and cat. no. R3156M)
and concentrated using a PCR Purification Kit. Subsequently, the
product was subjected to preparative 2.5% agarose (Nuisieve GTC
agarose, Cambrex, Invitrogen) gel electrophoresis and purified
using QIAGEN gel extraction Kit (QIAGEN, cat. no. 28706) according
to the supplier's recommendations.
[0440] The phagemid vector pAY02592 (essentially as pAffi1
described in Gronwall et al., supra) was restricted with the same
enzymes, purified using phenol/chloroform extraction and ethanol
precipitation. The restricted fragments and the restricted vector
were ligated in a molar ratio of 5:1 with T4 DNA ligase (Fermentas,
cat. no. EL0011) for 2 hours at RT, followed by overnight
incubation at 4.degree. C. The ligated DNA was recovered by
phenol/chloroform extraction and ethanol precipitation, followed by
dissolution in 10 mM Tris-HCl, pH 8.5. Thus, the resulting library
in vector pAY02592 encoded Z variants, each fused to an albumin
binding domain (ABD) derived from streptococcal protein G.
[0441] The ligation reactions (approximately 160 ng
DNA/transformation) were electroporated into electrocompetent E.
coli ER2738 cells (50 .mu.l, Lucigen, Middleton, Wis., USA).
Immediately after electroporation, approximately 1 ml of recovery
medium (supplied with the ER2738 cells) was added. The transformed
cells were incubated at 37.degree. C. for 60 min. Samples were
taken for titration and for determination of the number of
transformants. The cells were thereafter pooled and cultivated
overnight at 37.degree. C. in 1 l of TSB-YE medium, supplemented
with 2% glucose, 10 .mu.g/ml tetracycline and 100 .mu.g/ml
ampicillin. The cells were pelleted for 7 min at 4,000 g and
resuspended in a PBS/glycerol solution (approximately 40%
glycerol). The cells were aliquoted and stored at -80.degree. C.
Clones from the library of Z variants were sequenced in order to
verify the content and to evaluate the outcome of the constructed
library vis-a-vis the library design. Sequencing was performed as
described in Example 1 and the amino acid distribution was
verified.
[0442] Preparation of Phage Stock:
[0443] Phage stock containing the phagemid library was prepared in
a 20 l fermenter (Belach Bioteknik). Cells from a glycerol stock
containing the phagemid library were inoculated in 10 l of TSB-YE
(Tryptic Soy Broth-Yeast Extract; 30 g/l TSB, 5 g/l yeast extract)
supplemented with 1 g/l glucose, 100 mg/l ampicillin and 10 mg/l
tetracycline. When the cells reached an optical density at 600 nm
(OD600) of 0.6, approximately 1.5 l of the cultivation was infected
using a 5.times. molar excess of M13K07 helper phage. The cells
were incubated for 30 min, whereupon the fermenter was filled up to
10 l with complex fermentation medium [2.5 g/l
(NH.sub.4).sub.2SO.sub.4, 5.0 g/l yeast extract; 30 g/l tryptone, 2
g/l K2HPO.sub.4; 3 g/l KH.sub.2PO.sub.4, 1.25 g/l,
Na.sub.3C.sub.6H.sub.5O.sub.7.2 H.sub.2O; Breox FMT30 antifoaming
agent 0.1 ml/l]. The following components were added: 10 ml
carbenicillin 25 mg/ml, 5 ml kanamycin 50 mg/ml, 1 ml 1 M
isopropyl-.beta.-D-1-thiogalactopyranoside (IPTG); 17.5 ml/l of 300
g/l MgSO.sub.4, and 5 ml of a trace element solution [35 g/l
FeCl.sub.3. 6 H.sub.2O; 10.56 g/l ZnSO.sub.4.7 H.sub.2O; 2.64 g/l
CuSO.sub.4.5 H.sub.2O; 13.2 g/l MnSO.sub.4.H.sub.2O; 13.84 g/l
CaCl.sub.2.2 H.sub.2O, dissolved in 1.2 M HCl]. A glucose limited
fed-batch cultivation was started where a 600 g/l glucose solution
was fed to the reactor (3.5 g/h in the start, 37.5 g/h after 20 h
and until the end of the cultivation). pH was controlled at pH 7
through the automatic addition of 25% NH.sub.4OH, air was
supplemented (5 l/min), and the stirrer was set at 500 rpm. After
24 h of fed-batch cultivation the OD600 was 33.2. The cells in the
cultivation were pelleted by centrifugation at 15,900 g. The phage
particles were precipitated from the supernatant twice in PEG/NaCl,
filtered and dissolved in PBS and glycerol as in Example 2. Phage
stocks were stored at -80.degree. C. until use in selection.
Results
[0444] Library Construction:
[0445] The new library was designed based on a set of 16 FcRn
binding Z variants with verified binding properties (Example 2-6).
The theoretical size of the designed library was 5.3.times.10.sup.8
Z variants. The actual size of the library, determined by titration
after transformation to E. coli ER2738 cells, was
4.5.times.10.sup.9 transformants.
[0446] The library quality was tested by sequencing of 96
transformants and by comparing their actual sequences with the
theoretical design. The contents of the actual library compared to
the designed library were shown to be satisfying. A maturated
library of potential binders to FcRn was thus successfully
constructed.
Example 8
Selection and Screening of Z Variants from a Maturated Library
Materials and Methods
[0447] Phage Display Selection of Matured FcRn Binding Z
Variants:
[0448] The target proteins human FcRn (Biorbyt, cat. no. orb84388)
and murine FcRn (Biorbyt, cat. no. orb99076) were biotinylated
essentially as described in Example 2 using biotin at 10.times.
molar excess. Phage display selections, using the new library of Z
variant molecules described in Example 7, were performed in four
cycles against human FcRn or murine FcRn essentially as in Example
2 but with the following exceptions. Selection buffers were 0.1%
PCTG buffer, pH 5.5 (Mcllvaines phosphate-citrate buffer, pH 5.5,
supplemented with 0.1% Tween-20 and 0.1% gelatin) or 0.1% PCTG
buffer, pH 7.4, (Mcllvaines phosphate-citrate buffer, pH 7.4,
supplemented with 0.1% Tween-20 and 0.1% gelatin) respectively.
Prior to selection, HSA was added to the selection buffers to a
final concentration of 1.5 .mu.M. All tubes and beads used in the
selection were pre-blocked with either of the two different
selections buffers. A pre-selection step, by incubation of phage
stock with SA-beads for 45 min, was performed in cycle 1. For
capture of phage-target complexes, 1 mg beads per 1.1 .mu.g
biotinylated human FcRn or 1.6 .mu.g biotinylated murine FcRn was
used. Washes were performed with 0.1% PCT buffer pH 5.5 or pH 7.4
except for tracks 2-1-2-1 and 2-1-2-2 where 0.1% PCT supplemented
with 25 nM IgG (HERCEPTIN) or 10 nM IgG, respectively, was used as
outlined in Table 7.
[0449] The five tracks (1-5) in cycle 1 were divided in the second
to fourth cycles, resulting in totally seven tracks (1-1 to 5-1) in
cycle 2, eleven tracks (1-1-1 to 5-1-1) in cycle 3 and fourteen
tracks (1-1-1-1 to 5-1-1-1) in cycle 4. The bound phage particles
were eluted as described in Example 2.
[0450] An overview of the selection strategy, describing an
increased stringency in subsequent cycles, using a lowered target
concentration and an increased number of washes, is shown in Table
8.
TABLE-US-00020 TABLE 8 Overview of the maturation selection data.
Phage stock Target Number Selection from library or Target conc.
Selection Wash of Cycle track selection track species (nM) pH pH
washes 1 1 Zlib006FcRn.I human 100 7.4 7.4 2 1 2 Zlib006FcRn.I
human 100 7.4 5.5 2 1 3 Zlib006FcRn.I human 25 5.5 5.5 4 1 4
Zlib006FcRn.I murine 100 7.4 7.4 2 1 5 Zlib006FcRn.I murine 100 5.5
5.5 2 2 1-1 1 human 50 7.4 7.4 4 2 2-1 2 human 50 7.4 5.5 4 2 2-2 2
human 25 5.5 7.4 6 2 3-1 3 human 5 5.5 7.4 4 2 3-2 3 human 5 5.5
5.5 8 2 4-1 4 murine 50 7.4 5.5 2 2 5-1 5 murine 100 5.5 5.5 2 3
1-1-1 1-1 human 10 7.4 7.4 8 3 1-1-2 1-1 human 5 5.5 7.4 8 3 2-1-1
2-1 human 10 7.4 5.5 8 3 2-1-2 2-1 human 5 7.4 5.5 12 3 2-2-1 2-2
human 10 7.4 5.5 12 3 2-2-2 2-2 human 5 7.4 5.5 15 3 3-1-1 3-1
human 1 5.5 7.4 8 3 3-2-1 3-2 human 0.5 5.5 5.5 12 3 3-2-2 3-2
human 0.25 5.5 5.5 16 3 4-1-1 4-1 murine 10 7.4 5.5 6 3 5-1-1 5-1
murine 5 5.5 5.5 8 4 1-1-1-1 1-1-1 human 1 7.4 7.4 12 4 1-1-1-2
1-1-1 human 0.25 7.4 7.4 15 4 1-1-2-1 1-1-2 human 0.5 7.4 5.5 15 4
1-1-2-2 1-1-2 human 0.1 5.5 7.4 15 4 2-1-1-1 2-1-1 human 1 7.4 5.5
15 4 2-1-1-2 2-1-1 human 0.5 7.4 5.5 15 4 2-1-2-1 2-1-2 human 0.25
7.4 5.5 20 (+IgG) 4 2-1-2-2 2-1-2 human 0.1 7.4 5.5 20 (+IgG) 4
2-2-1-1 2-2-1 and 2-2-2 human 0.5 5.5 7.4 15 4 2-2-2-1 2-2-1 and
2-2-2 human 0.5 7.4 5.5 20 4 3-1-1-1 3-1-1 human 1 5.5 7.4 12 4
3-2-1-1 3-2-1 and 3-2-2 human 0.5 5.5 5.5 16 4 4-1-1-1 4-1-1 murine
1 7.4 5.5 12 4 5-1-1-1 5-1-1 murine 0.5 5.5 5.5 15
[0451] Amplification of Phew Particles:
[0452] Amplification of phage particles between selection cycle 1
and 2 was performed essentially as described in Example 2, with the
following exceptions. E. coli ER2738 was used for phage
amplification and M13K07 helper phage was used in 5.times. excess.
The amplification of phage particles between the selection cycles 2
and 4 was done by performing infection of bacteria in solution as
follows. After infection of log phase E. coli ER2738 with phage
particles, TSB supplemented with 2% glucose, 10 .mu.g/ml
tetracycline and 100 .mu.g/ml ampicillin was added, followed by
incubation with rotation for 30 min at 37.degree. C. Thereafter,
the bacteria were infected with M13K07 helper phage in 5.times.
excess. The infected bacteria were pelleted by centrifugation,
re-suspended in TSB-YE medium supplemented with 100 .mu.M IPTG, 25
.mu.g/ml kanamycin and 100 .mu.g/ml ampicillin, and grown overnight
at 30.degree. C. The overnight cultures were pelleted in a
centrifuge, and phage particles in the supernatant were
precipitated twice with PEG/NaCl buffer. Finally, the phage
particles were re-suspended in selection buffer before entering the
next selection cycle.
[0453] In the final selection cycle, log phase bacteria were
infected with eluate and diluted before spreading onto TBAB plates
(30 g/l tryptose blood agar base, Oxoid cat. no. CM0233B)
supplemented with 0.2 g/l ampicillin in order to form single
colonies to be used in ELISA screening.
[0454] Sequencing of Potential Binders:
[0455] Individual clones from the different selection tracks were
picked for sequencing. All clones run in the ELISA screening were
sequenced. Amplification of gene fragments and sequence analysis of
gene fragments were performed essentially as described in Example
2.
[0456] ELISA Screening of Z Variants:
[0457] Single colonies containing Z variants (expressed as Z
variant ABD fusion proteins as described in Example 2) were
randomly picked from the selected clones of the FcRn maturated
library and grown in 1 ml cultivations essentially as described in
Example 2. Preparation of the periplasmic supernatants was
performed as in Example 2 with eight freeze thawing cycles and the
periplasmic fractions were used undiluted in the ELISA screening.
ELISA screenings were performed at both pH 6.0 and pH 7.4
essentially as described in Example 2 using biotinylated human FcRn
at a concentration of 2 nM in each well. The periplasmic fraction
of the primary FcRn binder Z10193 (SEQ ID NO:708; assayed in above
experiments) was used as a positive control. Periplasm containing
the ABD moiety only was used as a negative control.
[0458] ELISA K.sub.D Analysis of FcRn Binding Z Variants:
[0459] A selection of FcRn binders was subjected to an analysis of
the response against a dilution series of biotinylated human FcRn
using ELISA at both pH 6.0 and pH 7.4 as described above.
Biotinylated human FcRn was added at a concentration of 30 nM and
diluted stepwise 1:3 down to 14 .mu.M. As a background control, all
Z variants were also assayed with no target protein added.
Periplasm samples containing the primary FcRn binder Z07918 (SEQ
ID. NO:707) was included and analyzed as a positive control.
Periplasm containing the ABD moiety only was used as a negative
control. Data were analyzed using Graph Pad Prism 5 and non-linear
regression and K.sub.D values (the half maximal effective
concentration) were calculated.
Results
[0460] Phage Display Selection of Maturated FcRn Binding Z
Variants:
[0461] Selection was performed in totally 14 parallel tracks
containing four cycles each. The different selection tracks
differed in target concentration, target type (human FcRn or murine
FcRn), selection time, and wash conditions.
[0462] Sequencing of Potential Binders:
[0463] Randomly picked clones were sequenced. Each individual Z
variant was given an identification number, Z #####, as described
in Example 2. In total, 445 new unique Z variant molecules were
identified.
[0464] The amino acid sequences of the 58 amino acid residues long
Z variants are listed in FIG. 1A-1MM and in the sequence listing as
SEQ ID NO:723-1058. The deduced FcRn binding motifs of these Z
variants are listed in FIG. 1A-1MM and in the sequence listing as
SEQ ID NO:17-352. The amino acid sequences of the 49 amino acid
residues long polypeptides predicted to constitute the complete
three-helix bundle within each of these Z variants are listed in
FIG. 1A-1MM and in the sequence listing as SEQ ID NO:370-705.
[0465] ELISA Screening of Z Variants:
[0466] Clones obtained after four selection cycles were produced in
96-well plates and screened for FcRn binding activity using ELISA.
All randomly picked clones were analyzed. At pH 6.0, 333 of the 445
unique Z variants were found to give a response of 0.3 AU or higher
(corresponding to at least 3.times. the negative control) against
human FcRn at a concentration of 2 nM. At pH 7.4, 278 of the 445
unique Z variants were found to give a response of 0.3 AU or higher
(corresponding to at least 3.times. the negative control) against
human FcRn at a concentration of 2 nM. Clones with a positive
signal against human FcRn were found in all tracks (including those
with murine target) except 1-1-1-1. The negative controls had
absorbances of 0.070-0.096 AU (pH 6.0) and 0.060-0.112 AU (pH 7.4),
respectively. The average response of the blank controls was 0.070
AU (pH 6.0) and 0.062 (pH 7.4).
[0467] ELISA K.sub.D Analysis of