U.S. patent application number 17/126838 was filed with the patent office on 2021-08-12 for fc-region variants with modified fcrn- and protein a-binding properties.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Tilman Schlothauer.
Application Number | 20210246228 17/126838 |
Document ID | / |
Family ID | 1000005541101 |
Filed Date | 2021-08-12 |
United States Patent
Application |
20210246228 |
Kind Code |
A1 |
Schlothauer; Tilman |
August 12, 2021 |
FC-REGION VARIANTS WITH MODIFIED FCRN- AND PROTEIN A-BINDING
PROPERTIES
Abstract
Herein is reported a heterodimeric polypeptide comprising a
first polypeptide comprising in N-terminal to C-terminal direction
at least a portion of an immunoglobulin hinge region, which
comprises one or more cysteine residues, an immunoglobulin
CH2-domain and an immunoglobulin CH3-domain, and a second
polypeptide comprising in N-terminal to C-terminal direction at
least a portion of an immunoglobulin hinge region, which comprises
one or more cysteine residues, an immunoglobulin CH2-domain and an
immunoglobulin CH3-domain, wherein the first polypeptide comprises
the mutations Y349C, T366S, L368A and Y407V (hole-chain) and the
second polypeptide comprises the mutations S354C and T366W
(knob-chain), and wherein the first polypeptide (hole-chain)
comprises the mutations i) I253A or I253G, and ii) L314A or L314G
or L314D, and wherein the first polypeptide and the second
polypeptide are connected by one or more disulfide bridges, and
wherein the CH3-domain of the first polypeptide and the CH3-domain
of the second polypeptide both bind or both do not bind to protein
A (numbering according to the Kabat EU index).
Inventors: |
Schlothauer; Tilman;
(Penzberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
1000005541101 |
Appl. No.: |
17/126838 |
Filed: |
December 18, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15586686 |
May 4, 2017 |
10899846 |
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17126838 |
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PCT/EP2015/075657 |
Nov 4, 2015 |
|
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15586686 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/52 20130101;
C07K 16/468 20130101; C07K 2317/524 20130101; C07K 2317/72
20130101; C07K 2317/14 20130101; C07K 16/1271 20130101; C07K
2317/53 20130101; C07K 1/22 20130101; C07K 2319/00 20130101; C07K
2317/71 20130101; C07K 2317/526 20130101; C07K 2317/31 20130101;
C07K 16/065 20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 16/06 20060101 C07K016/06; C07K 1/22 20060101
C07K001/22; C07K 16/12 20060101 C07K016/12 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 6, 2014 |
EP |
14192054.6 |
Claims
1-15. (canceled)
16. A method for treating an ocular vascular disease, the method
comprising administering to a subject in need thereof a composition
comprising an effective amount of a heterodimeric polypeptide to
transport a soluble receptor ligand from the eye to the blood,
wherein the heterodimeric polypeptide specifically binds the ligand
and wherein the heterodimeric polypeptide comprises: a first
polypeptide comprising in N-terminal to C-terminal direction at
least a portion of an immunoglobulin hinge region, which comprises
one or more cysteine residues, an immunoglobulin CH2-domain and an
immunoglobulin CH3-domain, and a second polypeptide comprising in
N-terminal to C-terminal direction at least a portion of an
immunoglobulin hinge region, which comprises one or more cysteine
residues, an immunoglobulin CH2-domain and an immunoglobulin
CH3-domain, wherein the first polypeptide comprises the mutations
Y349C, T366S, L368A and Y407V (hole-chain) and the second
polypeptide comprises the mutations S354C and T366W (knob-chain),
and wherein the first polypeptide (hole-chain) comprises one of the
mutations selected from each of i) and ii), wherein i) and ii) are:
(i) I253A or I253G, and (ii) L314A or L314G or L314D, and wherein
the first polypeptide and the second polypeptide are connected by
one or more disulfide bridges, and wherein the CH3-domain of the
first polypeptide and the CH3-domain of the second polypeptide both
bind or both do not bind to protein A, and wherein the knob-chain
does not comprise I253A, I253G, L314A, L314G or L314D mutations
(numbering according to the Kabat EU index).
17. The method according to claim 16, wherein the heterodimeric
polypeptide further comprises: a) the mutation T250Q, or b) the
mutations T250Q and one of the mutations T256E and T256A, or c) one
of the mutations T256E and T256A.
18. The method according to claim 16, wherein the heterodimeric
polypeptide further comprises: a) one of the mutations L251A,
L251G, and L251D, or b) one of the mutations H310A and H310G.
19. The method according to claim 16, wherein the heterodimeric
polypeptide further comprises at least one mutation selected from
one or more of i) to vi), wherein i) to vi) are: (i) T250Q, (ii)
M252Y, (iii) S254T, (iv) T256E or T256A, (v) T307A or T307H or
T307Q or T307P, and (vi) Q311H.
20. The method according to claim 16, wherein the immunoglobulin
hinge regions, the immunoglobulin CH2-domains, and the
immunoglobulin CH3-domains of the first and second polypeptides are
of the human IgG1 subclass.
21. The method according to claim 16, wherein the first polypeptide
and the second polypeptide further comprise the mutations L234A and
L235A.
22. The method according to claim 16, wherein the first polypeptide
and the second polypeptide further comprise the mutation P329G.
23. The method according to claim 16, wherein the immunoglobulin
hinge regions, the immunoglobulin CH2-domains, and the
immunoglobulin CH3-domains of the first and second polypeptides are
of the human IgG4 subclass.
24. The method according to claim 16, wherein the first polypeptide
and the second polypeptide further comprise the mutations S228P and
L235E.
25. The method according to claim 16, wherein the first polypeptide
and the second polypeptide further comprise the mutation P329G.
26. The method according to claim 16, wherein the heterodimeric
polypeptide is a full-length bispecific antibody.
27. The method according to claim 18, wherein the heterodimeric
polypeptide further comprises: a) the mutation T250Q, or b) the
mutation T250Q and one of the mutations T256E and T256A, or c) one
of the mutations T256E and T256A.
28. The method according to claim 18, wherein the heterodimeric
polypeptide further comprises: a) one of the mutations L251A,
L251G, and L251D, and b) one of the mutations H310A and H310G.
29. The method according to claim 27, wherein the heterodimeric
polypeptide further comprises: a) one of the mutations L251A,
L251G, and L251D, and b) one of the mutations H310A and H310G.
30. The method according to claim 27, wherein the heterodimeric
polypeptide further comprises: (i) a mutation selected from T307A,
T307H, T307Q, and T307P, or (ii) one or more of the mutations
selected from the group consisting of Q311H, M252Y, and S254T.
31. The method according to claim 30, wherein the heterodimeric
polypeptide further comprises: (i) a mutation selected from T307A,
T307H, T307Q, and T307P, and (ii) one or more of the mutations
selected from the group consisting of Q311H, M252Y, and S254T.
32. The method according to claim 16, wherein the composition is
administered by topical application to the cornea or by
intravitreal application.
33. The method according to claim 16, wherein the ocular vascular
disease is selected from the group consisting of wet age-related
macular degeneration (wet AMD), dry age-related macular
degeneration (dry AMD), diabetic macular edema (DME), cystoid
macular edema (CME), non-proliferative diabetic retinopathy (NPDR),
proliferative diabetic retinopathy (PDR), cystoid macular edema,
vasculitis (e.g. central retinal vein occlusion), papilloedema,
retinitis, conjunctivitis, uveitis, choroiditis, multifocal
choroiditis, ocular histoplasmosis, blepharitis, and dry eye
(Sjogren's disease).
34. The method according to claim 16, further comprising
administering one or more additional therapeutic agents for a
vascular eye disease.
35. The method according to claim 16, wherein the heterodimeric
polypeptide acts to inhibit angiogenesis in the eye of said
subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 15/586,686, filed May 4, 2017 which is a continuation of
International Application Number PCT/EP2015/075657, filed Nov. 4,
2015, which claims benefit under 35 U.S.C. .sctn. 119 to European
Application Number 14192054.6, filed Nov. 6, 2014 which are
incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0002] This application hereby incorporates by reference the
material of the electronic Sequence Listing filed concurrently
herewith. The material in the electronic Sequence Listing is
submitted as a text file entitled Sequence_Listing.txt created on
Dec. 15, 2020 which has a file size of 58.0 KB, and is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] Herein are reported IgG Fc-regions that have been modified
with respect to Fc-receptor as well as protein A binding.
BACKGROUND OF THE INVENTION
[0004] The demand for cost efficient production processes has led
to the necessity of optimization of the downstream purification,
including one or more affinity chromatography steps. Larger volumes
to be processed and harder requirements for the cleaning-in-place
(CIP) protocols are some of the features that need to be solved
(Hober, S., J. Chrom. B. 848 (2007) 40-47).
[0005] The purification of monoclonal antibodies by means of
selective Fc-region affinity ligands is the most promising
methodology for the large-scale production of therapeutic
monoclonal antibodies. In fact, this procedure does not require
establishing any interaction with the antigen specific part of the
antibody, i.e. the Fab domain, which is, thus, left intact and can
retain its properties (see Salvalaglio, M., et al., J. Chrom. A
1216 (2009) 8678-8686).
[0006] Due to its selectiveness, an affinity-purification step is
employed early in the purification chain and thereby the number of
successive unit operations can be reduced (see Hober supra;
MacLennan, J., Biotechnol. 13 (1995) 1180; Harakas, N. K.,
Bioprocess Technol. 18 (1994) 259).
[0007] The ligands most adopted to bind selectively IgG are
Staphylococcal protein A and protein G, which are able to establish
highly selective interactions with the Fc-region of most IgGs in a
region known as "consensus binding site" (CBS) (DeLano, W. L., et
al., Science 287 (2000) 1279), which is located at the hinge region
between the CH2 and CH3 domains of the Fc-region.
[0008] Staphylococcal protein A (SPA) is a cell wall associated
protein domain exposed on the surface of the Gram-positive
bacterium Staphylococcus aureus. SPA has high affinity to IgG from
various species, for instance human, rabbit and guinea pig IgG but
only weak interaction with bovine and mouse IgG (see the following
Table) (see Hober supra; Duhamel, R. C., et al., J. Immunol.
Methods 31 (1979) 211; Bjork, L. and Kronvall, G., Immunol. J. 133
(1984) 969; Richman, D. D., et al., J. Immunol. 128 (1982) 2300;
Amersham Pharmacia Biotech, Handbook, Antibody Purification
(2000)).
TABLE-US-00001 species subclass protein A binding human IgG1 ++
IgG2 ++ IgG3 -- IgG4 ++ IgA variable IgD - IgM variable rabbit no
distinction ++ guinea pig IgG1 ++ IgG2 ++ bovine + mouse IgG1 +
IgG2a ++ IgG2b + IgG3 + IgM variable chicken IgY - ++: strong
binding/ +: medium binding/ -: weak or no interaction
[0009] The heavy chain hinge-region between the CH2 and CH3 domains
of IgG is able to bind several proteins beyond protein A, such as
the neonatal Fc receptor (FcRn) (see DeLano and Salvalaglio
supra).
[0010] The SPA CBS comprehends a hydrophobic pocket on the surface
of the antibody. The residues composing the IgG CBS are Ile 253,
Ser 254, Met 252, Met 423, Tyr 326, His 435, Asn 434, His 433, Arg
255, and Glu 380 (numbering of the IgG heavy chain residues
according to the Kabat EU index numbering system). The charged
amino acids (Arg 255, Glu 380) are placed around a hydrophobic knob
formed by Ile 253 and Ser 254. This (can) result in the
establishment of polar and hydrophilic interactions (see
Salvalaglio supra).
[0011] In general, the protein A-IgG interaction can be described
using two main binding sites: the first is positioned in the heavy
chain CH2 domain and is characterized by hydrophobic interactions
between Phe 132, Leu 136, Ile 150 (of protein A) and the IgG
hydrophobic knob constituted by Ile 253 and Ser 254, and by one
electrostatic interaction between Lys 154 (protein A) and Thr 256
(IgG). The second site is located in the heavy chain CH3 domain and
is dominated by electrostatic interactions between Gln 129 and Tyr
133 (protein A) and His 433, Asn 434, and His 435 (IgG) (see
Salvalaglio supra).
[0012] Lindhofer, H., et al. (J. Immunol. 155 (1995) 219-225)
report preferential species-restricted heavy/light chain pairing in
rat/mouse quadromas.
[0013] Jedenberg, L., et al. (J. Immunol. Meth. 201 (1997) 25-34)
reported that SPA-binding analyses of two Fc variants (Fc13 and
Fc31, each containing an isotypic dipeptide substitution from the
respective other isotype) showed that Fc1 and Fc31 interact with
SPA, while Fc3 and Fc13 lack detectable SPA binding. The rendered
SPA binding of the Fc-region variant Fc31 is concluded to result
from the introduced dipeptide substitution R435H and F436Y.
[0014] Today the focus with respect to therapeutic monoclonal
antibodies is on the generation and use of bispecific or even
multispecific antibodies specifically binding to two or more
targets (antigens).
[0015] The basic challenge in generating multispecific
heterodimeric IgG antibodies from four antibody chains (two
different heavy chains and two different light chains) in one
expression cell line is the so-called chain association issue (see
Klein, C., et al., mAbs 4 (2012) 653-663). The required use of
different chains as the left and the right arm of the multispecific
antibody leads to antibody mixtures upon expression in one cell:
the two heavy chains are able to (theoretically) associate in four
different combinations (two thereof are identical), and each of
those can associate in a stochastic manner with the light chains,
resulting in 2.sup.4 (=a total of 16) theoretically possible chain
combinations. Of the 16 theoretically possible combinations ten can
be found of which only one corresponds to the desired functional
bispecific antibody (De Lau, W. B., et al., J. Immunol. 146 (1991)
906-914). The difficulties in isolating this desired bispecific
antibody out of complex mixtures and the inherent poor yield of
12.5% at a theoretical maximum make the production of a bispecific
antibody in one expression cell line extremely challenging.
[0016] To overcome the chain association issue and enforce the
correct association of the two different heavy chains, in the late
1990s Carter et al. from Genentech invented an approach termed
"knobs-into-holes" (KiH) (see Carter, P., J. Immunol. Meth. 248
(2001) 7-15; Merchant, A. M., et al., Nat. Biotechnol. 16 (1998)
677-681; Zhu, Z., et al., Prot. Sci. 6 (1997) 781-788; Ridgway, J.
B., et al., Prot. Eng. 9 (1996) 617-621; Atwell, S., et al., J.
Mol. Biol. 270(1997) 26-35; and U.S. Pat. No. 7,183,076).
Basically, the concept relies on modifications of the interface
between the two CH3 domains of the two heavy chains of an antibody
where most interactions occur. A bulky residue is introduced into
the CH3 domain of one antibody heavy chain and acts similarly to a
key ("knob"). In the other heavy chain, a "hole" is formed that is
able to accommodate this bulky residue, mimicking a lock. The
resulting heterodimeric Fc-region can be further stabilized by the
introduction/formation of artificial disulfide bridges. Notably,
all KiH mutations are buried within the CH3 domains and not
"visible" to the immune system. In addition, properties of
antibodies with KiH mutations such as (thermal) stability,
Fc.gamma.R binding and effector functions (e.g., ADCC, FcRn
binding) and pharmacokinetic (PK) behavior are not affected.
[0017] Correct heavy chain association with heterodimerization
yields above 97% can be achieved by introducing six mutations:
S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A,
Y407V in the "hole" heavy chain (see Carter supra; numbering of the
residues according to the Kabat EU index numbering system). While
hole-hole homodimers may occur, knob-knob homodimers typically are
not observed. Hole-hole dimers can either be depleted by selective
purification procedures or by procedures as outlined below.
[0018] While the issue of random heavy chain association has been
addressed, also correct light chain association has to be ensured.
Similar to the KiH CH3 domain approach, efforts have been
undertaken to investigate asymmetric light chain-heavy chain
interactions that might ultimately lead to full bispecific
IgGs.
[0019] Roche recently developed the CrossMab approach as a
possibility to enforce correct light chain pairing in bispecific
heterodimeric IgG antibodies when combining it with the KiH
technology (see Klein supra; Schaefer. W., et al., Proc. Natl.
Acad. Sci. USA 108 (2011) 11187-11192; Cain, C., SciBX 4 (2011)
1-4). This allows the generation of bispecific or even
multispecific antibodies in a generic fashion. In this format, one
arm of the intended bispecific antibody is left untouched. In the
second arm, the whole Fab region, or the VH-VL domains or the
CH1-CL domains are exchanged by domain crossover between the heavy
and light chain. As a consequence, the newly formed "crossed" light
chain does not associate with the (normal, i.e. not-crossed) heavy
chain Fab region of the other arm of the bispecific antibody any
longer. Thus, the correct "light chain" association can be enforced
by this minimal change in domain arrangement (see Schaefer
supra).
[0020] Zhu et al. introduced several sterically complementary
mutations, as well as disulfide bridges, in the two VL/VH
interfaces of diabody variants. When the mutations VL Y87A/F98M and
VH V37F/L45W were introduced into the anti-p185HER2 VL/VH
interface, a heterodimeric diabody was recovered with >90% yield
while maintaining overall yield and affinity compared with the
parental diabody (see Zhu supra).
[0021] Researchers from Chugai have similarly designed bispecific
diabodies by introduction of mutations into the VH-VL interfaces
(mainly conversion of Q39 in VH and Q38 in VL to charged residues)
to foster correct light chain association (WO 2006/106905; Igawa,
T., et al., Prot. Eng. Des. Sel. 23 (2010) 667-677).
[0022] In WO2011097603 a common light chain mouse is reported.
[0023] In WO2010151792 a bispecific antibody format providing ease
of isolation is provided, comprising immunoglobulin heavy chain
variable domains that are differentially modified, i.e.
heterodimeric, in the CH3 domain, wherein the differential
modifications are non-immunogenic or substantially non-immunogenic
with respect to the CH3 modifications, and at least one of the
modifications results in a differential affinity for the bispecific
antibody for an affinity reagent such as protein A, and the
bispecific antibody is isolable from a disrupted cell, from medium,
or from a mixture of antibodies based on its affinity for protein
A.
[0024] The neonatal Fc-receptor (FcRn) is important for the
metabolic fate of antibodies of the IgG class in vivo. The FcRn
functions to salvage IgG from the lysosomal degradation pathway,
resulting in reduced clearance and increased half-life. It is a
heterodimeric protein consisting of two polypeptides: a 50 kDa
class I major histocompatibility complex-like protein
(.alpha.-FcRn) and a 15 kDa .beta.2-microglobulin (.beta.2m). FcRn
binds with high affinity to the CH2-CH3 portion of the Fc-region of
an antibody of the class IgG. The interaction between an antibody
of the class IgG and the FcRn is pH dependent and occurs in a 1:2
stoichiometry, i.e. one IgG antibody molecule can interact with two
FcRn molecules via its two heavy chain Fc-region polypeptides (see
e.g. Huber, A. H., et al., J. Mol. Biol. 230 (1993) 1077-1083).
[0025] Thus, an IgGs in vitro FcRn binding
properties/characteristics are indicative of its in vivo
pharmacokinetic properties in the blood circulation.
[0026] In the interaction between the FcRn and the Fc-region of an
antibody of the IgG class different amino acid residues of the
heavy chain CH2- and CH3-domain are participating.
[0027] Different mutations that influence the FcRn binding and
therewith the half-live in the blood circulation are known.
Fc-region residues critical to the mouse Fc-region-mouse FcRn
interaction have been identified by site-directed mutagenesis (see
e.g. Dall'Acqua, W. F., et al. J. Immunol 169 (2002) 5171-5180).
Residues 1253, H310, H433, N434, and H435 (numbering according to
Kabat EU index numbering system) are involved in the interaction
(Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533-2536; Firan,
M., et al., Int. Immunol. 13 (2001) 993-1002; Kim, J. K., et al.,
Eur. J. Immunol. 24 (1994) 542-548). Residues 1253, H310, and H435
were found to be critical for the interaction of human Fc-region
with murine FcRn (Kim, J. K., et al., Eur. J. Immunol. 29 (1999)
2819-2885).
[0028] Methods to increase Fc-region (and likewise IgG) binding to
FcRn have been performed by mutating various amino acid residues in
the Fc-region: Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu
380, Met 428, His 433, and Asn 434 (see Kuo, T. T., et al., J.
Clin. Immunol. 30 (2010) 777-789; Ropeenian, D. C., et al., Nat.
Rev. Immunol. 7 (2007) 715-725).
[0029] The combination of the mutations M252Y, S254T, T256E has
been described by Dall'Acqua et al. to improve FcRn binding by
protein-protein interaction studies (Dall'Acqua, W. F., et al. J.
Biol. Chem. 281 (2006) 23514-23524). Studies of the human
Fc-region-human FcRn complex have shown that residues 1253, S254,
H435, and Y436 are crucial for the interaction (Firan, M., et al.,
Int. Immunol. 13 (2001) 993-1002; Shields, R. L., et al., J. Biol.
Chem. 276 (2001) 6591-6604). In Yeung, Y. A., et al. (J. Immunol.
182 (2009) 7667-7671) various mutants of residues 248 to 259 and
301 to 317 and 376 to 382 and 424 to 437 have been reported and
examined.
[0030] In US 2012/0009182 immunoglobulin variants with altered
binding to protein A are reported. The alteration of FcRn binding
affinities or serum half-lives of antibodies by mutagenesis is
reported in WO 2004/035752.
SUMMARY OF THE INVENTION
[0031] Herein are reported variant Fc-regions that specifically
bind to Staphylococcus protein A and that do or do not bind to
human FcRn. These variant Fc-regions contain specific amino acid
mutations in the CH2-domain whereas the CH3-domain is not changed
with respect to protein A binding. It has been found that these
mutations when used in the hole-chain of a heterodimeric Fc-region
allow for the purification of the heterodimeric Fc-region, i.e. the
separation of the heterodimeric Fc-region from the homodimeric
Fc-region by-product (hole-chain-hole-chain dimer).
[0032] One aspect as reported herein is a heterodimeric polypeptide
comprising [0033] a first polypeptide comprising in N-terminal to
C-terminal direction at least a portion of an immunoglobulin hinge
region, which comprises one or more cysteine residues, an
immunoglobulin CH2-domain and an immunoglobulin CH3-domain, and a
second polypeptide comprising in N-terminal to C-terminal direction
at least a portion of an immunoglobulin hinge region, which
comprises one or more cysteine residues, an immunoglobulin
CH2-domain and an immunoglobulin CH3-domain, [0034] wherein the
first polypeptide comprises the mutations Y349C, T366S, L368A and
Y407V (hole-chain) and the second polypeptide comprises the
mutations S354C and T366W (knob-chain), [0035] and [0036] wherein
the first polypeptide (hole-chain) comprises the mutations [0037]
i) I253A or I253G, and [0038] ii) L314A or L314G or L314D, [0039]
and [0040] wherein the first polypeptide and the second polypeptide
are connected by one, two or more disulfide bridges, [0041] and
[0042] wherein the CH3-domain of the first polypeptide and the
CH3-domain of the second polypeptide both bind or both do not bind
to protein A [0043] (numbering according to the Kabat EU
index).
[0044] In one embodiment the first polypeptide (hole-chain)
comprises the mutations [0045] i) I253A or I253G, and [0046] ii)
L314A or L314G or L314D, and [0047] iii) T250Q, and/or [0048] iv)
T256E or T256A.
[0049] In one embodiment the first polypeptide (hole-chain)
comprises the mutations [0050] i) I253A or I253G, and [0051] ii)
L314A or L314G or L314D, and [0052] iii) optionally a) T250Q,
and/or T256E or T256A, and [0053] iv) a) L251A or L251G or L251D,
and/or b) H310A or H310G.
[0054] In one embodiment the first polypeptide (hole-chain)
comprises the mutation [0055] i) I253A or I253G, and [0056] ii)
L314A or L314G or L314D, and [0057] iii) a) T250Q, and/or T256E or
T256A, and. [0058] iv) a) L251A or L251G or L251D, and/or b) H310A
or H310G. [0059] v) optionally a) T307A or T307H or T307Q or T307P,
and/or b) Q311H, and/or c) M252Y, and/or d) S254T.
[0060] In one embodiment the second polypeptide (knob-chain)
comprises the mutation [0061] i) T250Q, and/or [0062] ii) M252Y,
and/or [0063] iii) S254T, and/or [0064] iv) T256E or T256A, and/or
[0065] v) T307A or T307H or T307Q or T307P, and/or [0066] vi)
Q311H.
[0067] In one embodiment the immunoglobulin hinge region, the
immunoglobulin CH2-domain and the immunoglobulin CH3-domain of the
first and the second polypeptide are of the human IgG1 subclass. In
one embodiment the first polypeptide and the second polypeptide
each further comprise the mutations L234A and L235A. In one
embodiment the first polypeptide and the second polypeptide each
further comprise the mutation P329G. In one embodiment the first
polypeptide and the second polypeptide each further comprise the
mutations L234A, L235A and P329G.
[0068] In one embodiment the immunoglobulin hinge region, the
immunoglobulin CH2-domain and the immunoglobulin CH3-domain of the
first and the second polypeptide are of the human IgG4 subclass. In
one embodiment the first polypeptide and the second polypeptide
each further comprise the mutations S228P and L235E. In one
embodiment the first polypeptide and the second polypeptide each
further comprise the mutation P329G. In one embodiment the first
polypeptide and the second polypeptide each further comprise the
mutations S228P, L235E and P329G.
[0069] In one embodiment the heterodimeric polypeptide is an
Fc-region fusion polypeptide.
[0070] In one embodiment the heterodimeric polypeptide is a
full-length antibody.
[0071] In one embodiment the full-length antibody comprises a first
polypeptide (hole-chain) with in addition to the hole-mutations the
mutations I253A and L314A and a second polypeptide (knob-chain)
with the knob-mutations (numbering according to Kabat).
[0072] In one embodiment the full-length antibody comprises a first
polypeptide (hole-chain) with in addition to the hole-mutations the
mutations L251A and L314A and a second polypeptide (knob-chain)
with the knob-mutations (numbering according to Kabat).
[0073] In one embodiment the full-length antibody comprises a first
polypeptide (hole-chain) with in addition to the hole-mutations the
mutation L251A and a second polypeptide (knob-chain) with the
knob-mutations (numbering according to Kabat).
[0074] In one embodiment the full-length antibody comprises a first
polypeptide (hole-chain) with in addition to the hole-mutations the
mutation I253A and a second polypeptide (knob-chain) with the
knob-mutations (numbering according to Kabat).
[0075] In one embodiment the full-length antibody comprises a first
polypeptide (hole-chain) with in addition to the hole-mutations the
mutation L314 A and a second polypeptide (knob-chain) with the
knob-mutations (numbering according to Kabat).
[0076] In one embodiment the full-length antibody comprises a first
polypeptide (hole-chain) with in addition to the hole-mutations the
mutation H310A and a second polypeptide (knob-chain) with the
knob-mutations (numbering according to Kabat).
[0077] In one embodiment the full-length antibody comprises a first
polypeptide (hole-chain) with in addition to the hole-mutations the
mutations L251A, I253A and L314A and a second polypeptide
(knob-chain) with the knob-mutations (numbering according to
Kabat).
[0078] In one embodiment the full-length antibody comprises a first
polypeptide (hole-chain) with in addition to the hole-mutations the
mutations L251A, I253A and L314A and a second polypeptide
(knob-chain) with in addition to the knob-mutations the mutation
M252Y, S254T and T256E (numbering according to Kabat).
[0079] In one embodiment the full-length antibody comprises a first
polypeptide (hole-chain) with in addition to the hole-mutations the
mutations I253A, L314A, M428L and N434H and a second polypeptide
(knob-chain) with in addition to the knob-mutations the mutation
M252Y, S254T and T256E (numbering according to Kabat).
[0080] In one embodiment the full-length antibody further in
addition comprises one or more of the mutations selected from the
group comprising S17A, R19A, T57A, T57K, R66A, S70A, Y79A, Q81A,
N82aA and S82bA in the heavy chain variable domain (numbering
according to Kabat). In one embodiment the full-length antibody
comprises one or more of the mutations selected from the group
consisting of S17A, R19A, T57A, T57K, R66A, Q81A and N82aA in the
heavy chain variable domain and has reduced binding to protein A
compared to an antibody not having these mutations but having
otherwise the identical amino acid sequence (numbering according to
Kabat). In one embodiment the full-length antibody comprises one or
more of the mutations selected from the group consisting of S70A,
Y79A and S82bA in the heavy chain variable domain and has increased
binding to protein A compared to an antibody not having these
mutations but having otherwise the identical amino acid sequence
(numbering according to Kabat).
[0081] In one embodiment the full length antibody is a monospecific
antibody. In one embodiment the monospecific antibody is a
monovalent monospecific antibody. In one embodiment the
monospecific antibody is a bivalent monospecific antibody.
[0082] In one embodiment the full length antibody is a bispecific
antibody. In one embodiment the bispecific antibody is a bivalent
bispecific antibody. In one embodiment the bispecific antibody is a
tetravalent bispecific antibody.
[0083] In one embodiment the full length antibody is a trispecific
antibody. In one embodiment the trispecific antibody is a trivalent
trispecific antibody. In one embodiment the trispecific antibody is
a tetravalent trispecific antibody.
[0084] In one embodiment the heterodimeric polypeptide is a
bispecific full length antibody comprising [0085] a first
polypeptide comprising in N-terminal to C-terminal direction a
first heavy chain variable domain, an immunoglobulin CH1-domain of
the subclass IgG1, an immunoglobulin hinge region of the subclass
IgG1, an immunoglobulin CH2-domain of the subclass IgG1 and an
immunoglobulin CH3-domain of the subclass IgG1, [0086] a second
polypeptide comprising in N-terminal to C-terminal direction a
second heavy chain variable domain, an immunoglobulin CH1-domain of
the subclass IgG1, an immunoglobulin hinge region of the subclass
IgG1, an immunoglobulin CH2-domain of the subclass IgG1 and an
immunoglobulin CH3-domain of the subclass IgG1, [0087] a third
polypeptide comprising in N-terminal to C-terminal direction a
first light chain variable domain and a light chain constant
domain, [0088] a fourth polypeptide comprising in N-terminal to
C-terminal direction a second light chain variable domain and a
light chain constant domain, [0089] wherein the first heavy chain
variable domain and the first light chain variable domain form a
first binding site that specifically binds to a first antigen,
[0090] wherein the second heavy chain variable domain and the
second light chain variable domain form a second binding site that
specifically binds to a second antigen, [0091] wherein the first
polypeptide comprises the mutations Y349C, T366S, L368A, and Y407V,
L234A, L235A and P329G and the second polypeptide comprises the
mutations S354C, and T366W, L234A, L235A and P329G [0092] and
[0093] wherein the first polypeptide (hole-chain) comprises the
mutations [0094] i) I253A or I253G, and [0095] ii) L314A or L314G
or L314D, [0096] and [0097] wherein the first polypeptide and the
second polypeptide are connected by one or more disulfide bridges,
[0098] and [0099] wherein the CH3-domain of the first polypeptide
and the CH3-domain of the second polypeptide both bind or both do
not bind to protein A [0100] (numbering according to the Kabat EU
index).
[0101] In one embodiment the heterodimeric polypeptide is a
bispecific full length antibody comprising [0102] a first
polypeptide comprising in N-terminal to C-terminal direction a
first heavy chain variable domain, an immunoglobulin light chain
constant domain, an immunoglobulin hinge region of the subclass
IgG1, an immunoglobulin CH2-domain of the subclass IgG1 and an
immunoglobulin CH3-domain of the subclass IgG1, [0103] a second
polypeptide comprising in N-terminal to C-terminal direction a
second heavy chain variable domain, an immunoglobulin CH1-domain of
the subclass IgG1, an immunoglobulin hinge region of the subclass
IgG1, an immunoglobulin CH2-domain of the subclass IgG1 and an
immunoglobulin CH3-domain of the subclass IgG1, [0104] a third
polypeptide comprising in N-terminal to C-terminal direction a
first light chain variable domain and an immunoglobulin CH1-domain
of the subclass IgG1, [0105] a fourth polypeptide comprising in
N-terminal to C-terminal direction a second light chain variable
domain and a light chain constant domain, [0106] wherein the first
heavy chain variable domain and the first light chain variable
domain form a first binding site that specifically binds to a first
antigen, [0107] wherein the second heavy chain variable domain and
the second light chain variable domain form a second binding site
that specifically binds to a second antigen, [0108] wherein the
first polypeptide comprises the mutations Y349C, T366S, L368A, and
Y407V, L234A, L235A and P329G and the second polypeptide comprises
the mutations S354C, and T366W, L234A, L235A and P329G, [0109] and
[0110] wherein the first polypeptide (hole-chain) comprises the
mutations [0111] i) I253A or I253G, and [0112] ii) L314A or L314G
or L314D, [0113] and [0114] wherein the first polypeptide and the
second polypeptide are connected by one or more disulfide bridges,
[0115] and [0116] wherein the CH3-domain of the first polypeptide
and the CH3-domain of the second polypeptide both bind or both do
not bind to protein A [0117] (numbering according to the Kabat EU
index).
[0118] In one embodiment the heterodimeric polypeptide is a
bispecific full length antibody comprising [0119] a first
polypeptide comprising in N-terminal to C-terminal direction a
first heavy chain variable domain, an immunoglobulin CH1-domain of
the subclass IgG4, an immunoglobulin hinge region of the subclass
IgG4, an immunoglobulin CH2-domain of the subclass IgG4 and an
immunoglobulin CH3-domain of the subclass IgG4, [0120] a second
polypeptide comprising in N-terminal to C-terminal direction a
second heavy chain variable domain, an immunoglobulin CH1-domain of
the subclass IgG4, an immunoglobulin hinge region of the subclass
IgG4, an immunoglobulin CH2-domain of the subclass IgG4 and an
immunoglobulin CH3-domain of the subclass IgG4, [0121] a third
polypeptide comprising in N-terminal to C-terminal direction a
first light chain variable domain and a light chain constant
domain, [0122] a fourth polypeptide comprising in N-terminal to
C-terminal direction a second light chain variable domain and a
light chain constant domain, [0123] wherein the first heavy chain
variable domain and the first light chain variable domain form a
first binding site that specifically binds to a first antigen,
[0124] wherein the second heavy chain variable domain and the
second light chain variable domain form a second binding site that
specifically binds to a second antigen, [0125] wherein the first
polypeptide comprises the mutations Y349C, T366S, L368A, and Y407V,
S228P, L235E and P329G and the second polypeptide comprises the
mutations S354C, and T366W, S228P, L235E and P329G, [0126] and
[0127] wherein the first polypeptide (hole-chain) comprises the
mutations [0128] i) I253A or I253G, and [0129] ii) L314A or L314G
or L314D, [0130] and [0131] wherein the first polypeptide and the
second polypeptide are connected by one or more disulfide bridges,
[0132] and [0133] wherein the CH3-domain of the first polypeptide
and the CH3-domain of the second polypeptide both bind or both do
not bind to protein A [0134] (numbering according to the Kabat EU
index).
[0135] In one embodiment the heterodimeric polypeptide is a
bispecific full length antibody comprising [0136] a first
polypeptide comprising in N-terminal to C-terminal direction a
first heavy chain variable domain, an immunoglobulin light chain
constant domain, an immunoglobulin hinge region of the subclass
IgG4, an immunoglobulin CH2-domain of the subclass IgG4 and an
immunoglobulin CH3-domain of the subclass IgG4, [0137] a second
polypeptide comprising in N-terminal to C-terminal direction a
second heavy chain variable domain, an immunoglobulin CH1-domain of
the subclass IgG4, an immunoglobulin hinge region of the subclass
IgG4, an immunoglobulin CH2-domain of the subclass IgG4 and an
immunoglobulin CH3-domain of the subclass IgG4, [0138] a third
polypeptide comprising in N-terminal to C-terminal direction a
first light chain variable domain and an immunoglobulin CH1-domain
of the subclass IgG4, [0139] a fourth polypeptide comprising in
N-terminal to C-terminal direction a second light chain variable
domain and a light chain constant domain, [0140] wherein the first
heavy chain variable domain and the first light chain variable
domain form a first binding site that specifically binds to a first
antigen, [0141] wherein the second heavy chain variable domain and
the second light chain variable domain form a second binding site
that specifically binds to a second antigen, [0142] wherein the
first polypeptide comprises the mutations Y349C, T366S, L368A, and
Y407V, S228P, L235E and P329G and the second polypeptide comprises
the mutations S354C, and T366W, S228P, L235E and P329G, [0143] and
[0144] wherein the first polypeptide (hole-chain) comprises the
mutations [0145] i) I253A or I253G, and [0146] ii) L314A or L314G
or L314D, [0147] and [0148] wherein the first polypeptide and the
second polypeptide are connected by one or more disulfide bridges,
[0149] and [0150] wherein the CH3-domain of the first polypeptide
and the CH3-domain of the second polypeptide both bind or both do
not bind to protein A [0151] (numbering according to the Kabat EU
index).
[0152] In one embodiment the heterodimeric polypeptide is a
bispecific full length antibody comprising [0153] a first
polypeptide comprising in N-terminal to C-terminal direction a
first heavy chain variable domain, an immunoglobulin CH1-domain of
the subclass IgG1, an immunoglobulin hinge region of the subclass
IgG1, an immunoglobulin CH2-domain of the subclass IgG1, an
immunoglobulin CH3-domain of the subclass IgG1, a peptidic linker
and a first scFv, [0154] a second polypeptide comprising in
N-terminal to C-terminal direction a second heavy chain variable
domain, an immunoglobulin CH1-domain of the subclass IgG1, an
immunoglobulin hinge region of the subclass IgG1, an immunoglobulin
CH2-domain of the subclass IgG1, an immunoglobulin CH3-domain of
the subclass IgG1, a peptidic linker and a second scFv, [0155] a
third polypeptide comprising in N-terminal to C-terminal direction
a first light chain variable domain and a light chain constant
domain, [0156] a fourth polypeptide comprising in N-terminal to
C-terminal direction a second light chain variable domain and a
light chain constant domain, [0157] wherein the first heavy chain
variable domain and the first light chain variable domain form a
first binding site that specifically binds to a first antigen, and
the second heavy chain variable domain and the second light chain
variable domain form a second binding site that specifically binds
to a first antigen, and the first and the second scFv specifically
bind to a second antigen, [0158] wherein the first polypeptide
comprises the mutations Y349C, T366S, L368A, and Y407V, L234A,
L235A and P329G and the second polypeptide comprises the mutations
S354C, and T366W, L234A, L235A and P329G, [0159] and [0160] wherein
the first polypeptide (hole-chain) comprises the mutations [0161]
i) I253A or I253G, and [0162] ii) L314A or L314G or L314D, [0163]
and [0164] wherein the first polypeptide and the second polypeptide
are connected by one or more disulfide bridges, [0165] and [0166]
wherein the CH3-domain of the first polypeptide and the CH3-domain
of the second polypeptide both bind or both do not bind to protein
A [0167] (numbering according to the Kabat EU index).
[0168] In one embodiment the heterodimeric polypeptide is a
bispecific full length antibody comprising [0169] a first
polypeptide comprising in N-terminal to C-terminal direction a
first heavy chain variable domain, an immunoglobulin light chain
constant domain, an immunoglobulin hinge region of the subclass
IgG1, an immunoglobulin CH2-domain of the subclass IgG1, an
immunoglobulin CH3-domain of the subclass IgG1, a peptidic linker
and a first scFv, [0170] a second polypeptide comprising in
N-terminal to C-terminal direction a second heavy chain variable
domain, an immunoglobulin CH1-domain of the subclass IgG1, an
immunoglobulin hinge region of the subclass IgG1, an immunoglobulin
CH2-domain of the subclass IgG1, an immunoglobulin CH3-domain of
the subclass IgG1, a peptidic linker and a second scFv, [0171] a
third polypeptide comprising in N-terminal to C-terminal direction
a first light chain variable domain and an immunoglobulin
CH1-domain of the subclass IgG1, [0172] a fourth polypeptide
comprising in N-terminal to C-terminal direction a second light
chain variable domain and a light chain constant domain, [0173]
wherein the first heavy chain variable domain and the first light
chain variable domain form a first binding site that specifically
binds to a first antigen, and the second heavy chain variable
domain and the second light chain variable domain form a second
binding site that specifically binds to a first antigen, and the
first and the second scFv specifically bind to a second antigen,
[0174] wherein the first polypeptide comprises the mutations Y349C,
T366S, L368A, and Y407V, L234A, L235A and P329G and the second
polypeptide comprises the mutations S354C, and T366W, L234A, L235A
and P329G, [0175] and [0176] wherein the first polypeptide and the
second polypeptide are connected by one or more disulfide
bridges.
[0177] One aspect as reported herein is a method for producing a
heterodimeric polypeptide as reported herein comprising the
following steps: [0178] a) cultivating a mammalian cell comprising
one or more nucleic acids encoding the heterodimeric polypeptide,
[0179] b) recovering the heterodimeric polypeptide from the
cultivation medium, and [0180] c) purifying the heterodimeric
polypeptide with a protein A affinity chromatography and thereby
producing the dimeric polypeptide.
[0181] One aspect as reported herein is the use of the combination
of the mutations [0182] i) I253A or I253G, and [0183] ii) L314A or
L314G or L314D,
[0184] for separating heterodimeric polypeptides from homodimeric
polypeptides.
[0185] One aspect as reported herein is method of treatment of a
patient suffering from ocular vascular diseases by administering a
heterodimeric polypeptide as reported herein to a patient in the
need of such treatment.
[0186] One aspect as reported herein is a heterodimeric polypeptide
as reported herein for intravitreal application.
[0187] One aspect as reported herein is a heterodimeric polypeptide
as reported herein for use as a medicament.
[0188] One aspect as reported herein is a heterodimeric polypeptide
as reported herein for the treatment of vascular eye diseases.
[0189] One aspect as reported herein is a pharmaceutical
formulation comprising a heterodimeric polypeptide as reported
herein and optionally a pharmaceutically acceptable carrier.
[0190] For using an antibody that targets/binds to antigens not
only present in the eye but also in the remaining body a short
systemic half-live after passage of the blood-ocular-barrier from
the eye into the blood is beneficial in order to avoid systemic
side effects.
[0191] Additionally an antibody that specifically binds to ligands
of a receptor is only effective in the treatment of eye-diseases if
the antibody-antigen complex is removed from the eye, i.e. the
antibody functions as a transport vehicle for receptor ligands out
of the eye and thereby inhibits receptor signaling.
[0192] One aspect as reported herein is the use of a heterodimeric
polypeptide as reported herein for the transport of a soluble
receptor ligand from the eye over the blood-ocular-barrier into the
blood circulation.
[0193] One aspect as reported herein is the use of a heterodimeric
polypeptide as reported herein for the removal of one or more
soluble receptor ligands from the eye.
[0194] One aspect as reported herein is the use of a heterodimeric
polypeptide as reported herein for the treatment of eye diseases,
especially of ocular vascular diseases.
[0195] One aspect as reported herein is the use of a heterodimeric
polypeptide as reported herein for the transport of one or more
soluble receptor ligands from the intravitreal space to the blood
circulation.
[0196] One aspect as reported herein is a heterodimeric polypeptide
as reported herein for use in treating an eye disease.
[0197] One aspect as reported herein is a heterodimeric polypeptide
as reported herein for use in the transport of a soluble receptor
ligand from the eye over the blood-ocular-barrier into the blood
circulation.
[0198] One aspect as reported herein is a heterodimeric polypeptide
as reported herein for use in the removal of one or more soluble
receptor ligands from the eye.
[0199] One aspect as reported herein is a heterodimeric polypeptide
as reported herein for use in treating eye diseases, especially
ocular vascular diseases.
[0200] One aspect as reported herein is a heterodimeric polypeptide
as reported herein for use in the transport of one or more soluble
receptor ligands from the intravitreal space to the blood
circulation.
[0201] One aspect as reported herein is a method of treating an
individual having an ocular vascular disease comprising
administering to the individual an effective amount of a
heterodimeric polypeptide as reported herein.
[0202] One aspect as reported herein is a method for transporting a
soluble receptor ligand from the eye over the blood-ocular-barrier
into the blood circulation in an individual comprising
administering to the individual an effective amount of a
heterodimeric polypeptide as reported herein to transport a soluble
receptor ligand from the eye over the blood-ocular-barrier into the
blood circulation.
[0203] One aspect as reported herein is a method the removal of one
or more soluble receptor ligands from the eye in an individual
comprising administering to the individual an effective amount of a
heterodimeric polypeptide as reported herein to remove one or more
soluble receptor ligands from the eye.
[0204] One aspect as reported herein is a method for the transport
of one or more soluble receptor ligands from the intravitreal space
to the blood circulation in an individual comprising administering
to the individual an effective amount of a heterodimeric
polypeptide as reported herein to transport of one or more soluble
receptor ligands from the intravitreal space to the blood
circulation.
[0205] One aspect as reported herein is a method for transporting a
soluble receptor ligand from the intravitreal space or the eye over
the blood-ocular-barrier into the blood circulation in an
individual comprising administering to the individual an effective
amount of a heterodimeric polypeptide as reported herein to
transport a soluble receptor ligand from the eye over the
blood-ocular-barrier into the blood circulation.
[0206] In one embodiment the heterodimeric polypeptide is a
bispecific antibody. In one embodiment the bispecific antibody is a
bivalent bispecific antibody. In one embodiment the bispecific
antibody is a tetravalent bispecific antibody.
[0207] In one embodiment the heterodimeric polypeptide is a
trispecific antibody. In one embodiment the trispecific antibody is
a trivalent trispecific antibody. In one embodiment the trispecific
antibody is a tetravalent trispecific antibody.
[0208] In one embodiment the heterodimeric polypeptide is a
CrossMab.
[0209] In one embodiment the heterodimeric polypeptide is an
Fc-region fusion polypeptide.
[0210] In one embodiment the first polypeptide further comprises
the mutations Y349C, T366S, L368A and Y407V and the second
polypeptide further comprises the mutations S354C and T366W.
[0211] In one embodiment the antibody or the Fc-region fusion
polypeptide is of the subclass IgG1. In one embodiment the antibody
or the Fc-region fusion polypeptide further comprise the mutations
L234A and L235A. In one embodiment the antibody or the Fc-region
fusion polypeptide further comprise the mutation P329G.
[0212] In one embodiment the antibody or the Fc-region fusion
polypeptide is of the subclass IgG4. In one embodiment the antibody
or the Fc-region fusion polypeptide further comprise the mutations
S228P and L235E. In one embodiment the antibody or the Fc-region
fusion polypeptide further comprise the mutation P329G.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. Definitions
[0213] The term "about" denotes a range of +/-20% of the thereafter
following numerical value. In one embodiment the term about denotes
a range of +/-10% of the thereafter following numerical value. In
one embodiment the term about denotes a range of +/-5% of the
thereafter following numerical value.
[0214] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence alterations. In some
embodiments, the number of amino acid alterations are 10 or less, 9
or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3
or less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0215] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more hypervariable regions
(HVRs), compared to a parent antibody, which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
[0216] The term "alteration" denotes the mutation (substitution),
insertion (addition), or deletion of one or more amino acid
residues in a parent antibody or fusion polypeptide, e.g. a fusion
polypeptide comprising at least an FcRn binding portion of an
Fc-region, to obtain a modified antibody or fusion polypeptide. The
term, mutation" denotes that the specified amino acid residue is
substituted for a different amino acid residue. For example the
mutation L234A denotes that the amino acid residue lysine at
position 234 in an antibody Fc-region (polypeptide) is substituted
by the amino acid residue alanine (substitution of lysine with
alanine) (numbering according to the Kabat EU index numbering
system).
[0217] A "naturally occurring amino acid residues" denotes an amino
acid residue from the group consisting of alanine (three letter
code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn,
N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q),
glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H),
isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine
(Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser,
S), threonine (Thr, T), tryptophane (Trp, W), tyrosine (Tyr, Y),
and valine (Val, V).
[0218] The term "amino acid mutation" denotes the substitution of
at least one existing amino acid residue with another different
amino acid residue (=replacing amino acid residue). The replacing
amino acid residue may be a "naturally occurring amino acid
residues" and selected from the group consisting of alanine (three
letter code: Ala, one letter code: A), arginine (Arg, R),
asparagine (Asn, N), aspartic acid (asp, D), cysteine (Cys, C),
glutamine (Gln, Q), glutamic acid (Glu, E), glycine (glee, G),
histidine (his, H), isoleucine (lie, I), leucine (Leu, L), lysine
(lees, K), methionine (met, M), phenylalanine (Phe, F), proline
(pro, P), serine (seer, S), threonine (Thr, T), tryptophan (Trp,
W), tyrosine (try, Y), and valine (Val, V). The replacing amino
acid residue may be a "non-naturally occurring amino acid residue".
See e.g. U.S. Pat. No. 6,586,207, WO 98/48032, WO 03/073238, US
2004/0214988, WO 2005/35727, WO 2005/74524, Chin, J. W., et al., J.
Am. Chem. Soc. 124 (2002) 9026-9027; Chin, J. W. and Schultz, P.
G., ChemBioChem 11 (2002) 1135-1137; Chin, J. W., et al., PICAS
United States of America 99 (2002) 11020-11024; and, Wang, L. and
Schultz, P. G., Chem. (2002) 1-10 (all entirely incorporated by
reference herein).
[0219] The term "amino acid deletion" denotes the removal of at
least one amino acid residue at a predetermined position in an
amino acid sequence.
[0220] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, multispecific antibodies (e.g. bispecific
antibodies, trispecific antibodies), and antibody fragments so long
as they exhibit the desired antigen-, and/or protein A and/or
FcRn-binding activity.
[0221] The term "asymmetric Fc-region" denotes a pair of Fc-region
polypeptides that have different amino acid residues at
corresponding positions according to the Kabat EU index numbering
system.
[0222] The term "asymmetric Fc-region with respect to FcRn binding"
denotes an Fc-region that consists of two polypeptide chains that
have different amino acid residues at corresponding positions,
whereby the positions are determined according to the Kabat EU
index numbering system, whereby the different positions affect the
binding of the Fc-region to the human neonatal Fc-receptor (FcRn).
For the purpose herein the differences between the two polypeptide
chains of the Fc-region in an "asymmetric Fc-region with respect to
FcRn binding" do not include differences that have been introduced
to facilitate the formation of heterodimeric Fc-regions, e.g. for
the production of bispecific antibodies. These differences can also
be asymmetric, i.e. the two chains have differences at
non-corresponding amino acid residues according to the Kabat EU
index numbering system. These differences facilitate
heterodimerization and reduce homodimerization. Examples of such
differences are the so-called "knobs into holes" substitutions
(see, e.g., U.S. Pat. No. 7,695,936 and US 2003/0078385). The
following knobs and holes substitutions in the individual
polypeptide chains of an Fc-region of an IgG antibody of subclass
IgG1 have been found to increase heterodimer formation: 1) Y407T in
one chain and T366Y in the other chain; 2) Y407A in one chain and
T366W in the other chain; 3) F405A in one chain and T394W in the
other chain; 4) F405W in one chain and T394S in the other chain; 5)
Y407T in one chain and T366Y in the other chain; 6) T366Y and F405A
in one chain and T394W and Y407T in the other chain; 7) T366W and
F405W in one chain and T394S and Y407A in the other chain; 8) F405W
and Y407A in one chain and T366W and T394S in the other chain; and
9) T366W in one chain and T366S, L368A, and Y407V in the other
chain, whereby the last listed is especially suited. In addition,
changes creating new disulfide bridges between the two Fc-region
polypeptide chains facilitate heterodimer formation (see, e.g., US
2003/0078385). The following substitutions resulting in
appropriately spaced apart cysteine residues for the formation of
new intra-chain disulfide bonds in the individual polypeptide
chains of an Fc-region of an IgG antibody of subclass IgG1 have
been found to increase heterodimer formation: Y349C in one chain
and S354C in the other; Y349C in one chain and E356C in the other;
Y349C in one chain and E357C in the other; L351C in one chain and
S354C in the other; T394C in one chain and E397C in the other; or
D399C in one chain and K392C in the other. Further examples of
heterodimerization facilitating amino acid changes are the
so-called "charge pair substitutions" (see, e.g., WO 2009/089004).
The following charge pair substitutions in the individual
polypeptide chains of an Fc-region of an IgG antibody of subclass
IgG1 have been found to increase heterodimer formation: 1) K409D or
K409E in one chain and D399K or D399R in the other chain; 2) K392D
or K392E in one chain and D399K or D399R in the other chain; 3)
K439D or K439E in one chain and E356K or E356R in the other chain;
4) K370D or K370E in one chain and E357K or E357R in the other
chain; 5) K409D and K360D in one chain plus D399K and E356K in the
other chain; 6) K409D and K370D in one chain plus D399K and E357K
in the other chain; 7) K409D and K392D in one chain plus D399K,
E356K, and E357K in the other chain; 8) K409D and K392D in one
chain and D399K in the other chain; 9) K409D and K392D in one chain
and D399K and E356K in the other chain; 10) K409D and K392D in one
chain and D399K and D357K in the other chain; 11) K409D and K370D
in one chain and D399K and D357K in the other chain; 12) D399K in
one chain and K409D and K360D in the other chain; and 13) K409D and
K439D in one chain and D399K and E356K on the other.
[0223] The term "binding (to an antigen)" denotes the binding of an
antibody to its antigen in an in vitro assay, in one embodiment in
a binding assay in which the antibody is bound to a surface and
binding of the antigen to the antibody is measured by Surface
Plasmon Resonance (SPR). Binding means a binding affinity (K.sub.D)
of 10.sup.-8 M or less, in some embodiments of 10.sup.-13 to
10.sup.-8 M, in some embodiments of 10.sup.-13 to 10.sup.-9 M.
[0224] Binding can be investigated by a BIAcore assay (GE
Healthcare Biosensor AB, Uppsala, Sweden). The affinity of the
binding is defined by the terms k.sub.a (rate constant for the
association of the antibody from the antibody/antigen complex),
k.sub.d (dissociation constant), and K.sub.D(k.sub.d/k.sub.a).
[0225] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0226] The term "CH2-domain" denotes the part of an antibody heavy
chain polypeptide that extends approximately from EU position 231
to EU position 340 (EU numbering system according to Kabat). In one
embodiment a CH2 domain has the amino acid sequence of SEQ ID NO:
01: APELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA
KTKPREEQ E STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAK.
[0227] The term "CH3-domain" denotes the part of an antibody heavy
chain polypeptide that extends approximately from EU position 341
to EU position 446. In one embodiment the CH3 domain has the amino
acid sequence of SEQ ID NO: 02: GQPREPQ VYTLPPSRDE LTKNQVSLTC
LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV
MHEALHNHYT QKSLSLSPG.
[0228] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0229] The term "comparable length" denotes that two polypeptides
comprise the identical number of amino acid residues or can be
different in length by one or more and up to 10 amino acid residues
at most. In one embodiment the (Fc-region) polypeptides comprise
the identical number of amino acid residues or differ by a number
of from 1 to 10 amino acid residues. In one embodiment the
(Fc-region) polypeptides comprise the identical number of amino
acid residues or differ by a number of from 1 to 5 amino acid
residues. In one embodiment the (Fc-region) polypeptides comprise
the identical number of amino acid residues or differ by a number
of from 1 to 3 amino acid residues.
[0230] "Effector functions" refer to those biological activities
attributable to the Fc-region of an antibody, which vary with the
antibody class. Examples of antibody effector functions include: C1
q binding and complement dependent cytotoxicity (CDC); Fc receptor
binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g. B
cell receptor); and B-cell activation.
[0231] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0232] The term "Fc-fusion polypeptide" denotes a fusion of a
binding domain (e.g. an antigen binding domain such as a single
chain antibody, or a polypeptide such as a ligand of a receptor)
with an antibody Fc-region that exhibits the desired target-,
protein A- and FcRn-binding activity.
[0233] The term "Fc-region of human origin" denotes the C-terminal
region of an immunoglobulin heavy chain of human origin that
contains at least a part of the hinge region, the CH2 domain and
the CH3 domain. In one embodiment, a human IgG heavy chain
Fc-region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. In one embodiment the
Fc-region has the amino acid sequence of SEQ ID NO: 03. However,
the C-terminal lysine (Lys447) of the Fc-region may or may not be
present.
[0234] As used herein, the amino acid positions of all constant
regions and domains of the heavy and light chain are numbered
according to the Kabat numbering system described in Kabat, et al.,
Sequences of Proteins of Immunological Interest, 5th ed., Public
Health Service, National Institutes of Health, Bethesda, Md. (1991)
and is referred to as "numbering according to Kabat" herein.
Specifically the Kabat numbering system (see pages 647-660) of
Kabat, et al., Sequences of Proteins of Immunological Interest, 5th
ed., Public Health Service, National Institutes of Health,
Bethesda, Md. (1991) is used for the light chain constant domain CL
of kappa and lambda isotype and the Kabat EU index numbering system
(see pages 661-723) is used for the constant heavy chain domains
(CH1, Hinge, CH2 and CH3).
[0235] The term "FcRn" denotes the human neonatal Fc-receptor. FcRn
functions to salvage IgG from the lysosomal degradation pathway,
resulting in reduced clearance and increased half-life. The FcRn is
a heterodimeric protein consisting of two polypeptides: a 50 kDa
class I major histocompatibility complex-like protein
(.alpha.-FcRn) and a 15 kDa .beta.2-microglobulin .beta.2m). FcRn
binds with high affinity to the CH2-CH3 portion of the Fc-region of
IgG. The interaction between IgG and FcRn is strictly pH dependent
and occurs in a 1:2 stoichiometry, with one IgG binding to two FcRn
molecules via its two heavy chains (Huber, A. H., et al., J. Mol.
Biol. 230 (1993) 1077-1083). FcRn binding occurs in the endosome at
acidic pH (pH<6.5) and IgG is released at the neutral cell
surface (pH of about 7.4). The pH-sensitive nature of the
interaction facilitates the FcRn-mediated protection of IgGs
pinocytosed into cells from intracellular degradation by binding to
the receptor within the acidic environment of endosomes. FcRn then
facilitates the recycling of IgG to the cell surface and subsequent
release into the blood stream upon exposure of the FcRn-IgG complex
to the neutral pH environment outside the cell.
[0236] The term "FcRn binding portion of an Fc-region" denotes the
part of an antibody heavy chain polypeptide that extends
approximately from EU position 243 to EU position 261 and
approximately from EU position 275 to EU position 293 and
approximately from EU position 302 to EU position 319 and
approximately from EU position 336 to EU position 348 and
approximately from EU position 367 to EU position 393 and EU
position 408 and approximately from EU position 424 to EU position
440. In one embodiment one or more of the following amino acid
residues according to the EU numbering of Kabat are altered F243,
P244, P245 P, K246, P247, K248, D249, T250, L251, M252, 1253, S254,
R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278,
V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290,
P291, R292, E293, V302, V303, S304, V305, L306, T307, V308, L309,
H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, 1336,
S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347,
V348, C367, V369, F372, Y373, P374, S375, D376, 1377, A378, V379,
E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391,
T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432,
H433, N434, H435, Y436, T437, Q438, K439, and S440 (EU
numbering).
[0237] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0238] The term "full length antibody" denotes an antibody having a
structure substantially similar to a native antibody structure
comprising four polypeptides or having heavy chains that contain an
Fc-region as defined herein. A full-length antibody may comprise
further domains, such as e.g. a scFv or a scFab conjugated to one
or more of the chains of the full-length antibody. These conjugates
are also encompassed by the term full-length antibody.
[0239] The term "dimeric polypeptide" denotes a complex comprising
at least two polypeptides that are associated covalently. The
complex may comprise further polypeptides that are also associated
covalently or non-covalently with the other polypeptides. In one
embodiment the dimeric polypeptide comprises two or four
polypeptides.
[0240] The terms "heterodimer" or "heterodimeric" denote a molecule
that comprises two polypeptides (e.g. of comparable length),
wherein the two polypeptides have an amino acid sequence that have
at least one different amino acid residue in a corresponding
position, whereby corresponding position is determined according to
the Kabat EU index numbering system.
[0241] The terms "homodimer" and "homodimeric" denote a molecule
that comprises two polypeptides of comparable length, wherein the
two polypeptides have an amino acid sequence that is identical in
corresponding positions, whereby corresponding positions are
determined according to the Kabat EU index numbering system.
[0242] A heterodimeric polypeptide as reported herein is
heterodimeric determined with respect to mutations or properties in
focus. For example, with respect to FcRn and/or protein A binding
(i.e. the focused on properties) a dimeric polypeptide is
homodimeric (i.e. both polypeptides of the dimeric polypeptide
comprise these mutations) with respect to the mutations H310A,
H433A and Y436A (these mutations are in focus with respect to FcRn
and/or protein A binding property of the dimeric polypeptide) but
at the same time heterodimeric with respect to the mutations Y349C,
T366S, L368A and Y407V (these mutations are not in focus as these
mutations are directed to the heterodimerization of the dimeric
polypeptide and not to the FcRn/protein A binding properties) as
well as the mutations S354C and T366W, respectively (the first set
is comprised only in the first polypeptide whereas the second set
is comprised only in the second polypeptide). Further for example,
a dimeric polypeptide as reported herein can be heterodimeric with
respect to the mutations I253A, H310A, H433A, H435A and Y436A (i.e.
these mutations are directed all to the FcRn and/or protein A
binding properties of the dimeric polypeptide), i.e. one
polypeptide comprises the mutations I253A, H310A and H435A, whereas
the other polypeptide comprises the mutations H310A, H433A and
Y436A.
[0243] The terms "host cell", "host cell line", and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0244] A "human antibody" is one that possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0245] A "human consensus framework" is a framework, which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat, E. A. et al.,
Sequences of Proteins of Immunological Interest, 5th ed., Bethesda
Md. (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment,
for the VL, the subgroup is subgroup kappa I as in Kabat et al.,
supra. In one embodiment, for the VH, the subgroup is subgroup III
as in Kabat et al., supra.
[0246] The term "derived from" denotes that an amino acid sequence
is derived from a parent amino acid sequence by introducing
alterations at at least one position. Thus a derived amino acid
sequence differs from the corresponding parent amino acid sequence
at at least one corresponding position (numbering according to
Kabat EU index for antibody Fc-regions). In one embodiment an amino
acid sequence derived from a parent amino acid sequence differs by
one to fifteen amino acid residues at corresponding positions. In
one embodiment an amino acid sequence derived from a parent amino
acid sequence differs by one to ten amino acid residues at
corresponding positions. In one embodiment an amino acid sequence
derived from a parent amino acid sequence differs by one to six
amino acid residues at corresponding positions. Likewise a derived
amino acid sequence has a high amino acid sequence identity to its
parent amino acid sequence. In one embodiment an amino acid
sequence derived from a parent amino acid sequence has 80% or more
amino acid sequence identity. In one embodiment an amino acid
sequence derived from a parent amino acid sequence has 90% or more
amino acid sequence identity. In one embodiment an amino acid
sequence derived from a parent amino acid sequence has 95% or more
amino acid sequence identity.
[0247] The term "human Fc-region polypeptide" denotes an amino acid
sequence, which is identical to a "native" or "wild-type" human
Fc-region polypeptide. The term "variant (human) Fc-region
polypeptide" denotes an amino acid sequence, which is derived from
a "native" or "wild-type" human Fc-region polypeptide by virtue of
at least one "amino acid alteration". A "human Fc-region" is
consisting of two human Fc-region polypeptides. A "variant (human)
Fc-region" is consisting of two Fc-region polypeptides, whereby
both can be variant (human) Fc-region polypeptides or one is a
human Fc-region polypeptide and the other is a variant (human)
Fc-region polypeptide.
[0248] In one embodiment the human Fc-region polypeptide has the
amino acid sequence of a human IgG1 Fc-region polypeptide of SEQ ID
NO: 03, or of a human IgG2 Fc-region polypeptide of SEQ ID NO: 04,
or of a human IgG4 Fc-region polypeptide of SEQ ID NO: 06 with the
mutations as reported herein. In one embodiment the variant (human)
Fc-region polypeptide is derived from an Fc-region polypeptide of
SEQ ID NO: 03, or 04, or 06 and has at least one amino acid
mutation compared to the Fc-region polypeptide of SEQ ID NO: 03, or
04, or 06. In one embodiment the variant (human) Fc-region
polypeptide comprises/has from about one to about ten amino acid
mutations, and in one embodiment from about one to about five amino
acid mutations. In one embodiment the variant (human) Fc-region
polypeptide has at least about 80% homology with a human Fc-region
polypeptide of SEQ ID NO: 03, or 04, or 06. In one embodiment the
variant (human) Fc-region polypeptide has least about 90% homology
with a human Fc-region polypeptide of SEQ ID NO: 03, or 04, or 06.
In one embodiment the variant (human) Fc-region polypeptide has at
least about 95% homology with a human Fc-region polypeptide of SEQ
ID NO: 03, or 04, or 06.
[0249] The variant (human) Fc-region polypeptide derived from a
human Fc-region polypeptide of SEQ ID NO: 03, or 04, or 06 is
defined by the amino acid alterations that are contained. Thus, for
example, the term P329G denotes a variant (human) Fc-region
polypeptide derived human Fc-region polypeptide with the mutation
of proline to glycine at amino acid position 329 relative to the
human Fc-region polypeptide of SEQ ID NO: 03, or 04, or 06.
[0250] A human IgG1 Fc-region polypeptide has the following amino
acid sequence:
TABLE-US-00002 (SEQ ID NO: 03)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0251] A human IgG1 Fc-region derived Fc-region polypeptide with
the mutations L234A, L235A has the following amino acid
sequence:
TABLE-US-00003 (SEQ ID NO: 07)
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0252] A human IgG1 Fc-region derived Fc-region polypeptide with
Y349C, T366S, L368A and Y407V mutations has the following amino
acid sequence:
TABLE-US-00004 (SEQ ID NO: 08)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0253] A human IgG1 Fc-region derived Fc-region polypeptide with
S354C, T366W mutations has the following amino acid sequence:
TABLE-US-00005 (SEQ ID NO: 09)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0254] A human IgG1 Fc-region derived Fc-region polypeptide with
L234A, L235A mutations and Y349C, T366S, L368A, Y407V mutations has
the following amino acid sequence:
TABLE-US-00006 (SEQ ID NO: 10)
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0255] A human IgG1 Fc-region derived Fc-region polypeptide with a
L234A, L235A and S354C, T366W mutations has the following amino
acid sequence:
TABLE-US-00007 (SEQ ID NO: 11)
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0256] A human IgG1 Fc-region derived Fc-region polypeptide with a
P329G mutation has the following amino acid sequence:
TABLE-US-00008 (SEQ ID NO: 12)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0257] A human IgG1 Fc-region derived Fc-region polypeptide with
L234A, L235A mutations and P329G mutation has the following amino
acid sequence:
TABLE-US-00009 (SEQ ID NO: 13)
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0258] A human IgG1 Fc-region derived Fc-region polypeptide with a
P239G mutation and Y349C, T366S, L368A, Y407V mutations has the
following amino acid sequence:
TABLE-US-00010 (SEQ ID NO: 14)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0259] A human IgG1 Fc-region derived Fc-region polypeptide with a
P329G mutation and S354C, T366W mutation has the following amino
acid sequence:
TABLE-US-00011 (SEQ ID NO: 15)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0260] A human IgG1 Fc-region derived Fc-region polypeptide with
L234A, L235A, P329G and Y349C, T366S, L368A, Y407V mutations has
the following amino acid sequence:
TABLE-US-00012 (SEQ ID NO: 16)
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0261] A human IgG1 Fc-region derived Fc-region polypeptide with
L234A, L235A, P329G mutations and S354C, T366W mutations has the
following amino acid sequence:
TABLE-US-00013 (SEQ ID NO: 17)
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0262] A human IgG4 Fc-region polypeptide has the following amino
acid sequence:
TABLE-US-00014 (SEQ ID NO: 06)
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0263] A human IgG4 Fc-region derived Fc-region polypeptide with
S228P and L235E mutations has the following amino acid
sequence:
TABLE-US-00015 (SEQ ID NO: 18)
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0264] A human IgG4 Fc-region derived Fc-region polypeptide with
S228P, L235E mutations and P329G mutation has the following amino
acid sequence:
TABLE-US-00016 (SEQ ID NO: 19)
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0265] A human IgG4 Fc-region derived Fc-region polypeptide with
S354C, T366W mutations has the following amino acid sequence:
TABLE-US-00017 (SEQ ID NO: 20)
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
VVFSCSVMHEALHNHYTQKSLSLSLGK.
[0266] A human IgG4 Fc-region derived Fc-region polypeptide with
Y349C, T366S, L368A, Y407V mutations has the following amino acid
sequence:
TABLE-US-00018 (SEQ ID NO: 21)
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCA
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0267] A human IgG4 Fc-region derived Fc-region polypeptide with a
S228P, L235E and S354C, T366W mutations has the following amino
acid sequence:
TABLE-US-00019 (SEQ ID NO: 22)
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0268] A human IgG4 Fc-region derived Fc-region polypeptide with a
S228P, L235E and Y349C, T366S, L368A, Y407V mutations has the
following amino acid sequence:
TABLE-US-00020 (SEQ ID NO: 23)
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCA
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0269] A human IgG4 Fc-region derived Fc-region polypeptide with a
P329G mutation has the following amino acid sequence:
TABLE-US-00021 (SEQ ID NO: 24)
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0270] A human IgG4 Fc-region derived Fc-region polypeptide with a
P239G and Y349C, T366S, L368A, Y407V mutations has the following
amino acid sequence:
TABLE-US-00022 (SEQ ID NO: 25)
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCA
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0271] A human IgG4 Fc-region derived Fc-region polypeptide with a
P329G and S354C, T366W mutations has the following amino acid
sequence:
TABLE-US-00023 (SEQ ID NO: 26)
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0272] A human IgG4 Fc-region derived Fc-region polypeptide with a
S228P, L235E, P329G and Y349C, T366S, L368A, Y407V mutations has
the following amino acid sequence:
TABLE-US-00024 (SEQ ID NO: 27)
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCA
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0273] A human IgG4 Fc-region derived Fc-region polypeptide with a
S228P, L235E, P329G and S354C, T366W mutations has the following
amino acid sequence:
TABLE-US-00025 (SEQ ID NO: 28)
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0274] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., the CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0275] The term "hypervariable region" or "HVR", as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence ("complementarity determining
regions" or "CDRs") and form structurally defined loops
("hypervariable loops"), and/or contain the antigen-contacting
residues ("antigen contacts"). Generally, antibodies comprise six
HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2,
L3). HVRs as denoted herein include [0276] (a) hypervariable loops
occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96
(L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and
Lesk, A. M., J. Mol. Biol. 196 (1987) 901-917); [0277] (b) CDRs
occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97
(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, E. A. et
al., Sequences of Proteins of Immunological Interest, 5th ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991), NIH Publication 91-3242.); [0278] (c) antigen contacts
occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96
(L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J.
Mol. Biol. 262: 732-745 (1996)); and [0279] (d) combinations of
(a), (b), and/or (c), including HVR amino acid residues 46-56 (L2),
47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65
(H2), 93-102 (H3), and 94-102 (H3).
[0280] Unless otherwise indicated, HVR residues and other residues
in the variable domain (e.g., FR residues) are numbered herein
according to the Kabat EU index numbering system (Kabat et al.,
supra).
[0281] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g. cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0282] An "isolated" antibody is one, which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., size exclusion chromatography, ion exchange
or reverse phase HPLC). For review of methods for assessment of
antibody purity, see, e.g., Flatman, S. et al., J. Chrom. B 848
(2007) 79-87.
[0283] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0284] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0285] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 Da, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0286] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0287] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0288] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0289] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0290] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0291] The term "peptidic linker" as used herein denotes a peptide
with amino acid sequences, which is in one embodiment of synthetic
origin. The peptidic linker is in one embodiment a peptide with an
amino acid sequence with a length of at least 30 amino acids, in
one embodiment with a length of 32 to 50 amino acids. In one
embodiment the peptidic linker is a peptide with an amino acid
sequence with a length of 32 to 40 amino acids. In one embodiment
the peptidic linker is (G.times.S)n with G=glycine, S=serine, (x=3,
n=8, 9 or 10) or (x=4 and n=6, 7 or 8), in one embodiment with x=4,
n=6 or 7, in one embodiment with x=4, n=7. In one embodiment the
peptidic linker is (G.sub.4S).sub.6G.sub.2.
[0292] The term "recombinant antibody", as used herein, denotes all
antibodies (chimeric, humanized and human) that are prepared,
expressed, created or isolated by recombinant means. This includes
antibodies isolated from a host cell such as a NS0 or CHO cell, or
from an animal (e.g. a mouse) that is transgenic for human
immunoglobulin genes, or antibodies expressed using a recombinant
expression vector transfected into a host cell. Such recombinant
antibodies have variable and constant regions in a rearranged form.
The recombinant antibodies can be subjected to in vivo somatic
hypermutation. Thus, the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while
derived from and related to human germ line VH and VL sequences,
may not naturally exist within the human antibody germ line
repertoire in vivo.
[0293] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies or
Fc-region fusion polypeptides as reported herein are used to delay
development of a disease or to slow the progression of a
disease.
[0294] The term "valent" as used within the current application
denotes the presence of a specified number of binding sites in a
(antibody) molecule. As such, the terms "bivalent", "tetravalent",
and "hexavalent" denote the presence of two binding site, four
binding sites, and six binding sites, respectively, in a (antibody)
molecule. The bispecific antibodies as reported herein are in one
preferred embodiment "bivalent".
[0295] The term "variable region" or "variable domain" refer to the
domain of an antibody heavy or light chain that is involved in
binding of the antibody to its antigen. The variable domains of the
heavy chain and light chain (VH and VL, respectively) of an
antibody generally have similar structures, with each domain
comprising four framework regions (FRs) and three hypervariable
regions (HVRs) (see, e.g., Kindt, T. J. et al. Kuby Immunology, 6th
ed., W.H. Freeman and Co., N.Y. (2007), page 91). A single VH or VL
domain may be sufficient to confer antigen-binding specificity.
Furthermore, antibodies that bind a particular antigen may be
isolated using a VH or
[0296] VL domain from an antibody that binds the antigen to screen
a library of complementary VL or VH domains, respectively (see,
e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887;
Clackson, T. et al., Nature 352 (1991) 624-628).
[0297] The term "ocular vascular disease" includes, but is not
limited to intraocular neovascular syndromes such as diabetic
retinopathy, diabetic macular edema, retinopathy of prematurity,
neovascular glaucoma, retinal vein occlusions, central retinal vein
occlusions, macular degeneration, age-related macular degeneration,
retinitis pigmentosa, retinal angiomatous proliferation, macular
telangectasia, ischemic retinopathy, iris neovascularization,
intraocular neovascularization, comeal neovascularization, retinal
neovascularization, choroidal neovascularization, and retinal
degeneration (see e.g. Garner, A., Vascular diseases, In:
Pathobiology of ocular disease, A dynamic approach, Garner, A., and
Klintworth, G. K., (eds.), 2nd edition, Marcel Dekker, New York
(1994), pp. 1625-1710).
[0298] The term "vector", as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors".
[0299] The term "with (the) mutation IHH-AAA" as used herein refers
to the combination of the mutations I253A (Ile253A1a), H310A
(His310A1a), and H435A (His435A1a) and the term "with (the)
mutation HHY-AAA" as used herein refers to the combination of the
mutations H310A (His310A1a), H433A (His433A1a), and Y436A
(Tyr436A1a) and the term "with (the) mutation YTE" as used herein
refers to the combination of mutations M252Y (Met252Tyr), S254T
(Ser254Thr), and T256E (Thr256Glu) in the constant heavy chain
region of IgG1 or IgG4 subclass, wherein the numbering is according
to the Kabat EU index numbering system.
[0300] The term "with (the) mutations P329G LALA" as used herein
refers to the combination of the mutations L234A (Leu235A1a), L235A
(Leu234A1a) and P329G (Pro329Gly) in the constant heavy chain
region of IgG1 subclass, wherein the numbering is according to the
Kabat EU index numbering system. The term "with (the) mutation
SPLE" as used herein refers to the combination of the mutations
S228P (Ser228Pro) and L235E (Leu235Glu) in the constant heavy chain
region of IgG4 subclass, wherein the numbering is according to the
Kabat EU index numbering system. The term "with (the) mutation SPLE
and P329G" as used herein refers to the combination of the
mutations S228P (Ser228Pro), L235E (Leu235Glu) and P329G
(Pro329Gly) in the constant heavy chain region of IgG4 subclass,
wherein the numbering is according to the Kabat EU index numbering
system.
II. Compositions and Methods
[0301] In one aspect, the invention is based, in part, on the
finding that variant Fc-regions that specifically bind to
Staphylococcus protein A and that do or do not bind to human FcRn
when used in the hole-chain of a heterodimeric Fc-region allow for
the purification of the heterodimeric Fc-region. These variant
Fc-regions contain specific amino acid mutations in the CH2-domain
whereas the CH3-domain is not changed with respect to protein A
binding. It has been found that these mutations when used in the
hole-chain of a heterodimeric Fc-region allow for the purification
of the heterodimeric Fc-region, i.e. the separation of the
heterodimeric Fc-region from the homodimeric Fc-region by-product
(hole-chain-hole-chain dimer).
[0302] It has been found that this can be achieved by using the
combination of mutations in the first polypeptide (hole-chain)
[0303] i) I253A or I253G, and [0304] ii) L314A or L314G or
L314D.
[0305] The following table presents an exemplary overview of the
amino acid residues in an Fc-region that are involved in
interactions or have been changed to modify interactions.
TABLE-US-00026 interaction with KiH protein A effect of mutations
on residue protein A FcRn knob hole binding FcRn binding Pro238
P238A increase Thr250 T250Q/M428L increase Leu251 main-chain
contact Met252 hydrophobic M252W increase; packing M252Y increase;
M252Y/T256Q increase; M252F/T256D increase; M252Y/S254T/T256E
increase Ile253 main-chain interaction I253A reduction contact;
hydrogen bonding; significant binding reduction if mutated to Ala
Ser254 polar S254A reduction; interaction; M252Y/S254T/T256E
hydrogen increase bonding Arg255 salt-bridge R255A reduction Thr256
T256A increase; T256Q increase; T256P increase; M252Y/T256Q
reduction; M252F/T256D reduction; M252Y/S254T/T256E increase Pro257
P257I/Q311I increase; P257I/N434H increase Glu272 E272A increase
Asp280 D280K increase His285 reduction Lys288 K288A reduction;
K288A/N434A increase Val305 V305A increase Thr307 T307A increase;
T307A/E380A/N434A increase; T307Q/N434A increase; T307Q/N434S
increase; T307Q/E380A/N434A increase Val308 V308P/N434A increase
Leu309 L309A reduction His310 interaction H310A reduction;
H310Q/H433N reduction Gln311 polar or Q311A increase; charged
P257I/Q311I increase interaction Asp312 D312A increase Leu314
hydrophobic interaction Lys317 K317A increase Ala339 A339T increase
Tyr349 Y349C Ser354 S354C Thr366 T366W T366S Leu368 L368A Asp376
D376A increase; D376V/N434H increase Ala378 A378Q increase Glu380
salt-bridge E380A increase E380A/N434A increase; T307A/E380A/N434A
increase; T307Q/E380A/N434A increase Glu382 E382A increase Gly385
G385H increase; G385A/Q386P/N389S increase Gln386 G385A/Q386P/N389S
increase Asn389 G385A/Q386P/N389S increase Tyr407 Y407V Ser415
S415A reduction Ser424 S424A increase Met428 M428L increase;
T250Q/M428L increase Leu432 polar or charged interaction His433
polar or interaction H433A reduction; charged H310Q/H433N
interaction; reduction; salt-bridge H433K/N434F/Y436Hincrease;
H433R/N434Y/Y436Hincrease; H433K/N434F increase Asn434 hydrogen
interaction N434W/Y/F/A/H bonding; increase; significant
K288A/N434A increase; binding E380A/N434A increase; reduction if
T307A/E380A/N434A replaced by increase; Ala N434F/Y436H increase;
H433K/N434F/Y436Hincrease; H433R/N434Y/Y436Hincrease; H433K/N434F
increase; P257I/N434H increase; D376V/N434H increase; T307Q/N434A
increase; T307Q/N434S increase; V308P/N434A increase;
T307Q/E380A/N434A increase His435 hydrophobic interaction
H435R/Y436F H435A reduction; packing; eliminates H435R reduction
significant binding to binding protein A reduction if mutated to
Ala Tyr436 hydrophobic interaction H435R/Y436F Y436A reduction;
packing; eliminates N434F/Y436H increase; significant binding to
H433K/N434F/Y436Hincrease; binding protein A H433R/N434Y/Y436H
reduction if increase replaced by Ala
[0306] The modifications as reported herein alter the binding to
Staphylococcal protein A. The mutated residues are all located in
the CH2-domain. Although also residues in the CH3-domain do
interact with protein A the mutations of the residues in the
CH2-domain is sufficient to influence protein A binding. Thus, in
the heterodimeric molecules and antibodies reported herein both
CH3-domain have the same (identical) binding
properties/characteristics with respect to protein A. Thus, the
heterodimeric molecule as reported herein is heterodimeric
regarding protein A binding in the CH2-domain but homodimeric
regarding protein A binding in the CH3-domain.
[0307] In one embodiment the combination of mutations as reported
herein does alter or does substantially alter the serum half-life
of the heterodimeric polypeptide as compared with a corresponding
heterodimeric polypeptide that lacks this combination of
mutations.
[0308] In one embodiment the combination of mutations further does
not alter or does not substantially alter the serum half-life of
the heterodimeric polypeptide as compared with a corresponding
heterodimeric polypeptide that lacks this combination of
mutations.
[0309] A. The neonatal Fe-receptor (FeRn)
[0310] The neonatal Fc-receptor (FcRn) is important for the
metabolic fate of antibodies of the IgG class in vivo. The FcRn
functions to salvage wild-type IgG from the lysosomal degradation
pathway, resulting in reduced clearance and increased half-life. It
is a heterodimeric protein consisting of two polypeptides: a 50 kDa
class I major histocompatibility complex-like protein
(.alpha.-FcRn) and a 15 kDa .beta.2-microglobulin (.beta.2m). FcRn
binds with high affinity to the CH2-CH3 portion of the Fc-region of
an antibody of the class IgG. The interaction between an antibody
of the IgG class and the FcRn is pH dependent and occurs in a 1:2
stoichiometry, i.e. one IgG antibody molecule can interact with two
FcRn molecules via its two heavy chain Fc-region polypeptides (see
e.g. Huber, A. H., et al., J. Mol. Biol. 230 (1993) 1077-1083).
[0311] Thus, an IgGs in vitro FcRn binding
properties/characteristics are indicative of its in vivo
pharmacokinetic properties in the blood circulation.
[0312] In the interaction between the FcRn and the Fc-region of an
antibody of the IgG class different amino acid residues of the
heavy chain CH2- and CH3-domain are participating. The amino acid
residues interacting with the FcRn are located approximately
between EU position 243 and EU position 261, approximately between
EU position 275 and EU position 293, approximately between EU
position 302 and EU position 319, approximately between EU position
336 and EU position 348, approximately between EU position 367 and
EU position 393, at EU position 408, and approximately between EU
position 424 and EU position 440. More specifically the following
amino acid residues according to the EU numbering of Kabat are
involved in the interaction between the Fc-region and the FcRn:
F243, P244, P245 P, K246, P247, K248, D249, T250, L251, M252, 1253,
S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277,
Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289,
K290, P291, R292, E293, V302, V303, S304, V305, L306, T307, V308,
L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319,
1336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346,
Q347, V348, C367, V369, F372, Y373, P374, S375, D376, 1377, A378,
V379, E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389,
Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431,
L432, H433, N434, H435, Y436, T437, Q438, K439, and S440.
[0313] Site-directed mutagenesis studies have proven that the
critical binding sites in the Fc-region of IgGs for FcRn are
Histidine 310, Histidine 435, and Isoleucine 253 and to a lesser
extent Histidine 433 and Tyrosine 436 (see e.g. Kim, J. K., et al.,
Eur. J. Immunol. 29 (1999) 2819-2825; Raghavan, M., et al.,
Biochem. 34 (1995) 14649-14657; Medesan, C., et al., J Immunol. 158
(1997) 2211-2217).
[0314] Methods to increase IgG binding to FcRn have been performed
by mutating IgG at various amino acid residues: Threonine 250,
Methionine 252, Serine 254, Threonine 256, Threonine 307, Glutamic
acid 380, Methionine 428, Histidine 433, and Asparagine 434 (see
Kuo, T. T., et al., J. Clin. Immunol. 30 (2010) 777-789).
[0315] In some cases antibodies with reduced half-life in the blood
circulation are desired. For example, drugs for intravitreal
application should have a long half-live in the eye and a short
half-life in the blood circulation of the patient. Such antibodies
also have the advantage of increased exposure to a disease site,
e.g. in the eye.
[0316] Different mutations that influence the FcRn binding and
therewith the half-live in the blood circulation are known.
Fc-region residues critical to the mouse Fc-region--mouse FcRn
interaction have been identified by site-directed mutagenesis (see
e.g. Dall'Acqua, W. F., et al. J. Immunol 169 (2002) 5171-5180).
Residues 1253, H310, H433, N434, and H435 (EU numbering according
to Kabat) are involved in the interaction (Medesan, C., et al.,
Eur. J. Immunol. 26 (1996) 2533-2536; Firan, M., et al., Int.
Immunol. 13 (2001) 993-1002; Kim, J. K., et al., Eur. J. Immunol.
24 (1994) 542). Residues 1253, H310, and H435 were found to be
critical for the interaction of human Fc with murine FcRn (Kim, J.
K., et al., Eur. J. Immunol. 29 (1999) 2819-2855). Residues M252Y,
S254T, T256E have been described by Dall'Acqua et al. to improve
FcRn binding by protein-protein interaction studies (Dall'Acqua, W.
F., et al. J. Biol. Chem. 281 (2006) 23514-23524). Studies of the
human Fc-human FcRn complex have shown that residues 1253, S254,
H435, and Y436 are crucial for the interaction (Firan, M., et al.,
Int. Immunol. 13 (2001) 993-1002; Shields, R. L., et al., J. Biol.
Chem. 276 (2001) 6591-6604). In Yeung, Y. A., et al. (J. Immunol.
182 (2009) 7667-7671) various mutants of residues 248 to 259 and
301 to 317 and 376 to 382 and 424 to 437 have been reported and
examined. Exemplary mutations and their effect on FcRn binding are
listed in the following Table.
TABLE-US-00027 TABLE effect on half-live in the mutation FcRn
binding circulation reference H285 reduced reduced Kim, J. K.,
H310Q/H433N (murine) (in mouse) Scand. J. (murine IgG1) Immunol. 40
(1994) 457- 465 I253A reduced reduced Ghetie, V. and H310A (murine)
(in mouse) Ward, E. S., H435A Immunol. H436A Today 18 (murine IgG1)
(1997) 592- 598 T252L/T254S/T256F increased increased Ghetie, V.
and T252A/T254S/T256A (murine) (in mouse) Ward, E. S., (murine
IgG1) Immunol. Today 18 (1997) 592- 598 I253A reduced reduced
Medesan, C., H310A (murine) (in mouse) et al., J. H435A Immunol.
158 H436A (1997) 2211- H433A/N434Q 2217 (murine IgG1) I253A reduced
reduced Kim, J. K., H310A H310A: <0.1 (in mouse) Eur. J. H435A
rel. binding to Immunol. 29 H435R muFcRn (1999) 2819- (human IgG1)
(murine) 2825 H433A 1.1 rel. binding Kim, J. K., (human IgG1) to
muFcRn, Eur. J. 0.4 rel. binding Immunol. 29 Hu FcRn (1999) 2819-
(murine) 2825 I253A reduced <0.1 reduced Shields, R. L., S254A
relative et al., J. Biol. H435A binding to Chem. 276 Y436A huFcRn
(2001) 6591- (human IgG1) 6604 R255A reduced reduced Shields, R.
L., K288A (human) et al., J. Biol. L309A Chem. 276 S415A (2001)
6591- H433A 6604 (human IgG1) P238A increased increased Shields, R.
L., T256A (human) et al., J. Biol. E272A Chem. 276 V305A (2001)
6591- T307A 6604 Q311A D312A K317A D376A A378Q E380A E382A S424A
N434A K288A/N434A E380A/N434A T307A/E380A/N434A (human IgG1) H435A
reduced <0.1 reduced Firan, M., et (humanized IgG1) rel. al.,
Int. binding to Immunol. 13 huFcRn (2001) 993- 1002 I253A (no
binding) increased reduced Dall'Acqua, J. M252W (murine and (in
mouse) Immunol. 169 M252Y human) (2002) 5171- M252Y/T256Q 5180
M252F/T256D N434F/Y436H M252Y/S254T/T256E G385A/Q386P/N389S
H433K/N434F/Y436H H433R/N434Y/Y436H G385R/Q386T/P387R/N389P
M252Y/S254T/T256E/H433K/ N434F/Y436H M252Y/S254T/T256E/G385R/
Q386T/P387R/N389P (human IgG1) M428L increased increased Hinton, P.
R., T250Q/M428L (human) (in monkey) et al., J. Biol. (human IgG2)
Chem. 279 (2004) 6213- 6216 M252Y/S254T/T256E + increased increased
Vaccaro, C., et H433K/N434F (human) (in mouse) al., Nat. (human
IgG) Biotechnol. 23 (2005) 1283- 1288 T307A/E380A/N434A increased
increased in Pop, L. M., et (chimeric IgG1) transgenic mouse al.,
Int. Immunopharmacol. 5 (2005) 1279-1290 T250Q increased increased
in Petkova, S. B., E380A (human) transgenic mouse et al., Int.
M428L Immunol 18 N434A (2006) 1759- K288A/N434A 1769 E380A/N434A
T307A/E380A/N434A (human IgG1) I253A reduced reduced in Petkova, S.
B., (human IgG1) (human) transgenic mouse et al., Int. Immunol 18
(2006) 1759- 1769 S239D/A330L/I332E increased increased in
Dall'Acqua, M252Y/S254T/T256E (human and Cynomolgus W. F., et al.,
J. (humanized) Cynomolgus) Biol. Chem. 281 (2006) 23514-23524 T250Q
increased increased in Rhesus Hinton, P. R., M428L (human) apes et
al., J. T250Q/M428L Immunol. 176 (human IgG1) (2006) 346- 356
T250Q/M428L increased no change in Datta- P257I/Q311I (mouse and
Cynomolgus Mannan, A., et (humanized IgG1) Cynomolgus) increased in
mouse al., J. Biol. Chem. 282 (2007) 1709- 1717 P257I/Q311I
increased reduced in mice Datta- P257I/N434H at pH 6 P257I/N434H
Mannan, A., et D376V/N434H (human, reduced in al., Drug (humanized
IgG1) Cynomolgus, Cynomolgus Metab. mouse) Dispos. 35 (2007) 86-94
abrogate FcRn binding: increased and reducing the Ropeenian, I253
reduced binding ability of D. C. and H310 IgG for FcRn Akilesh, S.,
H433 reduces its serum Nat. Rev. H435 persistence; a Immunol. 7
reduce FcRn binding: higher-affinity (2007) 715- Y436 FcRn-IgG 725
increased FcRn binding: interaction prolongs T250 the half-lives of
IgG N252 and Fc-coupled S254 drugs in the serum T256 T307 M428 N434
N434A increased increased in Yeung, Y. A., T307Q/N434A (Cynomolgus
Cynomolgus et al., Cancer T307Q/N434S monkey) monkey Res. 70 (2010)
V308P/N434A 3269-3277 T307Q/E380A/N434A (human IgG1) 256P increased
at WO 2011/ 280K neutral pH 122011 339T 385H 428L 434W/Y/F/A/H
(human IgG)
[0317] The results of a symmetric engineering of an IgG1 Fc-region
to influence FcRn binding is shown in the following table
(alignment of mutations and retention time on an FcRn-affinity
chromatography column).
TABLE-US-00028 TABLE FcRn- affinity FcRn- FcRn- FcRn- column
effector function binding binding binding retention influencing
influencing influencing influencing time mutations mutation 1
mutation 2 mutation 3 [min] L234A/L235A/P329G -- -- -- 45.3
L234A/L235A/P329G I253A H310A H435A 2.3 L234A/L235A/P329G I253A --
-- 2.7 L234A/L235A/P329G -- H310A -- 2.4 L234A/L235A/P329G -- --
H435A 2.7 L234A/L235A/P329G I253A H310A -- 2.3 L234A/L235A/P329G
I253A -- H435A 2.3 L234A/L235A/P329G -- H310A H435A 2.4
L234A/L235A/P329G -- H310A Y436A 2.3 L234A/L235A/P329G H310A H433A
Y436A 2.4 L234A/L235A/P329G -- -- Y436A 41.3
[0318] Retention times below 3 minutes correspond to no binding as
the substance is in the flow-through (void peak).
[0319] The single mutation H310A is the most silent symmetrical
mutation to delete any FcRn-binding.
[0320] The symmetric single mutation I253A and H435A result in a
relative shift of retention time of 0.3 to 0.4 min. This can be
generally regarded as a non-detectable binding.
[0321] The single mutation Y436A results in detectable interaction
strength to the FcRn affinity column. Without being bound by this
theory this mutation could have an effect on FcRn mediated in vivo
half-life, which can be differentiated from a zero interaction such
as the combination of the I253A, H310A and H435A mutations (IHH-AAA
mutation).
[0322] The results obtained with a symmetrically modified anti-HER2
antibody are presented in the following table (see WO 2006/031370
for reference).
TABLE-US-00029 TABLE retention time mutation [min] I253H no binding
M252D no binding S254D no binding R255D 41.4 M252H 43.6 K288E 45.2
L309H 45.5 E258H 45.6 T256H 46.0 K290H 46.2 D98E 46.2 wild-type
46.3 K317H 46.3 Q311H 46.3 E430H 46.4 T307H 47.0 N434H 52.0
[0323] B. Ocular Vascular Diseases
[0324] Ocular vascular diseases are any pathological condition
characterized by altered or unregulated proliferation and invasion
of new blood vessels into the structures of ocular tissues such as
the retina or cornea.
[0325] In one embodiment the ocular vascular disease is selected
from the group consisting of wet age-related macular degeneration
(wet AMD), dry age-related macular degeneration (dry AMD), diabetic
macular edema (DME), cystoid macular edema (CME), non-proliferative
diabetic retinopathy (NPDR), proliferative diabetic retinopathy
(PDR), cystoid macular edema, vasculitis (e.g. central retinal vein
occlusion), papilloedema, retinitis, conjunctivitis, uveitis,
choroiditis, multifocal choroiditis, ocular histoplasmosis,
blepharitis, dry eye (Sjogren's disease) and other ophthalmic
diseases wherein the eye disease or disorder is associated with
ocular neovascularization, vascular leakage, and/or retinal
edema.
[0326] The antibody comprising the heterodimeric polypeptide as
reported herein is useful in the prevention and treatment of wet
AMD, dry AMD, CME, DME, NPDR, PDR, blepharitis, dry eye and
uveitis, in one preferred embodiment wet AMD, dry AMD, blepharitis,
and dry eye, also in one preferred embodiment CME, DME, NPDR and
PDR, also in one preferred embodiment blepharitis, and dry eye, in
particular wet AMD and dry AMD, and also particularly wet AMD.
[0327] In some embodiments, the ocular vascular disease is selected
from the group consisting of wet age-related macular degeneration
(wet AMD), macular edema, retinal vein occlusions, retinopathy of
prematurity, and diabetic retinopathy.
[0328] Other diseases associated with corneal neovascularization
include, but are not limited to, epidemic keratoconjunctivitis,
Vitamin A deficiency, contact lens overwear, atopic keratitis,
superior limbic keratitis, pterygium keratitis sicca, Sjogren's
disease, acne rosacea, phylectenulosis, syphilis, Mycobacteria
infections, lipid degeneration, chemical burns, bacterial ulcers,
fungal ulcers, Herpes simplex infections, Herpes zoster infections,
protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's
marginal degeneration, mariginal keratolysis, rheumatoid arthritis,
systemic lupus, polyarteritis, trauma, Wegener's sarcoidosis,
Scleritis, Steven's Johnson disease, periphigoid radial keratotomy,
and corneal graph rejection.
[0329] Diseases associated with retinal/choroidal
neovascularization include, but are not limited to, diabetic
retinopathy, macular degeneration, sickle cell anemia, sarcoid,
syphilis, pseudoxanthoma elasticum, Paget's disease, vein
occlusion, artery occlusion, carotid obstructive disease, chronic
uveitis/vitritis, mycobacterial infections, Lyme's disease,
systemic lupus erythematosis, retinopathy of prematurity, retinitis
pigmentosa, retina edema (including macular edema), Eale's disease,
Bechet's disease, infections causing a retinitis or choroiditis,
presumed ocular histoplasmosis, Best's disease, myopia, optic pits,
Stargart's disease, pars planitis, chronic retinal detachment,
hyperviscosity syndromes, toxoplasmosis, trauma and post-laser
complications.
[0330] Other diseases include, but are not limited to, diseases
associated with rubeosis (neovascularization of the angle) and
diseases caused by the abnormal proliferation of fibrovascular or
fibrous tissue including all forms of proliferative
vitreoretinopathy.
[0331] Retinopathy of prematurity (ROP) is a disease of the eye
that affects prematurely born babies. It is thought to be caused by
disorganized growth of retinal blood vessels, which may result in
scarring and retinal detachment. ROP can be mild and may resolve
spontaneously, but may lead to blindness in serious cases. As such,
all preterm babies are at risk for ROP, and very low birth weight
is an additional risk factor. Both oxygen toxicity and relative
hypoxia can contribute to the development of ROP.
[0332] Macular degeneration is a medical condition predominantly
found in elderly adults in which the center of the inner lining of
the eye, known as the macula area of the retina, suffers thinning,
atrophy, and in some cases, bleeding. This can result in loss of
central vision, which entails inability to see fine details, to
read, or to recognize faces. According to the American Academy of
Ophthalmology, it is the leading cause of central vision loss
(blindness) in the United States today for those over the age of
fifty years. Although some macular dystrophies that affect younger
individuals are sometimes referred to as macular degeneration, the
term generally refers to age-related macular degeneration (AMD or
ARMD).
[0333] Age-related macular degeneration begins with characteristic
yellow deposits in the macula (central area of the retina which
provides detailed central vision, called fovea) called drusen
between the retinal pigment epithelium and the underlying choroid.
Most people with these early changes (referred to as age-related
maculopathy) have good vision. People with drusen can go on to
develop advanced AMD. The risk is considerably higher when the
drusen are large and numerous and associated with disturbance in
the pigmented cell layer under the macula. Large and soft drusen
are related to elevated cholesterol deposits and may respond to
cholesterol lowering agents or the Rheo Procedure.
[0334] Advanced AMD, which is responsible for profound vision loss,
has two forms: dry and wet. Central geographic atrophy, the dry
form of advanced AMD, results from atrophy to the retinal pigment
epithelial layer below the retina, which causes vision loss through
loss of photoreceptors (rods and cones) in the central part of the
eye. While no treatment is available for this condition, vitamin
supplements with high doses of antioxidants, lutein and zeaxanthin,
have been demonstrated by the National Eye Institute and others to
slow the progression of dry macular degeneration and in some
patients, improve visual acuity.
[0335] Retinitis pigmentosa (RP) is a group of genetic eye
conditions. In the progression of symptoms for RP, night blindness
generally precedes tunnel vision by years or even decades. Many
people with RP do not become legally blind until their 40s or 50s
and retain some sight all their life. Others go completely blind
from RP, in some cases as early as childhood. Progression of RP is
different in each case. RP is a type of hereditary retinal
dystrophy, a group of inherited disorders in which abnormalities of
the photoreceptors (rods and cones) or the retinal pigment
epithelium (RPE) of the retina lead to progressive visual loss.
Affected individuals first experience defective dark adaptation or
nyctalopia (night blindness), followed by reduction of the
peripheral visual field (known as tunnel vision) and, sometimes,
loss of central vision late in the course of the disease.
[0336] Macular edema occurs when fluid and protein deposits collect
on or under the macula of the eye, a yellow central area of the
retina, causing it to thicken and swell. The swelling may distort a
person's central vision, as the macula is near the center of the
retina at the back of the eyeball. This area holds tightly packed
cones that provide sharp, clear central vision to enable a person
to see form, color, and detail that is directly in the line of
sight. Cystoid macular edema is a type of macular edema that
includes cyst formation.
[0337] C. The current invention
[0338] It has been found that one mutation one-sided in one
Fc-region polypeptide is sufficient to influence the binding
significantly. The more mutations are introduced into the Fc-region
the more the binding to Staphylococcal protein A and/or the FcRn is
changed, i.e. weakened or strengthened.
[0339] Herein is reported a method to deplete the hole-hole
mispairing by-product occurring in the production of knob-into-hole
(KiH) bispecific antibodies by CH2 domain design.
[0340] The amino acid positions L251, 1253, H310, L314 in the CH2
domain are interacting with protein A and the human neonatal Fc
receptor (FcRn).
[0341] By introducing an amino acid exchange at one or more of
these positions in the so call hole-chain of a KiH bispecific
antibody to an A, G or D the binding property to protein A and FcRn
can be silenced.
[0342] In one preferred embodiment the CH2 domain comprises the
mutations i) I253A or I253G, and ii) L314A or L314G or L314D.
[0343] By this design the hole-hole mispairing by-product can no
longer bind protein A and FcRn (no interaction possible with both
heavy chains). Thereby the hole-hole mispairing by-product will not
bind to a protein A and/or FcRn affinity chromatography column.
Thus, the hole-hole mispairing by-product will at least elute
earlier, i.e. in a separated detached peak, or will be not bind at
all and can be found in the flow-through of the protein A or FcRn
affinity column.
[0344] To compensate the impaired FcRn binding properties the
surrounding amino acids can be improved by introducing to the hole
side a T250Q and/or a T256E and/or a T307H mutation, either alone
or in a single, double or triple combination.
TABLE-US-00030 hole chain amino acid knob chain amino acid amino
acid and position mutation mutation resulting in a decrease of FcRn
binding in the final bispecific molecule L251 A, G, D L I253 A, G I
H310 A, G H L314 A, G, D L resulting in a decrease in protein A
binding with compensation of FcRn binding on the hole side in the
final bispecific molecule; can be used single or in combination
T250 Q T M252 Y M S254 T S T256 E, A T T307 A, H, Q, P T Q311 H Q
resulting in a decrease in protein A binding with compensation of
FcRn binding on the knob side in the final bispecific molecule; can
be used single or in combination T250 T Q M252 M Y S254 S T T256 T
E, A T307 T A, H, Q, P Q311 Q H
[0345] An overview on FcRn binding engineering is provided in the
following Table:
TABLE-US-00031 FcRn binding FcRn binding improved by decreased by
protein A protein G Position mutation to mutation to binding
binding CH2 T250 Q L251 D yes yes M252 Y L, D, (H) yes yes I253 A
yes yes S254 T A, D yes yes R255 A, D T256 E, A E258 (H) K288 E
T307 A, H, Q, P, H L309 A, H H310 A Q311 H L314 D yes no CH3 M428 L
(E) yes E430 H L432 D yes yes H433 A no yes N434 A, W, Y, H yes yes
H435 A yes Y436 A no yes
[0346] One aspect as reported herein is a heterodimeric polypeptide
comprising [0347] a first polypeptide comprising in N-terminal to
C-terminal direction at least a portion of an immunoglobulin hinge
region, which comprises one or more cysteine residues, an
immunoglobulin CH2-domain and an immunoglobulin CH3-domain, and a
second polypeptide comprising in N-terminal to C-terminal direction
at least a portion of an immunoglobulin hinge region, which
comprises one or more cysteine residues, an immunoglobulin
CH2-domain and an immunoglobulin CH3-domain, [0348] wherein the
first polypeptide comprises the mutations Y349C, T366S, L368A and
Y407V (hole-chain) and the second polypeptide comprises the
mutations S354C and T366W (knob-chain), [0349] and [0350] wherein
the first polypeptide (hole-chain) comprises the mutations [0351]
i) I253A or I253G, and [0352] ii) L314A or L314G or L314D, [0353]
and [0354] wherein the first polypeptide and the second polypeptide
are connected by one or more disulfide bridges, [0355] and [0356]
wherein the CH3-domain of the first polypeptide and the CH3-domain
of the second polypeptide both bind or both do not bind to protein
A [0357] (numbering according to the Kabat EU index).
[0358] In one embodiment the first polypeptide (hole-chain)
comprises the mutations [0359] i) I253A or I253G, and [0360] ii)
L314A or L314G or L314D, and [0361] iii) T250Q, and/or [0362] iv)
T256E or T256A.
[0363] In one embodiment the first polypeptide (hole-chain)
comprises the mutations [0364] i) I253A or I253G, and [0365] ii)
L314A or L314G or L314D, and [0366] iii) optionally a) T250Q,
and/or T256E or T256A, and. [0367] iv) a) L251A or L251G or L251D,
and/or b) H310A or H310G.
[0368] In one embodiment the first polypeptide (hole-chain)
comprises the mutation [0369] i) I253A or I253G, and [0370] ii)
L314A or L314G or L314D, and [0371] iii) a) T250Q, and/or T256E or
T256A, and. [0372] iv) a) L251A or L251G or L251D, and/or b) H310A
or H310G. [0373] v) optionally a) T307A or T307H or T307Q or T307P,
and/or b) [0374] Q311H, and/or c) M252Y, and/or d) S254T.
[0375] In one embodiment the second polypeptide (knob-chain)
comprises the mutation [0376] i) T250Q, and/or [0377] ii) M252Y,
and/or [0378] iii) S254T, and/or [0379] iv) T256E or T256A, and/or
[0380] v) T307A or T307H or T307Q or T307P, and/or [0381] vi)
Q311H.
[0382] In one embodiment the immunoglobulin hinge region, the
immunoglobulin CH2-domain and the immunoglobulin CH3-domain of the
first and the second polypeptide are of the human IgG1 subclass. In
one embodiment the first polypeptide and the second polypeptide
each further comprise the mutations L234A and L235A. In one
embodiment the first polypeptide and the second polypeptide each
further comprise the mutation P329G. In one embodiment the first
polypeptide and the second polypeptide each further comprise the
mutations L234A, L235A and P329G.
[0383] In one embodiment the immunoglobulin hinge region, the
immunoglobulin CH2-domain and the immunoglobulin CH3-domain of the
first and the second polypeptide are of the human IgG4 subclass. In
one embodiment the first polypeptide and the second polypeptide
each further comprise the mutations S228P and L235E. In one
embodiment the first polypeptide and the second polypeptide each
further comprise the mutation P329G. In one embodiment the first
polypeptide and the second polypeptide each further comprise the
mutations S228P, L235E and P329G.
[0384] In one embodiment the heterodimeric polypeptide is an
Fc-region fusion polypeptide.
[0385] In one embodiment the heterodimeric polypeptide is a
full-length antibody. In one embodiment the
[0386] In one embodiment the full length antibody is a monospecific
antibody. In one embodiment the monospecific antibody is a
monovalent monospecific antibody. In one embodiment the
monospecific antibody is a bivalent monospecific antibody.
[0387] In one embodiment the full length antibody is a bispecific
antibody. In one embodiment the bispecific antibody is a bivalent
bispecific antibody. In one embodiment the bispecific antibody is a
tetravalent bispecific antibody.
[0388] In one embodiment the full length antibody is a trispecific
antibody. In one embodiment the trispecific antibody is a trivalent
trispecific antibody. In one embodiment the trispecific antibody is
a tetravalent trispecific antibody.
[0389] One aspect as reported herein is an antibody comprising the
heterodimeric polypeptide (variant human IgG class Fc-region) as
reported herein.
[0390] The Fc-region (heterodimeric polypeptide) as reported herein
when contained in a full length antibody confers the above
described characteristics to the molecule.
[0391] Antibodies, e.g. full-length antibodies or CrossMabs, can
comprise a variant (human) human IgG class Fc-region as reported
herein.
[0392] The heterodimeric polypeptides have due to the mutations the
properties of not binding to Staphylococcal protein A in one chain
(the hole-chain) and of binding to Staphylococcal protein A in the
other chain (the knob-chain).
[0393] Thus, these antibodies can be purified, i.e. separated from
unwanted hole-chain dimeric by-products by using conventional
protein A affinity materials, such as MabSelectSure. It is not
required to use highly sophisticated but species limited affinity
materials, such as e.g. KappaSelect, which is only useable with
antibodies comprising a light chain of the kappa subclass.
Additionally it is not required to adopt the purification method if
a modification/exchange of the light chain subclass is made.
[0394] One aspect as reported herein is a method for producing a
heterodimeric polypeptide as reported herein comprising the
following steps: [0395] a) cultivating a mammalian cell comprising
one or more nucleic acids encoding a heterodimeric polypeptide as
reported herein, [0396] b) recovering the heterodimeric polypeptide
from the cultivation medium, and [0397] c) purifying the
heterodimeric polypeptide with a protein A affinity chromatography
and thereby producing the dimeric polypeptide.
[0398] One aspect as reported herein is the use of the combination
of mutations i) I253A or I253G, and ii) L314A or L314G or L314D for
separating heterodimeric polypeptides from homodimeric
polypeptides.
[0399] One aspect as reported herein is a bispecific antibody
providing ease of isolation/purification comprising immunoglobulin
heavy chain Fc-regions that are differentially modified, wherein at
least one of the modifications results in i) a differential
affinity of the bispecific antibody for protein A, and the
heterodimeric bispecific antibody is isolable from a disrupted
cell, from medium, or from a mixture of antibodies based on its
affinity for protein A.
[0400] In one embodiment the bispecific antibody elutes at a pH
value above pH 4.0.
[0401] In one embodiment the bispecific antibody is isolated using
a protein A affinity chromatography and a pH gradient or pH step,
wherein the pH gradient or pH step includes the addition of a salt.
In a specific embodiment, the salt is present at a concentration of
about 0.5 molar to about 1 molar. In one embodiment, the salt is
selected from the group consisting of lithium, sodium, and
potassium salts of acetate; sodium and potassium bicarbonates;
lithium, sodium, and potassium carbonates; lithium, sodium,
potassium, and magnesium chlorides; sodium and potassium fluorides;
sodium, potassium, and calcium nitrates; sodium and potassium
phosphates; and calcium and magnesium sulfates. In one embodiment
the salt is a halide salt of an alkaline metal or alkaline earth
metal. In one preferred embodiment the salt is sodium chloride.
[0402] In one aspect the dimeric polypeptide comprises a first
polypeptide that is modified as reported herein and a second
polypeptide that is not modified regarding protein A or FcRn
binding, so as to form a heterodimeric polypeptide, wherein the
differential modification results in the dimeric polypeptide
eluting from a protein A affinity material at 0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.2, 1.3, or 1.4 pH unit(s) higher than a corresponding
dimeric polypeptide that lacks the differential modification. In
one embodiment, the differentially modified dimeric polypeptide
elutes at a pH of 4 or higher, whereas the unmodified dimeric
polypeptide elutes at a pH of 3.5 or lower. In one embodiment, the
differentially modified dimeric polypeptide elutes at a pH of about
4, whereas the unmodified dimeric polypeptide elutes at a pH of
about 2.8-3.5, 2.8-3.2, or 2.8-3. In these embodiments,
"unmodified" refers to lack of the modification i) I253A and I253G,
and ii) L314A and L314G and L314D (Kabat EU index numbering system)
in both of the polypeptides.
[0403] For chromatographic runs the addition of 0.5 molar to 1
molar salt (e.g. NaCl) may improve the separation of homodimeric
polypeptide and heterodimeric polypeptide, especially if derived
from the human IgG1 subclass. The addition of salt to the elution
solution increasing the pH value can broaden the pH range for
elution such that e.g. a pH step gradient could successfully
separate the two species.
[0404] Accordingly, in one embodiment a method for separating a
bispecific antibody comprising a heterodimeric IgG Fc-region with
one chain comprising mutations as reported herein, comprises a step
of employing a pH gradient in the presence of a salt. In one
embodiment, the salt is present at a concentration sufficient to
maximize the pH difference between elution from a protein A
chromatography material of an IgG Fc-region homodimer and an IgG
Fc-region heterodimer. In one embodiment the salt is present at a
concentration of about 0.5 molar to about 1 molar. In one
embodiment the salt is a salt of an alkaline metal or an alkaline
earth metal and a halogen. In one embodiment the salt is a chloride
salt of an alkaline metal or an alkaline earth metal, such as e.g.
NaCl, KCl, LiCl, CaCl.sub.2, or MgCl.sub.2. In one embodiment the
pH gradient is from about pH 4 to about pH 5. In one embodiment the
gradient is a linear gradient. In one embodiment, the pH gradient
is a step gradient. In one embodiment the method comprises applying
to an equilibrated protein A affinity column a solution of about pH
4. In one embodiment the bispecific antibody comprising the
heterodimeric IgG Fc-region with respect to the modifications as
reported herein elutes from the protein A affinity chromatography
material in one or more fractions substantially free of
non-heterodimeric bispecific antibody.
[0405] The heterodimeric polypeptide as reported herein is produced
by recombinant means. Thus, one aspect of the current invention is
a nucleic acid encoding the heterodimeric polypeptide as reported
herein and a further aspect is a cell comprising the nucleic acid
encoding the heterodimeric polypeptide as reported herein.
[0406] Methods for recombinant production are widely known in the
state of the art and comprise protein expression in prokaryotic and
eukaryotic cells with subsequent isolation of the heterodimeric
polypeptide and usually purification to a pharmaceutically
acceptable purity. For the expression of the heterodimeric
polypeptides as aforementioned in a host cell, nucleic acids
encoding the respective first and second polypeptides are inserted
into expression vectors by standard methods. Expression is
performed in appropriate prokaryotic or eukaryotic host cells like
CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6
cells, yeast, or E. coli cells, and the heterodimeric polypeptide
is recovered from the cells (cultivation supernatant or cells after
lysis).
[0407] General methods for recombinant production of antibodies are
well known in the state of the art and described, for example, in
the review articles of Makrides, S. C., Protein Expr. Purif. 17
(1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996)
271-282; Kaufman, R. J., Mol. Biotechnol. 16 (2000) 151-160;
Werner, R. G., Drug Res. 48 (1998) 870-880.
[0408] Accordingly one aspect as reported herein is a method for
the production of a heterodimeric polypeptide as reported herein,
comprising the steps of [0409] a) transforming a host cell with one
or more vectors comprising nucleic acid molecules encoding the
heterodimeric polypeptide as reported herein, [0410] b) culturing
the host cell to express the heterodimeric polypeptide, and c)
recovering the heterodimeric polypeptide from the culture and
thereby producing the heterodimeric polypeptide.
[0411] In one embodiment the recovering step under c) includes the
use of an immunoglobulin Fc-region specific capture reagent. In one
embodiment this Fc-region specific capture reagent is used in a
bind-and-elute-mode. Examples of such
[0412] Fc-region specific capture reagents are e.g. Staphylococcus
protein A-based affinity chromatography columns, which are based on
a highly rigid agarose base matrix that allows high flow rates and
low back pressure at large scale. They feature a ligand that binds
to the heterodimeric polypeptide, i.e. its Fc-region. The ligands
are attached to the matrix via a long hydrophilic spacer arm to
make it easily available for binding to the target molecule.
[0413] The heterodimeric polypeptides as reported herein are
suitably separated from the culture medium by conventional
immunoglobulin purification procedures such as, for example,
protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0414] DNA and RNA encoding the monoclonal antibodies are readily
isolated and sequenced using conventional procedures. Once
isolated, the DNA may be inserted into expression vectors, which
are then transfected into host cells such as HEK 293 cells, CHO
cells, or myeloma cells that do not otherwise produce heterodimeric
polypeptides, to obtain the synthesis of recombinant monoclonal
heterodimeric polypeptides in the host cells.
[0415] Purification of antibodies is performed in order to
eliminate cellular components or other contaminants, e.g. other
cellular nucleic acids or proteins, by standard techniques,
including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis, and others well known
in the art (see Ausubel, F., et al., ed. Current Protocols in
Molecular Biology, Greene Publishing and Wiley Interscience, New
York (1987)). Different methods are well established and widespread
used for protein purification, such as affinity chromatography with
microbial proteins (e.g. protein A or protein G affinity
chromatography), ion exchange chromatography (e.g. cation exchange
(carboxymethyl resins), anion exchange (amino ethyl resins) and
mixed-mode exchange), thiophilic adsorption (e.g. with
beta-mercaptoethanol and other SH ligands), hydrophobic interaction
or aromatic adsorption chromatography (e.g. with phenyl-Sepharose,
aza-arenophilic resins, or m-aminophenylboronic acid), metal
chelate affinity chromatography (e.g. with Ni(II)- and
Cu(II)-affinity material), size exclusion chromatography, and
electrophoretical methods (such as gel electrophoresis, capillary
electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75
(1998) 93-102).
[0416] One aspect of the invention is a pharmaceutical formulation
comprising a heterodimeric polypeptide as reported herein. Another
aspect of the invention is the use of a heterodimeric polypeptide
as reported herein for the manufacture of a pharmaceutical
formulation. A further aspect of the invention is a method for the
manufacture of a pharmaceutical formulation comprising a
heterodimeric polypeptide as reported herein. In another aspect,
the present invention provides a formulation, e.g. a pharmaceutical
formulation, containing a heterodimeric polypeptide as reported
herein, formulated together with a pharmaceutical carrier.
[0417] A formulation as reported herein can be administered by a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. To administer a compound of the
invention by certain routes of administration, it may be necessary
to coat the compound with, or co-administer the compound with, a
material to prevent its inactivation. For example, the compound may
be administered to a subject in an appropriate carrier, for
example, liposomes, or a diluent. Pharmaceutically acceptable
diluents include saline and aqueous buffer solutions.
Pharmaceutical carriers include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. The use of such
media and agents for pharmaceutically active substances is known in
the art.
[0418] Many possible modes of delivery can be used, including, but
not limited to intraocular application or topical application. In
one embodiment the application is intraocular and includes, but
it's not limited to subconjunctival injection, intracanieral
injection, injection into the anterior chamber via the termporai
limbus, intrastromal injection, intracorneal injection, subretinal
injection, aqueous humor injection, subtenon injection or sustained
delivery device, intravitreal injection (e.g., front, mid or back
vitreal injection). In one embodiment the application is topical
and includes, but it's not limited to eye drops to the cornea.
[0419] In one embodiment the heterodimeric polypeptide as reported
herein or the pharmaceutical formulation as reported herein is
administered via intravitreal application, e.g. via intravitreal
injection. This can be performed in accordance with standard
procedures known in the art. See, e.g., Ritter et al., J. Clin.
Invest. 116 (2006) 3266-3276; Russelakis-Cameiro et al.,
Neuropathol. Appl. Neurobiol. 25 (1999) 196-206; and Wray et al.,
Arch. Neurol. 33 (1976) 183-185.
[0420] In some embodiments, therapeutic kits of the invention can
contain one or more doses of a heterodimeric polypeptide as
reported herein present in a pharmaceutical formulation as
described herein, a suitable device for intravitreal injection of
the pharmaceutical formulation, and an instruction detailing
suitable subjects and protocols for carrying out the injection. In
these embodiments, the formulations are typically administered to
the subject in need of treatment via intravitreal injection. This
can be performed in accordance with standard procedures known in
the art (see, e.g., Ritter et al., J. Clin. Invest. 116 (2006)
3266-3276; Russelakis-Cameiro et al., Neuropathol. Appl. Neurobiol.
25 (1999) 196-206; and Wray et al., Arch. Neurol. 33 (1976)
183-185).
[0421] The formulation may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the formulations. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents, which delay absorption,
such as aluminum monostearate and gelatin.
[0422] Regardless of the route of administration selected, the
compounds as reported herein, which may be used in a suitable
hydrated form, and/or the pharmaceutical formulations as reported
herein, are formulated into pharmaceutically acceptable dosage
forms by conventional methods known to those of skill in the
art.
[0423] Actual dosage levels of the active ingredients in the
pharmaceutical formulation as reported herein may be varied so as
to obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient. The selected dosage level will depend upon a variety of
pharmacokinetic factors including the activity of the particular
compositions of the present invention employed, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0424] The formulation must be sterile and fluid to the extent that
the formulation is deliverable by syringe. In addition to water,
the carrier in one preferred embodiment is an isotonic buffered
saline solution.
[0425] Proper fluidity can be maintained, for example, by use of
coating such as lecithin, by maintenance of required particle size
in the case of dispersion and by use of surfactants. In many cases,
it is preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in
the composition.
[0426] The formulation can comprise an ophthalmic depot formulation
comprising an active agent for subconjunctival administration. The
ophthalmic depot formulation comprises microparticles of
essentially pure active agent, e.g., a heterodimeric polypeptide as
reported herein. The microparticles comprising a heterodimeric
polypeptide as reported herein can be embedded in a biocompatible
pharmaceutically acceptable polymer or a lipid-encapsulating agent.
The depot formulations may be adapted to release all of
substantially all the active material over an extended period of
time. The polymer or lipid matrix, if present, may be adapted to
degrade sufficiently to be transported from the site of
administration after release of all or substantially all the active
agent. The depot formulation can be liquid formulation, comprising
a pharmaceutical acceptable polymer and a dissolved or dispersed
active agent. Upon injection, the polymer forms a depot at the
injections site, e.g. by gelifying or precipitating.
[0427] Another aspect of the invention is a heterodimeric
polypeptide as reported herein for use in the treatment of ocular
vascular diseases.
[0428] One embodiment of the invention is a heterodimeric
polypeptide as reported herein for use in the treatment of ocular
vascular diseases.
[0429] Another aspect of the invention is the pharmaceutical
formulation for use in the treatment of ocular vascular
diseases.
[0430] Another aspect of the invention is the use of a
heterodimeric polypeptide as reported herein for the manufacture of
a medicament for the treatment of ocular vascular disease.
[0431] Another aspect of the invention is method of treatment of
patient suffering from ocular vascular diseases by administering a
heterodimeric polypeptide as reported herein to a patient in the
need of such treatment.
[0432] It is herewith expressly stated that the term "comprising"
as used herein comprises the term "consisting of". Thus, all
aspects and embodiments that contain the term "comprising" are
likewise disclosed with the term "consisting of".
[0433] D. Modifications
[0434] In a further aspect, a heterodimeric polypeptide according
to any of the above embodiments may incorporate any of the
features, singly or in combination, as described in Sections 1-6
below:
[0435] 1. Antibody Affinity
[0436] In one embodiment, Kd is measured using a BIACORE.RTM.
surface plasmon resonance assay. For example, an assay using a
BIACORE.RTM.-2000 or a BIACORE.RTM.-3000 (GE Healthcare Inc.,
Piscataway, N.J.) is performed at 25.degree. C. with immobilized
binding partner CM5 chips at .about.10 response units (RU). In one
embodiment, carboxymethylated dextran biosensor chips (CM5, GE
Healthcare Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Binding partner is diluted with 10 mM sodium acetate,
pH 4.8, to 5 .mu.g/mL (.about.0.2 .mu.M) before injection at a flow
rate of 5 .mu.l/minute to achieve approximately 10 response units
(RU) of coupled binding partner. Following the injection of the
binding partner, 1 M ethanolamine is injected to block non-reacted
groups. For kinetics measurements, two-fold serial dilutions of the
dimeric polypeptide containing fusion polypeptide or antibody (0.78
nM to 500 nM) are injected in PBS with 0.05% polysorbate 20
(TWEEN-20.TM.) surfactant (PBST) at 25.degree. C. at a flow rate of
approximately 25 .mu.L/min. Association rates (k.sub.on) and
dissociation rates (k.sub.off) are calculated using a simple
one-to-one Langmuir binding model (BIACORE.RTM. Evaluation Software
version 3.2) by simultaneously fitting the association and
dissociation sensorgrams. The equilibrium dissociation constant
(Kd) is calculated as the ratio k.sub.off/k.sub.on (see, e.g.,
Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). If the on-rate
exceeds 10.sup.6 M.sup.-1 s.sup.-1 by the surface plasmon resonance
assay above, then the on-rate can be determined by using a
fluorescent quenching technique that measures the increase or
decrease in fluorescence emission intensity (excitation=295 nm;
emission=340 nm, 16 nm band-pass) at 25.degree. C. of a 20 nM
anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of
increasing concentrations of antigen as measured in a spectrometer,
such as a stop-flow equipped spectrophotometer (Aviv Instruments)
or a 8000-series SLM-AMINCO.TM. spectrophotometer
(ThermoSpectronic) with a stirred cuvette.
[0437] 2. Chimeric and Humanized Antibodies
[0438] In certain embodiments, a heterodimeric polypeptide as
reported herein is comprised in a chimeric antibody. Certain
chimeric antibodies are described, e.g., in U.S. Pat. No.
4,816,567; and Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA
81 (1984) 6851-6855). In one example, a chimeric antibody comprises
a non-human variable region (e.g., a variable region derived from a
mouse, rat, hamster, rabbit, or non-human primate, such as a
monkey) and a human constant region. In a further example, a
chimeric antibody is a "class switched" antibody in which the class
or subclass has been changed from that of the parent antibody.
Chimeric antibodies include antigen-binding fragments thereof.
[0439] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region.
[0440] In some embodiments, some FR residues in a humanized
antibody are substituted with corresponding residues from a
non-human antibody (e.g., the antibody from which the HVR residues
are derived), e.g., to restore or improve antibody specificity or
affinity.
[0441] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro, J. C. and Fransson, J., Front. Biosci.
13 (2008) 1619-1633, and are further described, e.g., in Riechmann,
I., et al., Nature 332 (1988) 323-329; Queen, C., et al., Proc.
Natl. Acad. Sci. USA 86 (1989) 10029-10033; U.S. Pat. Nos.
5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri, S. V., et
al., Methods 36 (2005) 25-34 (describing specificity determining
region (SDR) grafting); Padlan, E. A., Mol. Immunol. 28 (1991)
489-498 (describing "resurfacing"); Dall'Acqua, W. F. et al.,
Methods 36 (2005) 43-60 (describing "FR shuffling"); Osbourn, J. et
al., Methods 36 (2005) 61-68; and Klimka, A. et al., Br. J. Cancer
83 (2000) 252-260 (describing the "guided selection" approach to FR
shuffling).
[0442] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims, M. J., et al., J. Immunol.
151 (1993) 2296-2308; framework regions derived from the consensus
sequence of human antibodies of a particular subgroup of light or
heavy chain variable regions (see, e.g., Carter, P., et al., Proc.
Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L. G., et
al., J. Immunol. 151 (1993) 2623-2632); human mature (somatically
mutated) framework regions or human germline framework regions
(see, e.g., Almagro, J. C. and Fransson, J., Front. Biosci. 13
(2008) 1619-1633); and framework regions derived from screening FR
libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 (1997)
10678-10684 and Rosok, M. J. et al., J. Biol. Chem. 271 (19969
22611-22618).
[0443] 3. Human Antibodies
[0444] In certain embodiments, a dimeric polypeptide as reported
herein is derived from a human antibody. Human antibodies can be
produced using various techniques known in the art. Human
antibodies are described generally in van Dijk, M. A. and van de
Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg,
N., Curr. Opin. Immunol. 20 (2008) 450-459.
[0445] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
N., Nat. Biotech. 23 (2005) 1117-1125. See also, e.g., U.S. Pat.
Nos. 6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology;
U.S. Pat. No. 5,770,429 describing HuMAB.RTM. technology; U.S. Pat.
No. 7,041,870 describing K-M MOUSE.RTM. technology, and US
2007/0061900, describing VELoCIMOUSE.RTM. technology). Human
variable regions from intact antibodies generated by such animals
may be further modified, e.g., by combining with a different human
constant region.
[0446] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor, D., J. Immuno1.133 (1984) 3001-3005; Brodeur,
B. R., et al., Monoclonal Antibody Production Techniques and
Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and
Boemer, P., et al., J. Immunol. 147 (1991) 86-95). Human antibodies
generated via human B-cell hybridoma technology are also described
in Li, J., et al., Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562.
Additional methods include those described, for example, in U.S.
Pat. No. 7,189,826 (describing production of monoclonal human IgM
antibodies from hybridoma cell lines) and Ni, J., Xiandai Mianyixue
26 (2006) 265-268 (describing human-human hybridomas). Human
hybridoma technology (Trioma technology) is also described in
Vollmers, H. P. and Brandlein, S., Histology and Histopathology 20
(2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods and
Findings in Experimental and Clinical Pharmacology 27 (2005)
185-191.
[0447] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0448] 4. Library-Derived Antibodies
[0449] In certain embodiments a heterodimeric polypeptide as
reported herein is comprised in a library-derived antibody.
Library-derived antibodies may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities.
[0450] For example, a variety of methods are known in the art for
generating phage display libraries and screening such libraries for
antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom, H. R. et al., Methods in
Molecular Biology 178 (2001) 1-37 and further described, e.g., in
the McCafferty, J. et al., Nature348 (1990) 552-554; Clackson, T.
et al., Nature 352 (1991) 624-628; Marks, J. D. et al., J. Mol.
Biol. 222 (1992) 581-597; Marks, J. D. and Bradbury, A., Methods in
Molecular Biology 248 (2003) 161-175; Sidhu, S. S. et al., J. Mol.
Biol. 338 (2004) 299-310; Lee, C. V. et al., J. Mol. Biol. 340
(2004) 1073-1093; Fellouse, F. A., Proc. Natl. Acad. Sci. USA 101
(2004) 12467-12472; and Lee, C. V. et al., J. Immunol. Methods 284
(2004) 119-132.
[0451] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter, G., et al., Ann.
Rev. Immunol. 12 (1994) 433-455. Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self-antigens without any
immunization as described by Griffiths, A. D., et al., EMBO J. 12
(1993) 725-734. Finally, naive libraries can also be made
synthetically by cloning non-rearranged V-gene segments from stem
cells, and using PCR primers containing random sequence to encode
the highly variable CDR3 regions and to accomplish rearrangement in
vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.
Biol. 227 (1992) 381-388. Patent publications describing human
antibody phage libraries include, for example: U.S. Pat. No.
5,750,373, and US 2005/0079574, US 2005/0119455, US 2005/0266000,
US 2007/0117126, US 2007/0160598, US 2007/0237764, US 2007/0292936,
and US 2009/0002360.
[0452] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0453] 5. Multispecific Antibodies
[0454] In certain embodiments, a heterodimeric polypeptide as
reported herein is comprised in a multispecific antibody, e.g. a
bispecific antibody. Multispecific antibodies are monoclonal
antibodies that have binding specificities for at least two
different sites. In certain embodiments, one of the binding
specificities is for a first antigen and the other is for a
different second antigen. In certain embodiments, bispecific
antibodies may bind to two different epitopes of the same antigen.
Bispecific antibodies may also be used to localize cytotoxic agents
to cells, which express at least one of the antigens. Bispecific
antibodies can be prepared as full-length antibodies or antibody
fragments.
[0455] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO
93/08829, and Traunecker, A., et al., EMBO J. 10 (1991) 3655-3659),
and "knob-in-hole" engineering (see, e.g., U.S. Pat. No.
5,731,168). Multi-specific antibodies may also be made by
engineering electrostatic steering effects for making antibody
Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or
more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980,
and Brennan, M. et al., Science229 (1985) 81-83); using leucine
zippers to produce bi-specific antibodies (see, e.g., Kostelny, S.
A., et al., J. Immunol. 148 (1992) 1547-1553; using "diabody"
technology for making bispecific antibody fragments (see, e.g.,
Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993)
6444-6448); and using single-chain Fv (scFv) dimers (see, e.g.
Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and preparing
trispecific antibodies as described, e.g., in Tuft, A. et al., J.
Immunol. 147 (1991) 60-69).
[0456] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576).
[0457] The antibody or fragment herein also includes a "Dual Acting
Fab" or "DAF" (see, US 2008/0069820, for example).
[0458] The antibody or fragment herein also includes multispecific
antibodies described in WO 2009/080251, WO 2009/080252, WO
2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO
2010/136172, WO 2010/145792, and WO 2010/145793.
[0459] 6. Antibody Variants
[0460] In certain embodiments, a heterodimeric polypeptide as
reported herein is comprised in an antibody. In further embodiment
amino acid sequence variants of the antibodies provided herein are
contemplated. For example, it may be desirable to improve the
binding affinity and/or other biological properties of the
antibody. Amino acid sequence variants of an antibody may be
prepared by introducing appropriate modifications into the
nucleotide sequence encoding the antibody, or by peptide synthesis.
Such modifications include, for example, deletions from, and/or
insertions into and/or substitutions of residues within the amino
acid sequences of the antibody.
[0461] Any combination of deletion, insertion, and substitution can
be made to arrive at the final construct, provided that the final
construct possesses the desired characteristics, e.g.,
antigen-binding.
[0462] a) Substitution, Insertion, and Deletion Variants
[0463] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in the Table below under the heading of
"preferred substitutions". More substantial changes are provided in
the following Table under the heading of "exemplary substitutions",
and as further described below in reference to amino acid side
chain classes. Amino acid substitutions may be introduced into an
antibody of interest and the products screened for a desired
activity, e.g., retained/improved antigen binding, decreased
immunogenicity, or improved ADCC or CDC.
TABLE-US-00032 TABLE Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Leu norleucine Leu (L) norleucine; Ile; Val; Met;
Ile Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu norleucine
[0464] Amino acids may be grouped according to common side-chain
properties: [0465] (1) hydrophobic: norleucine, Met, Ala, Val, Leu,
Ile; [0466] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0467] (3) acidic: Asp, Glu; [0468] (4) basic: His, Lys, Arg;
[0469] (5) residues that influence chain orientation: Gly, Pro;
[0470] (6) aromatic: Trp, Tyr, Phe.
[0471] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0472] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity-matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0473] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, P. S., Methods Mol. Biol. 207 (2008) 179-196), and/or
residues that contact antigen, with the resulting variant VH or VL
being tested for binding affinity. Affinity maturation by
constructing and reselecting from secondary libraries has been
described, e.g., in Hoogenboom, H. R. et al. in Methods in
Molecular Biology 178 (2002) 1-37. In some embodiments of affinity
maturation, diversity is introduced into the variable genes chosen
for maturation by any of a variety of methods (e.g., error-prone
PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A
secondary library is then created. The library is then screened to
identify any antibody variants with the desired affinity. Another
method to introduce diversity involves HVR-directed approaches, in
which several HVR residues (e.g., 4-6 residues at a time) are
randomized. HVR residues involved in antigen binding may be
specifically identified, e.g., using alanine scanning mutagenesis
or modeling. CDR-H3 and CDR-L3 in particular are often
targeted.
[0474] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may, for example, be outside of antigen contacting
residues in the HVRs. In certain embodiments of the variant VH and
VL sequences provided above, each HVR either is unaltered, or
contains no more than one, two or three amino acid
substitutions.
[0475] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham, B. C. and Wells,
J. A., Science 244 (1989) 1081-1085. In this method, a residue or
group of target residues (e.g., charged residues such as Arg, Asp,
His, Lys, and Glu) are identified and replaced by a neutral or
negatively charged amino acid (e.g., alanine or polyalanine) to
determine whether the interaction of the antibody with antigen is
affected. Further substitutions may be introduced at the amino acid
locations demonstrating functional sensitivity to the initial
substitutions. Alternatively, or additionally, a crystal structure
of an antigen-antibody complex to identify contact points between
the antibody and antigen can be used. Such contact residues and
neighboring residues may be targeted or eliminated as candidates
for substitution. Variants may be screened to determine whether
they contain the desired properties.
[0476] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide, which
increases the serum half-life of the antibody.
[0477] b) Glycosylation Variants
[0478] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0479] Where the antibody comprises an Fc-region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc-region. See, e.g., Wright, A.
and Morrison, S. L., TIBTECH 15 (1997) 26-32. The oligosaccharide
may include various carbohydrates, e.g., mannose, N-acetyl
glucosamine (GlcNAc), galactose, and sialic acid, as well as a
fucose attached to a GlcNAc in the "stem" of the biantennary
oligosaccharide structure. In some embodiments, modifications of
the oligosaccharide in an antibody of the invention may be made in
order to create antibody variants with certain improved
properties.
[0480] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc-region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e. g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc-region (EU numbering of Fc-region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
fucosylation variants may have improved ADCC function. See, e.g.,
US 2003/0157108; US 2004/0093621. Examples of publications related
to "defucosylated" or "fucose-deficient" antibody variants include:
US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US
2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US
2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO
2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140;
Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249;
Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622.
Examples of cell lines capable of producing defucosylated
antibodies include Lec13 CHO cells deficient in protein
fucosylation (Ripka, J., et al., Arch. Biochem. Biophys. 249 (1986)
533-545; US 2003/0157108; and WO 2004/056312, especially at Example
11), and knockout cell lines, such as alpha-1,6-fucosyltransferase
gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki, N., et
al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y., et al.,
Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).
[0481] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc-region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878; U.S. Pat. No. 6,602,684; and US
2005/0123546. Antibody variants with at least one galactose residue
in the oligosaccharide attached to the Fc-region are also provided.
Such antibody variants may have improved CDC function. Such
antibody variants are described, e.g., in WO 1997/30087; WO
1998/58964; and WO 1999/22764.
[0482] c) Fe-Region Variants
[0483] In certain embodiments, one or more further amino acid
modifications may be introduced into a heterodimeric polypeptide as
reported herein, thereby generating an Fc-region variant. The
Fc-region variant may be derived from a human Fc-region sequence
(e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc-region) comprising an
amino acid modification (e.g. a substitution/mutation) at one or
more amino acid positions.
[0484] In certain embodiments, the invention contemplates a
heterodimeric polypeptide that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half-life of the dimeric polypeptide in vivo is important
yet certain effector functions (such as CDC and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the heterodimeric
polypeptide antibody lacks Fc.gamma.R binding (hence likely lacking
ADCC activity), but retains FcRn binding ability. The primary cells
for mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas
monocytes express Fc.gamma.R1, Fc.gamma.RII and Fc.gamma.RIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page
464 of Ravetch, J. V. and Kinet, J. P., Annu. Rev. Immunol. 9
(1991) 457-492. Non-limiting examples of in vitro assays to assess
ADCC activity of a molecule of interest are described in U.S. Pat.
No. 5,500,362 (see, e.g. Hellstrom, I. et al., Proc. Natl. Acad.
Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl.
Acad. Sci. USA 82 (1985) 1499-1502); U.S. Pat. No. 5,821,337 (see
Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361).
Alternatively, non-radioactive assays methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.RTM. non-radioactive cytotoxicity assay (Promega, Madison,
Wis.). Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95
(1998) 652-656. C1q binding assays may also be carried out to
confirm that the dimeric polypeptide is unable to bind C1q and
hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in
WO 2006/029879 and WO 2005/100402. To assess complement activation,
a CDC assay may be performed (see, for example, Gazzano-Santoro, H.
et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, M. S. et
al., Blood 101 (2003) 1045-1052; and Cragg, M. S. and M. J.
Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo
clearance/half-life determinations can also be performed using
methods known in the art (see, e.g., Petkova, S. B. et al., Int.
Immunol. 18 (2006) 1759-1769).
[0485] Heterodimeric polypeptides with reduced effector function
include those with substitution of one or more of Fc-region
residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.
6,737,056). Such Fc-region variants include Fc-regions with
substitutions at two or more of amino acid positions 265, 269, 270,
297 and 327, including the so-called "DANA" Fc-region mutant with
substitution of residues 265 and 297 to alanine (U.S. Pat. No.
7,332,581).
[0486] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276
(2001) 6591-6604).
[0487] In certain embodiments, a heterodimeric polypeptide variant
comprises an Fc-region with one or more amino acid substitutions,
which improve ADCC, e.g., substitutions at positions 298, 333,
and/or 334 of the Fc-region (EU numbering of residues).
[0488] In some embodiments, alterations are made in the Fc-region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie, E.
E. et al., J. Immunol. 164 (2000) 4178-4184.
[0489] Antibodies with increased half-lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer, R. L. et al., J.
Immunol. 117 (1976) 587-593, and Kim, J. K. et al., J. Immunol. 24
(1994) 2429-2434), are described in US 2005/0014934. Those
antibodies comprise an Fc-region with one or more substitutions
therein, which improve binding of the Fc-region to FcRn. Such
Fc-region variants include those with substitutions at one or more
of Fc-region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311,
312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,
e.g., substitution of Fc-region residue 434 (U.S. Pat. No.
7,371,826).
[0490] See also Duncan, A. R. and Winter, G., Nature 322 (1988)
738-740; U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351
concerning other examples of Fc-region variants.
[0491] d) Cysteine Engineered Antibody Variants
[0492] In certain embodiments, it may be desirable to create
cysteine engineered heterodimeric polypeptides, e.g., in analogy to
"thioMAbs," in which one or more residues of an antibody are
substituted with cysteine residues. In particular embodiments, the
substituted residues occur at accessible sites of the heterodimeric
polypeptide. By substituting those residues with cysteine, reactive
thiol groups are thereby positioned at accessible sites of the
heterodimeric polypeptide and may be used to conjugate the
heterodimeric polypeptide to other moieties, such as drug moieties
or linker-drug moieties, to create an immunoconjugate, as described
further herein. In certain embodiments, any one or more of the
following residues may be substituted with cysteine: V205 (Kabat
numbering) of the light chain; A118 (EU numbering) of the heavy
chain; and 5400 (EU numbering) of the heavy chain Fc-region.
Cysteine engineered dimeric polypeptides may be generated as
described, e.g., in U.S. Pat. No. 7,521,541.
[0493] e) Derivatives
[0494] In certain embodiments, a heterodimeric polypeptide as
reported herein may be further modified to contain additional
non-proteinaceous moieties that are known in the art and readily
available. The moieties suitable for derivatization of the
heterodimeric polypeptide include but are not limited to
water-soluble polymers. Non-limiting examples of water soluble
polymers include, but are not limited to, polyethylene glycol
(PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyamino acids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, pro-propylene glycol homopolymers,
polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or non-branched. The
number of polymers attached to the dimeric polypeptide may vary,
and if more than one polymer is attached, they can be the same or
different molecules. In general, the number and/or type of polymers
used for derivatization can be determined based on considerations
including, but not limited to, the particular properties or
functions of the dimeric polypeptide to be improved, whether the
dimeric polypeptide derivative will be used in a therapy under
defined conditions, etc.
[0495] In another embodiment, conjugates of a heterodimeric
polypeptide as reported herein and non-proteinaceous moiety that
may be selectively heated by exposure to radiation are provided. In
one embodiment, the non-proteinaceous moiety is a carbon nanotube
(Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)
11600-11605). The radiation may be of any wavelength, and includes,
but is not limited to, wavelengths that do not harm ordinary cells,
but which heat the non-proteinaceous moiety to a temperature at
which cells proximal to the dimeric polypeptide-non-proteinaceous
moiety are killed.
[0496] f) Heterodimerization
[0497] There exist several approaches for CH3-modifications to
enforce the heterodimerization, which are well described e.g. in WO
96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901,
WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO
2012058768, WO 2013157954, WO 2013096291. Typically in all such
approaches the first CH3 domain and the second CH3 domains are both
engineered in a complementary manner so that each CH3 domain (or
the heavy chain comprising it) cannot longer homodimerize with
itself but is forced to heterodimerize with the complementary
engineered other CH3 domain (so that the first and second CH3
domain heterodimerize and no homodimers between the two first or
the two second CH3 domains are formed). These different approaches
for improved heavy chain heterodimerization are contemplated as
different alternatives in combination with the heavy-light chain
modifications (VH and VL exchange/replacement in one binding arm
and the introduction of substitutions of charged amino acids with
opposite charges in the CH1/CL interface) in the multispecific
antibodies according to the invention which reduce light chain
mispairing an Bence-Jones type side products.
[0498] In one preferred embodiment of the invention (in case the
multispecific antibody comprises CH3 domains in the heavy chains)
the CH3 domains of said heterodimeric polypeptide according to the
invention can be altered by the "knob-into-holes" technology which
is described in detail with several examples in e.g. WO 96/027011,
Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621; and
Merchant, A. M., et al., Nat. Biotechnol. 16 (1998) 677-681; WO
98/050431. In this method the interaction surfaces of the two CH3
domains are altered to increase the heterodimerization of both
heavy chains containing these two CH3 domains. Each of the two CH3
domains (of the two heavy chains) can be the "knob", while the
other is the "hole". The introduction of a disulfide bridge further
stabilizes the heterodimers (Merchant, A. M., et al., Nature
Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270
(1997) 26-35) and increases the yield.
[0499] Thus in one embodiment of the invention said heterodimeric
polypeptide (comprises a CH3 domain in each heavy chain and) is
further characterized in that [0500] the first CH3 domain of the
first heavy chain of the antibody under a) and the second CH3
domain of the second heavy chain of the antibody under b) each meet
at an interface, which comprises an original interface between the
antibody CH3 domains. [0501] wherein said interface is altered to
promote the formation of the multispecific antibody, wherein the
alteration is characterized in that: [0502] i) the CH3 domain of
one heavy chain is altered, so that within the original interface
of the CH3 domain of one heavy chain that meets the original
interface of the CH3 domain of the other heavy chain within the
multispecific antibody, an amino acid residue is replaced with an
amino acid residue having a larger side chain volume, thereby
generating a protuberance within the interface of the CH3 domain of
one heavy chain, which is positionable in a cavity within the
interface of the CH3 domain of the other heavy chain and [0503] ii)
the CH3 domain of the other heavy chain is altered, so that within
the original interface of the second CH3 domain that meets the
original interface of the first CH3 domain within the multispecific
antibody [0504] an amino acid residue is replaced with an amino
acid residue having a smaller side chain volume, thereby generating
a cavity within the interface of the second CH3 domain within which
a protuberance within the interface of the first CH3 domain is
positionable.
[0505] Preferably said amino acid residue having a larger side
chain volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), tryptophan (W).
[0506] Preferably said amino acid residue having a smaller side
chain volume is selected from the group consisting of alanine (A),
serine (S), threonine (T), valine (V).
[0507] In one aspect of the invention both CH3 domains are further
altered by the introduction of cysteine (C) as amino acid in the
corresponding positions of each CH3 domain such that a disulfide
bridge between both CH3 domains can be formed.
[0508] In one preferred embodiment, said heterodimeric polypeptide
comprises an amino acid T366W mutation in the first CH3 domain of
the "knobs-chain" and amino acid T366S, L368A, Y407V mutations in
the second CH3 domain of the "hole-chain". An additional interchain
disulfide bridge between the CH3 domains can also be used
(Merchant, A. M., et al., Nature Biotech. 16 (1998) 677-681) e.g.
by introducing an amino acid Y349C mutation into the CH3 domain of
the "hole-chain" and an amino acid E356C mutation or an amino acid
S354C mutation into the CH3 domain of the "knobs-chain".
[0509] In one preferred embodiment, said heterodimeric polypeptide
(which comprises a CH3 domain in each heavy chain) comprises amino
acid S354C, T366W mutations in one of the two CH3 domains and amino
acid Y349C, T366S, L368A, Y407V mutations in the other of the two
CH3 domains (the additional amino acid S354C mutation in one CH3
domain and the additional amino acid Y349C mutation in the other
CH3 domain forming an interchain disulfide bridge) (numbering
according to Kabat).
[0510] Other techniques for CH3-modifications to enforcing the
heterodimerization are contemplated as alternatives of the
invention and described e.g. in WO 96/27011, WO 98/050431, EP
1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO
2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO
2013/157954, WO 2013/096291.
[0511] In one embodiment the heterodimerization approach described
in EP 1 870 459A1, can be used alternatively. This approach is
based on the by the introduction of substitutions/mutations of
charged amino acids with the opposite charge at specific amino acid
positions of the in the CH3/CH3 domain interface between both heavy
chains. One preferred embodiment for said heterodimeric polypeptide
are amino acid R409D; K370E mutations in the first CH3 domain of
the multispecific antibody and amino acid D399K; E357K mutations in
the seconds CH3 domain of the multispecific antibody (numbering
according to Kabat).
[0512] In another embodiment said heterodimeric polypeptide
comprises a amino acid T366W mutation in the CH3 domain of the
"knobs chain" and amino acid T366S, L368A, Y407V mutations in the
CH3 domain of the "hole-chain" and additionally amino acid R409D;
K370E mutations in the CH3 domain of the "knobs-chain" and amino
acid D399K; E357K mutations in the CH3 domain of the
"hole-chain".
[0513] In another embodiment said heterodimeric polypeptide
comprises amino acid S354C, T366W mutations in one of the two CH3
domains and amino acid Y349C, T366S, L368A, Y407V mutations in the
other of the two CH3 domains or said multispecific antibody
comprises amino acid Y349C, T366W mutations in one of the two CH3
domains and amino acid S354C, T366S, L368A, Y407V mutations in the
other of the two CH3 domains and additionally amino acid R409D;
K370E mutations in the CH3 domain of the "knobs-chain" and amino
acid D399K; E357K mutations in the CH3 domain of the
"hole-chain".
[0514] In one embodiment the heterodimerization approach described
in WO2013/157953 can be used alternatively. In one embodiment a
first CH3 domain comprises amino acid T366K mutation and a second
CH3 domain polypeptide comprises amino acid L351D mutation. In a
further embodiment the first CH3 domain comprises further amino
acid L351K mutation. In a further embodiment the second CH3 domain
comprises further amino acid mutation selected from Y349E, Y349D
and L368E (preferably L368E).
[0515] In one embodiment the heterodimerization approach described
in WO2012/058768 can be used alternatively. In one embodiment a
first CH3 domain comprises amino acid L351Y, Y407A mutations and a
second CH3 domain comprises amino acid T366A, K409F mutations. In a
further embodiment the second CH3 domain comprises a further amino
acid mutation at position T411, D399, 5400, F405, N390, or K392
e.g. selected from a) T411 N, T411 R, T411Q, T411 K, T411D, T411E
or T411W, b) D399R, D399W, D399Y or D399K, c S400E, S400D, S400R,
or S400K F4051, F405M, F405T, F405S, F405V or F405W N390R, N390K or
N390D K392V, K392M, K392R, K392L, K392F or K392E. In a further
embodiment a first CH3 domain comprises amino acid L351Y, Y407A
mutations and a second CH3 domain comprises amino acid T366V, K409F
mutations. In a further embodiment a first CH3 domain comprises
amino acid Y407A mutations and a second CH3 domain comprises amino
acid T366A, K409F mutations. In a further embodiment the second CH3
domain comprises a further amino acid K392E, T411E, D399R and S400R
mutations.
[0516] In one embodiment the heterodimerization approach described
in WO2011/143545 can be used alternatively e.g. with the amino acid
modification at a position selected from the group consisting of
368 and 409.
[0517] In one embodiment the heterodimerization approach described
in WO2011/090762, which also uses the knobs-into-holes technology
described above, can be used alternatively. In one embodiment a
first CH3 domain comprises amino acid T366W mutations and a second
CH3 domain comprises amino acid Y407A mutations. In one embodiment
a first CH3 domain comprises amino acid T366Y mutations and a
second CH3 domain comprises amino acid Y407T mutations.
[0518] In one embodiment the multispecific antibody is of IgG2
isotype and the heterodimerization approach described in
WO2010/129304 can be used alternatively.
[0519] In one embodiment the heterodimerization approach described
in WO2009/089004 can be used alternatively. In one embodiment a
first CH3 domain comprises amino acid substitution of K392 or N392
with a negative-charged amino acid (e.g. glutamic acid (E), or
aspartic acid (D), preferably K392D or N392D) and a second CH3
domain comprises amino acid substitution of D399, E356, D356, or
E357 with a positive-charged amino acid (e.g. Lysine (K) or
arginine (R), preferably D399K, E356K, D356K, or E357K and more
preferably D399K and E356K. In a further embodiment the first CH3
domain further comprises amino acid substitution of K409 or R409
with a negative-charged amino acid (e.g. glutamic acid (E), or
aspartic acid (D), preferably K409D or R409D). In a further
embodiment the first CH3 domain further or alternatively comprises
amino acid substitution of K439 and/or K370 with a negative-charged
amino acid (e.g. glutamic acid (E), or aspartic acid (D)).
[0520] In one embodiment the heterodimerization approach described
in WO2007/147901 can be used alternatively. In one embodiment a
first CH3 domain comprises amino acid K253E, D282K, and K322D
mutations and a second CH3 domain comprises amino acid D239K,
E240K, and K292D mutations.
[0521] In one embodiment the heterodimerization approach described
in WO2007/110205 can be used alternatively.
[0522] E. Recombinant Methods and Compositions
[0523] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid(s) encoding a heterodimeric
polypeptide as reported herein is(are) provided. Such nucleic acid
may encode an amino acid sequence comprising the first polypeptide
and/or an amino acid sequence comprising the second polypeptide of
the heterodimeric polypeptide. In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the first polypeptide of the heterodimeric polypeptide
and an amino acid sequence comprising the second polypeptide of the
heterodimeric polypeptide, or (2) a first vector comprising a
nucleic acid that encodes an amino acid sequence comprising the
first polypeptide of the heterodimeric polypeptide and a second
vector comprising a nucleic acid that encodes an amino acid
sequence comprising the second polypeptide of the heterodimeric
polypeptide. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making a heterodimeric
polypeptide as reported herein is provided, wherein the method
comprises culturing a host cell comprising a nucleic acid encoding
the heterodimeric polypeptide, as provided above, under conditions
suitable for expression of the heterodimeric polypeptide, and
optionally recovering the antibody from the host cell (or host cell
culture medium).
[0524] For recombinant production of a heterodimeric polypeptide as
reported herein, nucleic acid encoding a heterodimeric polypeptide,
e.g., as described above, is isolated and inserted into one or more
vectors for further cloning and/or expression in a host cell. Such
nucleic acid may be readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the variant
Fc-region polypeptide(s) and the heavy and light chains of the
antibody).
[0525] Suitable host cells for cloning or expression of
heterodimeric polypeptide-encoding vectors include prokaryotic or
eukaryotic cells described herein. For example, heterodimeric
polypeptides may be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed. For
expression of antibody fragments and polypeptides in bacteria, see,
e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also
Charlton, K. A., In: Methods in Molecular Biology, Vol. 248, Lo, B.
K. C. (ed.), Humana Press, Totowa, N.J. (2003), pp. 245-254,
describing expression of antibody fragments in E. coli.). After
expression, the heterodimeric polypeptide may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0526] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for heterodimeric polypeptide-encoding vectors, including fungi and
yeast strains whose glycosylation pathways have been "humanized"
resulting in the production of a dimeric polypeptide with a
partially or fully human glycosylation pattern. See Gemgross, T.
U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat.
Biotech. 24 (2006) 210-215.
[0527] Suitable host cells for the expression of glycosylated
heterodimeric polypeptide are also derived from multicellular
organisms (invertebrates and vertebrates). Examples of invertebrate
cells include plant and insect cells. Numerous baculoviral strains
have been identified which may be used in conjunction with insect
cells, particularly for transfection of Spodoptera frugiperda
cells.
[0528] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0529] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (HEK293 or 293 cells as described, e.g., in Graham, F.
L., et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney
cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in
Mather, J. P., Biol. Reprod. 23 (1980) 243-252); monkey kidney
cells (CV1); African green monkey kidney cells (VERO-76); human
cervical carcinoma cells (HELA); canine kidney cells (MDCK);
buffalo rat liver cells (BRL 3A); human lung cells (W138); human
liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells,
as described, e.g., in Mather, J. P., et al., Annals N.Y. Acad.
Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful
mammalian host cell lines include Chinese hamster ovary (CHO)
cells, including DHFR.sup.- CHO cells (Urlaub, G., et al., Proc.
Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines
such as Y0, NS0 and Sp2/0. For a review of certain mammalian host
cell lines suitable for antibody production, see, e.g., Yazaki, P.
and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C.
(ed.), Humana Press, Totowa, N.J. (2004), pp. 255-268.
[0530] F. Combination Treatment
[0531] In certain embodiments the heterodimeric polypeptide as
reported herein or pharmaceutical formulation as reported herein is
administered alone (without an additional therapeutic agent) for
the treatment of one or more ocular vascular diseases described
herein.
[0532] In other embodiments the heterodimeric polypeptide antibody
or pharmaceutical formulation as reported herein is administered in
combination with one or more additional therapeutic agents or
methods for the treatment of one or more vascular eye diseases
described herein.
[0533] In other embodiments, the heterodimeric polypeptide or
pharmaceutical formulation as reported herein is formulated in
combination with one or more additional therapeutic agents and
administered for the treatment of one or more vascular eye diseases
described herein.
[0534] In certain embodiments, the combination treatments provided
herein include that the heterodimeric polypeptide or pharmaceutical
formulation as reported herein is administered sequentially with
one or more additional therapeutic agents for the treatment of one
or more ocular vascular diseases described herein.
[0535] The additional therapeutic agents include, but are not
limited to, Tryptophanyl-tRNA synthetase (TrpRS), EyeOOl (anti-VEGF
PEGylated aptamer), squalamine, RETAANE.TM. (anecortave acetate for
depot suspension; Alcon, Inc.), Combretastatin A4 Prodrug (CA4P),
MACUGEN.TM., MIFEPREX.TM. (mifepristone-ru486), subtenon
triamcinolone acetonide, intravitreal crystalline triamcinolone
acetonide, Prinomastat (AG3340-synthetic matrix metalloproteinase
inhibitor, Pfizer), fluocinolone acetonide (including fluocinolone
intraocular implant, Bausch & Lomb/Control Delivery Systems),
VEGFR inhibitors (Sugen), VEGF-Trap (Regeneron/Aventis), VEGF
receptor tyrosine kinase inhibitors such as
4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylme-
thoxy)quinazoline (ZD6474),
4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)-
quinazoline (AZD2171), vatalanib (PTK787) and SU1 1248 (sunitinib),
linomide, and inhibitors of integrin v.beta.3 function and
angiostatin.
[0536] Other pharmaceutical therapies that can be used in
combination with the heterodimeric polypeptide or pharmaceutical
formulation as reported herein, including, but are not limited to,
VISUDYNE.TM. with use of a non-thermal laser, PKC 412, Endovion
(NeuroSearch A/S), neurotrophic factors, including by way of
example Glial Derived Neurotrophic Factor and Ciliary Neurotrophic
Factor, diatazem, dorzolamide, Phototrop, 9-cis-retinal, eye
medication (including Echo Therapy) including phospholine iodide or
echothiophate or carbonic anhydrase inhibitors, AE-941 (AEtema
Laboratories, Inc.), Sima-027 (Sima Therapeutics, Inc.), pegaptanib
(NeXstar Pharmaceuticals/Gilead Sciences), neurotrophins
(including, by way of example only, NT-4/5, Genentech), CandS
(Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals),
integrin antagonists (including those from Jerini AG and Abbott
Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem
Ltd.), thalidomide (as used, for example, by EntreMed, Inc.),
cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan/Oculex),
DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate
(University of Michigan), LYN-002 (Lynkeus Biotech), microalgal
compound (Aquasearch/Albany, Mera Pharmaceuticals), D-9120
(Celltech Group plc.), ATX-S10 (Hamamatsu Photonics), TGF-beta 2
(Genzyme/Celtrix), tyrosine kinase inhibitors (Allergan, SUGEN,
Pfizer), NX-278-L (NeXstar Pharmaceuticals/Gilead Sciences), Opt-24
(OPTIS France SA), retinal cell ganglion neuroprotectants (Cogent
Neurosciences), N-nitropyrazole derivatives (Texas A&M
University System), KP-102 (Krenitsky Pharmaceuticals),
cyclosporine A, timited retinal translocation, photodynamic
therapy, (including, by way of example only, receptor-targeted PDT,
Bristol-Myers Squibb, Co.; porfimer sodium for injection with PDT;
verteporfin, QLT Inc.; rostaporfin with PDT, Miravent Medical
Technologies; talaporfin sodium with PDT, Nippon Petroleum;
motexafin lutetium, Pharmacyclics, Inc.), antisense
oligonucleotides (including, by way of example, products tested by
Novagali Pharma SA and ISIS-13650, Isis Pharmaceuticals), laser
photocoagulation, drusen lasering, macular hole surgery, macular
translocation surgery, implantable miniature telescopes, Phi-Motion
Angiography (also known as Micro-Laser Therapy and Feeder Vessel
Treatment), Proton Beam Therapy, microstimulation therapy, Retinal
Detachment and Vitreous Surgery, Scleral Buckle, Submacular
Surgery, Transpupillary Thermotherapy, Photosystem I therapy, use
of RNA interference (RNAi), extracorporeal rheopheresis (also known
as membrane differential filtration and Rheotherapy), microchip
implantation, stem cell therapy, gene replacement therapy, ribozyme
gene therapy (including gene therapy for hypoxia response element,
Oxford Biomedica; Lentipak, Genetix; PDEF gene therapy, GenVec),
photoreceptor/retinal cells transplantation (including
transplantable retinal epithelial cells, Diacrin, Inc.; retinal
cell transplant, Cell Genesys, Inc.), and acupuncture.
[0537] Any anti-angiogenic agent can be used in combination with
the heterodimeric polypeptide or pharmaceutical formulation as
reported herein, including, but not limited to, those listed by
Carmeliet and Jain (Nature 407 (2000) 249-257). In certain
embodiments, the anti-angiogenic agent is another VEGF antagonist
or a VEGF receptor antagonist such as VEGF variants, soluble VEGF
receptor fragments, aptamers capable of blocking VEGF or VEGFR,
neutralizing anti-VEGFR antibodies, low molecule weight inhibitors
of VEGFR tyrosine kinases and any combinations thereof and these
include anti-VEGF aptamers (e.g. Pegaptanib), soluble recombinant
decoy receptors (e.g. VEGF Trap). In certain embodiments, the
anti-angiogenic agent is include corticosteroids, angiostatic
steroids, anecortave acetate, angiostatin, endostatin, small
interfering RNA's decreasing expression of VEGFR or VEGF ligand,
post-VEGFR blockade with tyrosine kinase inhibitors, MMP
inhibitors, IGFBP3, SDF-1 blockers, PEDF, gamma-secretase,
Delta-like ligand 4, integrin antagonists, HIF-1 alpha blockade,
protein kinase CK2 blockade, and inhibition of stem cell (i.e.
endothelial progenitor cell) homing to the site of
neovascularization using vascular endothelial cadherin (CD-144) and
stromal derived factor (SDF)-I antibodies. Small molecule RTK
inhibitors targeting VEGF receptors including PTK787 can also be
used. Agents that have activity against neovascularization that are
not necessarily anti-VEGF compounds can also be used and include
anti-inflammatory drugs, m-Tor inhibitors, rapamycin, everolismus,
temsirolismus, cyclospohne, anti-TNF agents, anti-complement
agents, and non-steroidal anti-inflammatory agents. Agents that are
neuroprotective and can potentially reduce the progression of dry
macular degeneration can also be used, such as the class of drugs
called the ,neurosteroids". These include drugs such as
dehydroepiandrosterone (DHEA) (Brand names: Prastera(R) and
Fidelin(R)), dehydroepiandrosterone sulfate, and pregnenolone
sulfate. Any AMD (age-related macular degeneration) therapeutic
agent can be used in combination with the dimeric polypeptide or
pharmaceutical formulation as reported herein, including but not
limited to verteporfin in combination with PDT, pegaptanib sodium,
zinc, or an antioxidant(s), alone or in any combination.
[0538] G. Pharmaceutical Formulations
[0539] Pharmaceutical formulations of a heterodimeric polypeptide
as reported herein are prepared by mixing such heterodimeric
polypeptide having the desired degree of purity with one or more
optional pharmaceutically acceptable carriers (Remington's
Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)), in
the form of lyophilized formulations or aqueous solutions.
Pharmaceutically acceptable carriers are generally nontoxic to
recipients at the dosages and concentrations employed, and include,
but are not limited to: buffers such as phosphate, citrate, and
other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyl dimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride;
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
poly(vinylpyrrolidone); amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as polyethylene glycol (PEG). Exemplary
pharmaceutically acceptable carriers herein further include
interstitial drug dispersion agents such as soluble neutral-active
hyaluronidase glycoproteins (sHASEGP), for example, human soluble
PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX.RTM.,
Baxter International, Inc.). Certain exemplary sHASEGPs and methods
of use, including rhuPH20, are described in US 2005/0260186 and US
2006/0104968. In one aspect, a sHASEGP is combined with one or more
additional glycosaminoglycanases such as chondroitinases.
[0540] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the
latter formulations including a histidine-acetate buffer.
[0541] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such active ingredients are suitably
present in combination in amounts that are effective for the
purpose intended.
[0542] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethyl cellulose or
gelatin-microcapsules and poly-(methyl methacrylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nanoparticles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.
(ed.) (1980).
[0543] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0544] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0545] H. Therapeutic Methods and Compositions
[0546] Any of the heterodimeric polypeptides as reported herein may
be used in therapeutic methods.
[0547] In one aspect, a heterodimeric polypeptide as reported
herein for use as a medicament is provided. In further aspects, a
heterodimeric polypeptide for use in treating ocular vascular
diseases is provided. In certain embodiments, a heterodimeric
polypeptide for use in a method of treatment is provided. In
certain embodiments, the invention provides a heterodimeric
polypeptide for use in a method of treating an individual having an
ocular vascular disease comprising administering to the individual
an effective amount of the heterodimeric polypeptide as reported
herein. In one such embodiment, the method further comprises
administering to the individual an effective amount of at least one
additional therapeutic agent, e.g., as described above in section
D. In further embodiments, the invention provides a heterodimeric
polypeptide for use in inhibiting angiogenesis in the eye. In
certain embodiments, the invention provides a heterodimeric
polypeptide for use in a method of inhibiting angiogenesis in an
individual comprising administering to the individual an effective
of the heterodimeric polypeptide to inhibit angiogenesis. An
"individual" according to any of the above embodiments is in one
preferred embodiment a human.
[0548] In a further aspect, the invention provides for the use of a
heterodimeric polypeptide in the manufacture or preparation of a
medicament. In one embodiment, the medicament is for treatment of
an ocular vascular disease. In a further embodiment, the medicament
is for use in a method of treating an ocular vascular disease
comprising administering to an individual having an ocular vascular
disease an effective amount of the medicament. In one such
embodiment, the method further comprises administering to the
individual an effective amount of at least one additional
therapeutic agent, e.g., as described above. In a further
embodiment, the medicament is for inhibiting angiogenesis. In a
further embodiment, the medicament is for use in a method of
inhibiting angiogenesis in an individual comprising administering
to the individual an amount effective of the medicament to inhibit
angiogenesis. An "individual" according to any of the above
embodiments may be a human.
[0549] In a further aspect, the invention provides a method for
treating a vascular eye disease. In one embodiment, the method
comprises administering to an individual having such a vascular eye
disease an effective amount of a heterodimeric polypeptide as
reported herein. In one such embodiment, the method further
comprises administering to the individual an effective amount of at
least one additional therapeutic agent, as described below. An
"individual" according to any of the above embodiments may be a
human.
[0550] In a further aspect, the invention provides a method for
inhibiting angiogenesis in the eye in an individual. In one
embodiment, the method comprises administering to the individual an
effective amount of a heterodimeric polypeptide as reported herein
to inhibit angiogenesis. In one embodiment, an "individual" is a
human.
[0551] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the heterodimeric polypeptides as
reported herein, e.g., for use in any of the above therapeutic
methods. In one embodiment, a pharmaceutical formulation comprises
any of the heterodimeric polypeptides as reported herein and a
pharmaceutically acceptable carrier. In another embodiment, a
pharmaceutical formulation comprises any of the heterodimeric
polypeptides as reported herein and at least one additional
therapeutic agent, e.g., as described below.
[0552] Heterodimeric polypeptide as reported herein can be used
either alone or in combination with other agents in a therapy. For
instance, a heterodimeric polypeptide as reported herein may be
co-administered with at least one additional therapeutic agent
[0553] A heterodimeric polypeptide as reported herein (and any
additional therapeutic agent) can be administered by any suitable
means, including parenteral, intrapulmonary, and intranasal, and,
if desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
Dosing can be by any suitable route, e.g. by injections, such as
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic. Various dosing
schedules including but not limited to single or multiple
administrations over various time-points, bolus administration, and
pulse infusion are contemplated herein.
[0554] Heterodimeric polypeptides as reported herein would be
formulated, dosed, and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The heterodimeric polypeptide need not be, but is optionally
formulated with one or more agents currently used to prevent or
treat the disorder in question. The effective amount of such other
agents depends on the amount of heterodimeric polypeptide present
in the formulation, the type of disorder or treatment, and other
factors discussed above. These are generally used in the same
dosages and with administration routes as described herein, or
about from 1 to 99% of the dosages described herein, or in any
dosage and by any route that is empirically/clinically determined
to be appropriate.
[0555] For the prevention or treatment of disease, the appropriate
dosage of a heterodimeric polypeptide as reported herein (when used
alone or in combination with one or more other additional
therapeutic agents) will depend on the type of disease to be
treated, the type of heterodimeric polypeptide, the severity and
course of the disease, whether the heterodimeric polypeptide is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the
heterodimeric polypeptide, and the discretion of the attending
physician. The heterodimeric polypeptide is suitably administered
to the patient at one time or over a series of treatments.
Depending on the type and severity of the disease, about 1 .mu.g/kg
to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg) of heterodimeric polypeptide
can be an initial candidate dosage for administration to the
patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the heterodimeric polypeptide would be in the range from
about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of
about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any
combination thereof) may be administered to the patient. Such doses
may be administered intermittently, e.g. every week or every three
weeks (e.g. such that the patient receives from about two to about
twenty, or e.g. about six doses of the dimeric polypeptide). An
initial higher loading dose, followed by one or more lower doses
may be administered. The progress of this therapy is easily
monitored by conventional techniques and assays.
III. Articles of Manufacture
[0556] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition, which is by itself or combined with another
composition effective for treating, preventing and/or diagnosing
the condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is a heterodimeric polypeptide as
reported herein. The label or package insert indicates that the
composition is used for treating the condition of choice. Moreover,
the article of manufacture may comprise (a) a first container with
a composition contained therein, wherein the composition comprises
a heterodimeric polypeptide as reported herein; and (b) a second
container with a composition contained therein, wherein the
composition comprises a further otherwise therapeutic agent. The
article of manufacture in this embodiment of the invention may
further comprise a package insert indicating that the compositions
can be used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a pharmaceutically
acceptable buffer, such as bacteriostatic water for injection
(BWFI), phosphate-buffered saline, Ringer's solution and dextrose
solution. It may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
[0557] It is understood that any of the above articles of
manufacture may include an immunoconjugate as reported herein in
place of or in addition to a heterodimeric polypeptide as reported
herein.
IV. EXAMPLES
[0558] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
[0559] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
[0560] Methods
[0561] Electrospray ionization mass spectrometry (ESI-MS)
[0562] Protein aliquots (50 .mu.g) are deglycosylated by adding 0.5
.mu.L N-Glycanase plus (Roche) and sodium phosphate buffer (0.1 M,
pH 7.1) to obtain a final sample volume of 115 .mu.L. The mixture
is incubated at 37.degree. C. for 18 h. Afterwards for reduction
and denaturing 60 .mu.L 0.5 M TCEP (Pierce) in 4 M guanidine*HCl
(Pierce) and 50 .mu.L 8 M guanidine*HCl are added. The mixture is
incubated at 37.degree. C. for 30 min. Samples are desalted by size
exclusion chromatography (Sepharose G-25, isocratic, 40%
acetonitrile with 2% formic acid). ESI mass spectra (+ve) are
recorded on a Q-TOF instrument (maXis, Bruker) equipped with a nano
ESI source (TriVersa NanoMate, Advion). MS parameter settings are
as follows: Transfer: Funnel RF, 400 Vpp; ISCID Energy, 0 eV;
Multipole RF, 400 Vpp; Quadrupole: Ion Energy, 4.0 eV; Low Mass,
600 m/z; Source: Dry Gas, 8 L/min; Dry Gas Temperature, 160.degree.
C.; Collision Cell: Collision Energy, 10 eV; Collision RF: 2000
Vpp; Ion Cooler: Ion Cooler RF, 300 Vpp; Transfer Time: 120 .mu.s;
Pre Puls Storage, 10 .mu.s; scan range m/z 600 to 2000. For data
evaluation in-house developed software (MassAnalyzer) is used.
[0563] FcRn surface plasmon resonance (SPR) analysis
[0564] The binding properties of wild-type antibody and the mutants
to FcRn are analyzed by surface plasmon resonance (SPR) technology
using a BIAcore T100 instrument (BIAcore AB, Uppsala, Sweden). This
system is well established for the study of molecular interactions.
It allows a continuous real-time monitoring of ligand/analyte
bindings and thus the determination of kinetic parameters in
various assay settings. SPR-technology is based on the measurement
of the refractive index close to the surface of a gold-coated
biosensor chip. Changes in the refractive index indicate mass
changes on the surface caused by the interaction of immobilized
ligand with analyte injected in solution. If molecules bind to an
immobilized ligand on the surface the mass increases, in case of
dissociation the mass decreases. In the current assay, the FcRn
receptor is immobilized onto a BIAcore CM5-biosensor chip (GE
Healthcare Bioscience, Uppsala, Sweden) via amine coupling to a
level of 400 Response units (RU). The assay is carried out at room
temperature with PBS, 0.05% Tween20 pH 6.0 (GE Healthcare
Bioscience) as running and dilution buffer. 200 nM of samples are
injected at a flow rate of 50 .mu.L/min at room temperature.
Association time is 180 sec., dissociation phase took 360 sec.
Regeneration of the chip surface is reached by a short injection of
HBS-P, pH 8.0. Evaluation of SPR-data is performed by comparison of
the biological response signal height at 180 sec. after injection
and at 300 sec. after injection. The corresponding parameters are
the RU max level (180 sec. after injection) and late stability (300
sec. after end of injection).
[0565] Protein A surface plasmon resonance (SPR) analysis
[0566] The assay is based on surface plasmon resonance
spectroscopy. Protein A is immobilized onto the surface of a SPR
biosensor. By injecting the sample into the flow cells of the SPR
spectrometer it forms a complex with the immobilized protein A
resulting in an increasing mass on the sensor chip surface, and
therefore to a higher response (as 1 RU is defined as 1
pg/mm.sup.2). Afterwards the sensor chip is regenerated by
dissolving the sample-protein A-complex. The gained responses are
then evaluated for the signal high in response units (RU) and the
dissociation behavior.
[0567] Around 3500 response units (RU) of protein A (20 .mu.g/mL)
are coupled onto a CM5 chip (GE Healthcare) at pH 4.0 by using the
amine coupling kit of GE Healthcare.
[0568] The sample and system buffer is HBS-P+(0.01 M HEPES, 0.15 M
NaCl, 0.005% Surfactant P20 Sterile-filtered, pH 7.4). Flow cell
temperature is set to 25.degree. C. and sample compartment
temperature to 12.degree. C. The system is primed with running
buffer. Then, a 5 nM solutions of the sample constructs are
injected for 120 seconds with a flow rate of 30 .mu.L/min, followed
by a 300 seconds dissociation phase. Then the sensor chip surface
is regenerated by two 30 seconds long injections of Glycine-HCl pH
1.5 at a flow rate of 30 .mu.L/min. Each sample is measured as a
triplicate.
[0569] The term "with (the) mutation IHH-AAA" as used herein refers
the combination of the mutations I253A (Ile253A1a), H310A
(His310A1a), and H435A (His435A1a) in a constant heavy chain region
of IgG1 or IgG4 subclass (numbering according to the Kabat EU index
numbering system), the term "with (the) mutation HHY-AAA" as used
herein refers the combination of the mutations H310A (His310A1a),
H433A (His433A1a) and Y436A (Tyr436A1a) in a constant heavy chain
region of IgG1 or IgG4 subclass (numbering according to the Kabat
EU index numbering system), the term "with (the) mutation P329G
LALA" as used herein refers to the combination of the mutations
L234A (Leu234A1a), L235A (Leu235A1a) and P329G (Pro329Gly) in a
constant heavy chain region of IgG1 subclass (numbering according
to the Kabat EU index numbering system), and the term "with (the)
mutation SPLE" as used herein refers to the combination of the
mutations S228P (Ser228Pro) and L235E (Leu235Glu) in a constant
heavy chain region of IgG4 subclass (numbering according to the
Kabat EU index numbering system).
[0570] General
[0571] General information regarding the nucleotide sequences of
human immunoglobulin light and heavy chains is given in: Kabat, E.
A., et al., Sequences of Proteins of Immunological Interest, 5th
ed., Public Health Service, National Institutes of Health,
Bethesda, Md. (1991). Amino acid residues of antibody chains are
numbered and referred to according to EU numbering (Edelman, G. M.,
et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E. A.,
et al., Sequences of Proteins of Immunological Interest, 5th ed.,
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)).
[0572] Recombinant DNA Techniques
[0573] Standard methods are used to manipulate DNA as described in
Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
The molecular biological reagents are used according to the
manufacturer's instructions.
[0574] Gene Synthesis
[0575] Desired gene segments are ordered according to given
specifications at Geneart (Regensburg, Germany).
[0576] DNA Sequence Determination
[0577] DNA sequences are determined by double strand sequencing
performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe
GmbH (Vaterstetten, Germany).
[0578] DNA and protein sequence analysis and sequence data
management
[0579] The GCG's (Genetics Computer Group, Madison, Wis.) software
package version 10.2 and Infomax's Vector NT1 Advance suite version
8.0 is used for sequence creation, mapping, analysis, annotation
and illustration.
[0580] Expression Vectors
[0581] For the expression of the described antibodies expression
vectors for transient expression (e.g. in HEK293-F cells) based
either on a cDNA organization with or without a CMV-Intron A
promoter or on a genomic organization with a CMV promoter are
used.
[0582] Beside the antibody expression cassette the vectors
contains: [0583] an origin of replication which allows replication
of this vector in E. coli, [0584] a .beta.-lactamase gene which
confers ampicillin resistance in E. coli, and [0585] the
dihydrofolate reductase gene from Mus musculus as a selectable
marker in eukaryotic cells.
[0586] The transcription unit of the antibody gene is composed of
the following elements: [0587] unique restriction site(s) at the 5'
end, [0588] the immediate early enhancer and promoter from the
human cytomegalovirus, [0589] in the case of the cDNA organization
followed by the Intron A sequence, [0590] a 5'-untranslated region
of a human immunoglobulin gene, [0591] a nucleic acid encoding an
immunoglobulin heavy chain signal sequence, [0592] a nucleic acid
encoding the human antibody chain (wild-type or with domain
exchange) either as cDNA or in genomic organization with the
immunoglobulin exon-intron organization, [0593] a 3' non-translated
region with a polyadenylation signal sequence, and [0594] unique
restriction site(s) at the 3' end.
[0595] The nucleic acids encoding the antibody chains are generated
by PCR and/or gene synthesis and assembled by known recombinant
methods and techniques by connection of the according nucleic acid
segments e.g. using unique restriction sites in the respective
vectors. The subcloned nucleic acid sequences are verified by DNA
sequencing. For transient transfections larger quantities of the
vectors are prepared by vector preparation from transformed E. coli
cultures (Nucleobond AX, Macherey-Nagel).
[0596] Cell Culture Techniques
[0597] Standard cell culture techniques are used as described in
Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso,
M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M.
(eds.), John Wiley & Sons, Inc.
[0598] The bispecific antibodies are expressed by transient
co-transfection of the respective expression vectors in HEK29-F
cells growing in suspension as described below.
Example 1
Expression and Purification
[0599] Transient Transfections in HEK293-F System
[0600] The monospecific and bispecific antibodies are generated by
transient transfection with the respective vectors (e.g. encoding
the heavy and modified heavy chain, as well as the corresponding
light and modified light chain) using the HEK293-F system
(Invitrogen) according to the manufacturer's instruction. Briefly,
HEK293-F cells (Invitrogen) growing in suspension either in a shake
flask or in a stirred fermenter in serum-free FreeStyle.TM. 293
expression medium (Invitrogen) are transfected with a mix of the
respective expression vectors and 293fectin.TM. or fectin
(Invitrogen). For 2 L shake flask (Corning) HEK293-F cells are
seeded at a density of 1*10.sup.6 cells/mL in 600 mL and incubated
at 120 rpm, 8% CO.sub.2. The day after the cells are transfected at
a cell density of approx. 1.5*10.sup.6 cells/mL with approx. 42 mL
mix of A) 20 mL Opti-MEM (Invitrogen) with 600 .mu.g total vector
DNA (1 .mu.g/mL) encoding the heavy or modified heavy chain,
respectively and the corresponding light chain in an equimolar
ratio and B) 20 ml Opti-MEM with 1.2 mL 293 fectin or fectin (2
.mu.L/mL). According to the glucose consumption glucose solution is
added during the course of the fermentation. The supernatant
containing the secreted antibody is harvested after 5-10 days and
antibodies are either directly purified from the supernatant or the
supernatant is frozen and stored.
[0601] Purification
[0602] Bispecific antibodies are purified from cell culture
supernatants by affinity chromatography using
MabSelectSure-Sepharose.TM., hydrophobic interaction chromatography
using butyl-Sepharose (GE Healthcare, Sweden) and Superdex 200 size
exclusion (GE Healthcare, Sweden) chromatography.
[0603] Briefly, sterile filtered cell culture supernatants are
captured on a MabSelectSure resin equilibrated with PBS buffer (10
mM Na.sub.2HPO.sub.4, 1 mM KH.sub.2PO.sub.4, 137 mM NaCl and 2.7 mM
KCl, pH 7.4), washed with equilibration buffer and eluted with 25
mM sodium citrate at pH 3.0. The eluted antibody fractions are
pooled and neutralized with 2 M Tris, pH 9.0. The antibody pools
are prepared for hydrophobic interaction chromatography by adding
1.6 M ammonium sulfate solution to a final concentration of 0.8 M
ammonium sulfate and the pH adjusted to pH 5.0 using acetic acid.
After equilibration of the butyl-Sepharose resin with 35 mM sodium
acetate, 0.8 M ammonium sulfate, pH 5.0, the antibodies are applied
to the resin, washed with equilibration buffer and eluted with a
linear gradient to 35 mM sodium acetate pH 5.0. The (monospecific
or bispecific) antibody containing fractions were pooled and
further purified by size exclusion chromatography using a Superdex
200 26/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM
histidine, 140 mM NaCl, pH 6.0. The (monospecific or bispecific)
antibody containing fractions were pooled, concentrated to the
required concentration using Vivaspin ultrafiltration devices
(Sartorius Stedim Biotech S. A., France) and stored at -80.degree.
C.
[0604] Purity and antibody integrity can be analyzed after each
purification step by CE-SDS using microfluidic Labchip technology
(Caliper Life Science, USA). Five .mu.L of protein solution is
prepared for CE-SDS analysis using the HT Protein Express Reagent
Kit according manufacturer's instructions and analyzed on Labchip
GXII system using a HT Protein Express Chip. Data are analyzed
using Labchip GX Software.
[0605] The aggregate content of antibody samples can be analyzed by
high-performance SEC using a Superdex 200 analytical size-exclusion
column (GE Healthcare, Sweden) in 2xPBS (20 mM Na.sub.2HPO.sub.4, 2
mM KH.sub.2PO.sub.4, 274 mM NaCl and 5.4 mM KCl, pH 7.4) running
buffer at 25.degree. C. 25 pg protein are injected on the column at
a flow rate of 0.75 mL/min and eluted isocratic over 50
minutes.
Example 2
FeRn Chromatography
[0606] Coupling to Streptavidin Sepharose:
[0607] One gram streptavidin Sepharose (GE Healthcare) is added to
the biotinylated and dialyzed receptor and incubated for two hours
with shaking. The receptor derivatized Sepharose is filled in a 1
mL XK column (GE Healthcare).
[0608] Chromatography Using the FcRn Affinity Column:
[0609] Conditions:
[0610] column dimensions: 50 mm.times.5 mm
[0611] bed height: 5 cm
[0612] loading: 50 .mu.g sample
[0613] equilibration buffer: 20 mM MES, with 150 mM NaCl, adjusted
to pH 5.5
[0614] elution buffer: 20 mM Tris/HCl, with 150 mM NaCl, adjusted
to pH 8.8
[0615] elution: 7.5 CV equilibration buffer, in 30 CV to 100%
elution buffer, 10 CV elution buffer
Sequence CWU 1
1
301107PRTHomo sapiens 1Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 20 25 30Trp Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val 35 40 45Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60Glu Ser Thr Tyr Arg Trp Ser
Val Leu Thr Val Leu His Gln Asp Trp65 70 75 80Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 85 90 95Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys 100 1052106PRTHomo sapiens 2Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40
45Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
50 55 60Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100
1053227PRTHomo sapiens 3Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120
125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys2254223PRTHomo sapiens 4Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
Val Ala Gly Pro Ser Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Phe Arg Val Val Ser65 70 75 80Val Leu Thr Val
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val
Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile 100 105 110Ser
Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120
125Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
130 135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu
Ser Asn145 150 155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg 180 185 190Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 195 200 205His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 2205227PRTHomo
sapiens 5Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu
Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val
Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Phe65 70 75 80Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser
Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150
155 160Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro
Pro 165 170 175Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe
Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn Arg Phe Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys2256229PRTHomo
sapiens 6Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
Glu Phe1 5 10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln Val Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Lys2257227PRTArtificial Sequencehuman IgG1 Fc-region derived
Fc-region polypeptide with the mutations L234A, L235A 7Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly1 5 10 15Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220Pro Gly Lys2258227PRTArtificial Sequencehuman
IgG1 Fc-region derived Fc-region polypeptide with Y349C, T366S,
L368A and Y407V mutations 8Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120
125Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys2259227PRTArtificial Sequencehuman IgG1 Fc-region derived
Fc-region polypeptide with S354C, T366W mutations 9Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65
70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser 130 135 140Leu Trp Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200
205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220Pro Gly Lys22510227PRTArtificial Sequencehuman IgG1
Fc-region derived Fc-region polypeptide with L234A, L235A mutations
and Y349C, T366S, L368A, Y407V mutations 10Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly1 5 10 15Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75
80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140Leu Ser Cys Ala Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200
205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220Pro Gly Lys22511227PRTArtificial Sequencehuman IgG1
Fc-region derived Fc-region polypeptide with a L234A, L235A and
S354C, T366W mutations 11Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120
125Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys22512227PRTArtificial Sequencehuman IgG1 Fc-region derived
Fc-region polypeptide with a P329G mutation 12Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75
80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro
Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120
125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys22513227PRTArtificial Sequencehuman IgG1 Fc-region derived
Fc-region polypeptide with L234A, L235A mutations and P329G
mutation 13Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150
155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys22514227PRTArtificial Sequencehuman IgG1 Fc-region derived
Fc-region polypeptide with a P239G mutation and Y349C, T366S,
L368A, Y407V mutations 14Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120
125Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys22515227PRTArtificial Sequencehuman IgG1 Fc-region derived
Fc-region polypeptide with a P329G mutation and S354C, T366W
mutation 15Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150
155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys22516227PRTArtificial Sequencehuman IgG1 Fc-region derived
Fc-region polypeptide with L234A, L235A, P329G and Y349C, T366S,
L368A, Y407V mutations 16Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120
125Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys22517227PRTArtificial Sequencehuman IgG1 Fc-region derived
Fc-region polypeptide with L234A, L235A, P329G mutations and S354C,
T366W mutations 17Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Ala Ala Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys22518229PRTArtificial Sequencehuman IgG4 Fc-region derived
Fc-region polypeptide with S228P and L235E mutations 18Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe1 5 10 15Glu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40
45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser65 70 75 80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ser 100 105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 115 120 125Gln Val Tyr Thr Leu Pro Pro Ser
Gln Glu Glu Met Thr Lys Asn Gln 130 135 140Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150 155 160Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185
190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 210 215 220Leu Ser Leu Gly Lys22519229PRTArtificial
Sequencehuman IgG4 Fc-region derived Fc-region polypeptide with
S228P, L235E mutations and P329G mutation 19Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe1 5 10 15Glu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65 70 75
80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Gly
Ser 100 105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 115 120 125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln 130 135 140Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala145 150 155 160Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220Leu Ser Leu Gly Lys22520229PRTArtificial Sequencehuman
IgG4 Fc-region derived Fc-region polypeptide with S354C, T366W
mutations 20Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
Glu Phe1 5 10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln Val Tyr
Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys Asn Gln 130 135 140Val
Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Lys22521229PRTArtificial Sequencehuman IgG4 Fc-region derived
Fc-region polypeptide with Y349C, T366S, L368A, Y407V mutations
21Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe1
5 10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln Val Cys Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140Val Ser Leu
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150 155
160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Lys22522229PRTArtificial Sequencehuman IgG4 Fc-region derived
Fc-region polypeptide with a S228P, L235E and S354C, T366W
mutations 22Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe1 5 10 15Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln Val Tyr
Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys Asn Gln 130 135 140Val
Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Lys22523229PRTArtificial Sequencehuman IgG4 Fc-region derived
Fc-region polypeptide with a S228P, L235E and Y349C, T366S, L368A,
Y407V mutations 23Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe1 5 10 15Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln
Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Val Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Lys22524229PRTArtificial Sequencehuman IgG4 Fc-region derived
Fc-region polypeptide with a P329G mutation 24Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe1 5 10 15Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45Ser Gln
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65 70 75
80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Gly
Ser 100 105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 115 120 125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln 130 135 140Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala145 150 155 160Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220Leu Ser Leu Gly Lys22525229PRTArtificial Sequencehuman
IgG4 Fc-region derived Fc-region polypeptide with a P239G and
Y349C, T366S, L368A, Y407V mutations 25Glu Ser Lys Tyr Gly Pro Pro
Cys Pro Ser Cys Pro Ala Pro Glu Phe1 5 10 15Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45Ser Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90
95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Gly Ser
100 105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 115 120 125Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln 130 135 140Val Ser Leu Ser Cys Ala Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu 180 185 190Thr Val Asp
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215
220Leu Ser Leu Gly Lys22526229PRTArtificial Sequencehuman IgG4
Fc-region derived Fc-region polypeptide with a P329G and S354C,
T366W mutations 26Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
Ala Pro Glu Phe1 5 10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Gly Ser 100 105 110Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln
Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Lys22527229PRTArtificial Sequencehuman IgG4 Fc-region derived
Fc-region polypeptide with a S228P, L235E, P329G and Y349C, T366S,
L368A, Y407V mutations 27Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
Cys Pro Ala Pro Glu Phe1 5 10 15Glu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Gly Ser 100 105 110Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120
125Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Val Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu
Gly Lys22528229PRTArtificial Sequencehuman IgG4 Fc-region derived
Fc-region polypeptide with a S228P, L235E, P329G and S354C, T366W
mutations 28Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe1 5 10 15Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Gly Ser 100 105 110Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln Val Tyr
Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys Asn Gln 130 135 140Val
Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Lys22529105PRThomo sapiens 29Gln Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu1 5 10 15Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 20 25 30Tyr Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45Lys Ala Gly Val Glu Thr
Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60Tyr Ala Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser65 70 75 80His Arg Ser
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95Lys Thr
Val Ala Pro Thr Glu Cys Ser 100 10530107PRTHomo sapiens 30Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25
30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
* * * * *